Patent Application
20050277204
Biological, chemical, and environmental studies often require the separation of particular targets from amongst a heterogeneous population of materials. Often, the separation of a particular target, as well as its further analysis, are hindered by factors including (a) a very low concentration of the target within the heterogeneous starting mixture of materials, (b) the presence of agents which degrade the target, (c) the presence of agents which interfere with the isolation of the target, and (d) the presence of agents which interfere with the analysis of target following its isolation. The most advantageous methods and compositions facilitate the separation of low concentrations of target from a wide range of either liquid or solid samples containing a heterogeneous mixtures of non-target materials. Such methods and compositions may be further modified or combined with existing methodologies to help maintain the integrity of the target (e.g., prevent its degradation or contamination) and/or to inhibit the activity of agents which interfere with the further analysis of the target (e.g., agents which interfere with PCR analysis of DNA samples, agents which interfere with mass spectroscopic analysis of protein samples, or agents which interfere with cytological analysis of bacteria or viruses).
Advances in fields including cell biology, molecular biology, chemistry, toxicology, and pharmacology have spawned a variety of techniques for analyzing biological materials, chemical materials, and environmental materials including, but not limited to, DNA, RNA, protein, bacterial cells and spores (including gram+ and gram−), viruses (including DNA based and RNA based), small organic molecules, and large chemical compounds. However, the efficient application of many powerful analytical tools is often hindered by an inability to separate a target material of interest away from a heterogeneous population of materials contained in a sample. The present invention provides methods, compositions, and apparatuses to facilitate the separation and/or identification of targets from environmental, biological, and chemical samples.
The present invention provides methods, compositions, and apparatuses which can be used to separate and/or identify a target from a heterogeneous mixture of agents. Separation of a target, which may be DNA, RNA, protein, bacterial cells or spores, viruses, small organic molecules, or chemical compounds, facilitates further analysis and identification of the target. The present invention has a wide range of forensic, medical, environmental, industrial, public health, and anti-bioterrorism applications, and is suitable for use in separating targets from a wide range of gaseous, liquid, and solid samples.
In a first aspect, the present invention provides an improved method for separating a target from a heterogeneous sample. In one embodiment, the method comprises contacting the sample containing a target of interest with a substrate capable of binding the target with a higher affinity than the affinity of the substrate for non-target materials. In another embodiment, the surface of the substrate is coated with a modifying agent that further increases the affinity of the substrate for one or more particular targets. In another embodiment, the substrate is coated with one or more of the amine containing modifying agents disclosed herein. The use of either magnetic or non-magnetic substrates coated with one or more simple modifying agents is a significant advance over separation technologies that rely on separation or detection of targets using beads coated with antibodies that are immunoreactive with a particular target. Not only are the simple modifying agents disclosed herein cheaper and easier to produce than antibody coated beads, but they are also of more general applicability and do not require identification and production of antibodies immunoreactive with each and every possible target of interest. The need for such extensive information of possible targets is a significant limitation to the general applicability and cost effectiveness of previously available technologies. Furthermore, antibodies are prone to denaturing and degradation when exposed to chemicals and components present in environmental samples such as soils, whereas the simple modifying agents disclosed herein are more robust than antibodies against such degradation.
The target can be DNA, RNA, protein, bacterial cells or spores, viruses, small organic molecules, or chemical compounds. Furthermore, target DNA, RNA, or protein can be derived from human or non-human animals, plants, bacteria, viruses, fungi, or protozoa. The invention contemplates the use of this method alone or in combination with the previously disclosed SNAP/MITLL methodology for analyzing nucleic acids under conditions which inhibit the degradation of the nucleic acid or the contamination of the nucleic acid sample with agents that inhibit the further analysis of the target nucleic acid.
Following separation of target using either methodology, the target can be further analyzed using routine techniques in cell biology, molecular biology, chemistry, or toxicology. The particular technique can be selected based on the target, and one of skill in the art can readily select an appropriate technique(s). In one embodiment, the target is DNA obtained from a particular biological or environmental sample, and further analysis of the DNA may involve PCR analysis of the DNA. The DNA may be of human, animal, bacterial, plant, fungal, protozoan, or viral origin depending on the particular application of the technology. In another embodiment, the target is RNA obtained from a particular biological or environmental sample, and further analysis of the RNA may involve RT-PCR analysis of the RNA or in situ hybridization analysis of RNA. The RNA may be of human, animal, bacterial, plant, fungal, protozoan, or viral origin. In still another embodiment, the target is a bacterial cell or spore obtained from a particular biological or environmental sample. Further analysis may involve analysis of the bacterial cell or spore itself. Exemplary methods for analyzing the cells or spores include, but are not limited to, microscopy, culture, cytological testing, and the analysis of bacterial cell surface markers. Additionally, analysis of the target bacterial cell or spore may involve analysis of DNA or RNA prepared from the target cell or spore, as well as analysis of both the cell or spore itself and DNA or RNA prepared from the target cell or spore. In yet another embodiment, the target is a protein obtained from a particular biological or environmental sample. The protein may be of human, animal, bacterial, plant, fungal, protozoan, or viral origin depending on the particular application of the technology. Further analysis of the protein may involve peptide sequencing, mass spectroscopy, and 1 or 2-dimensional gel electrophoresis.
In a second aspect, the present invention provides particular surface modifying agents that can be coupled to the surface of a substrate. Substrates modified with one or more surface modifying agents have an increased affinity for particular targets in comparison to either unmodified substrates or substrates modified with other surface modifying agents. The invention contemplates modification of a wide range of substrates including, but not limited to plates, chips, coverslips, culture vessels, tubes, beads, probes, fiber-optics, filters, cartridges, strips, and the like. Furthermore, the invention contemplates that such substrates can be composed of any of a wide range of materials including, but not limited to, plastic, glass, metal, and silica, and furthermore that the materials may possess magnetic or paramagnetic characteristics. As can be construed from the list of exemplary substrates, a suitable substrate can be virtually any size or shape, and one of skill in the art can readily select a suitable substrate based on the particular target as well as the particular materials from which the target must be analyzed.
In one embodiment, a substrate is modified with one surface modifying agent. In another embodiment, a substrate is modified with two or more surface modifying agents. In still another embodiment, the surface modifying agent is coupled to the substrate via a cleavable linker which allows the release of the modifying agent from the substrate. When multiple surface modifying agents are used, the agents may each have an increased affinity for the same target, or the agents may have an increased affinity for different targets so that the modified substrates are capable of separating more than one target. Furthermore, when multiple surface modifying agents are used, the agents may each have the same affinity for a particular target or the agents may have varying affinities for a particular target.
In a third aspect, the present invention provides apparatuses which can be used to separate targets from biological, chemical or environmental samples. The invention includes two classes of apparatuses. The first class includes apparatuses which facilitate the interaction between substrates and samples. Such apparatuses are particularly important for large scale implementation of the methods of the present invention. By way of example, when separating targets from small samples of soil, water, air, or bodily fluids, the efficient delivery of modified substrate to the sample containing the target is straightforward. In such settings, it is relatively easy to insure that the entire sample is contacted with substrate, and thus the substrate has an opportunity to interact with target throughout the entire sample. However, when larger samples are involved, it is a less straightforward process to ensure that the substrate contacts target which may be distributed evenly or unevenly throughout the large sample. For such applications, the invention provides a device for facilitating the even mixing of substrate throughout large samples containing target. One example which illustrates an application of this apparatus is in industrial food-processing facilities. Large vessels containing food, beverage, or ingredients for the production of various foods or beverages may become contaminated with bacteria, viruses, or chemicals during processing or storage. However, the efficient detection of such potentially harmful contaminants may be hindered by the large volumes of sample. One application of this first class of apparatus is in the food-processing industry where the apparatus could be used to regularly and efficiently evaluate the quality of large volumes of food or ingredients.
The second class of apparatuses provides alternative coated substrates, such as filters and cartridges, which can be used to readily process a sample containing a target. These apparatuses have a wide range of biological, environmental, and industrial applications, and can be used to efficiently analyze solid, liquid, or gaseous samples. Of particular note, filters and cartridges which analyze sample based on the Affinity Protocol can be used alone or can be used in combination with other available filters and cartridges. Filters and cartridges can be used in any of a variety of settings.
Of particular note, the methods, compositions, and apparatuses of the present invention can be used in a traditional laboratory or hospital setting, or in the field where access to other laboratory equipment and supplies may be limited. Furthermore, using the compositions and apparatuses of the present invention, the separation methods can be performed in less time than other traditional separation methodologies. The ability to perform rapid analysis of samples is crucial in any of a number of laboratory and field applications. By way of example, decreased sample analysis time can allow doctors and hospitals to provide immediately to patients the results of diagnostic tests. This shortens the time prior to which treatment can begin and decreases the risk of patient flight and noncompliance. By way of further example, rapid analysis facilitates crime scene investigations. By way of still further analysis, rapid analysis of environmental pollution facilitates correlating the pollution with particular industrial or natural events.
In any of the foregoing, the separation methods of the present invention (whether implemented using filters, cartridges, or other substrates) can be performed in less than 30 minutes. In another embodiment, the separation methods can be performed in less than or equal to 25, 20, 15, 14, 13, 12, 11, 10, 9, or 8 minutes. In yet another embodiment, the separation methods can be performed in less than or equal to 7, 6, 5, or 4 minutes. Targets separated using the methods of the present invention can, optionally, be further analyzed using other rapid analytical techniques.
In any of the foregoing, the time required to carry out the separation methods of the present invention (whether implemented using filters, cartridges, or other substrates) includes the time required for binding of target to substrate (e.g., capture time) and may also include the time required to release the target from the substrate (e.g., elution time). In one embodiment, the capture time can be less than or equal to 30, 25, 20, 15, 14, 13, 12, 11, 10, 9, or 8 minutes. In another embodiment, the capture time can be less than or equal to 7, 6, 5, 4, 3, 2, or 1 minutes. In another embodiment, the capture time can be 5-10 minutes, 1-5 minutes, 1 minute, or less than 1 minute. Targets captured by the methods of the present invention can, optionally, be eluted from the substrate. Eluted targets can, optionally, be further analyzed using other rapid analytical techniques.
In another embodiment, the elution time can be less than or equal to 30, 25, 20, 15, 14, 13, 12, 11, 10, 9, or 8 minutes. In another embodiment, the elution time can be less than or equal to 7, 6, 5, 4, 3, 2, or I minutes. In another embodiment, the elution time can be 5-10 minutes, 1-5 minutes, 1 minute, or less than 1 minute. Targets eluted by the methods of the present invention can, optionally, be further analyzed using other rapid analytical techniques.
In any of the foregoing, the separation methods of the present invention may require the use of an effective amount of a substrate. Although the use of a larger concentration of substrate may be advantageous in certain applications, the use of a minimal concentration of substrate helps reduce the cost of the method and helps increase its ease of use in the field (e.g., reduces the amount of consumable reagents required for use). In one embodiment, the amount of substrate is greater than 10 mg/mL of sample. In one embodiment, the amount of substrate is less than or equal to 10 mg/mL of sample. In another embodiment, the amount of substrate is less than or equal or 7, 6, or 5 mg/mL of sample. In still another embodiment, the amount of substrate is less than or equal to 4, 3, 2, or 1 mg/mL of sample. In still another example, the amount of substrate is 5-10 mg/ml of sample or 1-5 mg/mL of sample.
In a fourth aspect, the invention provides particular cartridges or devices that can be used to separate a target from a heterogeneous sample. Exemplary devices are multi-chambered devices. Further exemplary devices comprise two or three chambers. Such devices can optionally contain one or more valves that reversibly modulate the passage of material between the chambers.
In one embodiment, the device comprises an input chamber. The input chamber may have or otherwise contain a substrate that binds to a target to form a target-substrate complex. Exemplary substrates are described in detail in the application. For example, exemplary substrates are coated or uncoated substrates which bind to the target with higher affinity than to non-target materials. The device may further comprises a collector element that attracts or associates with the substrate; and an eluate chamber. The collector element attracts or associates with the substrate, thereby separating the target-substrate complex away from the remainder of the heterogeneous sample.
In one embodiment, the device can be used to separate multiple targets from a heterogeneous sample, for example, two or more targets.
In one embodiment, the device can be manufactured to include substrate within the input chamber. In another embodiment, the device can be manufactured without substrate in the input chamber. For such embodiments in which the device is manufactured without the substrate, substrate can be added prior to device use. Substrate added prior to device use can be supplied along with the device as a kit comprising the device and one or more substrates. Alternatively, substrate can be supplied separately by the end-user or a third party.
In one embodiment, the eluate chamber comprises an amount of an elution buffer sufficient to elute the target from the substrate, thereby separating the target from the substrate.
In one embodiment, the substrate is a magnetic or paramagnetic substrate. In another embodiment, the substrate is modified with one or more surface modifying agents. In still another embodiment, the one or more surface modifying agents are appended to the substrate via a cleavable linker. In yet another embodiment, the substrate comprises one or more magnetic beads.
In one embodiment, the collector element is included within a processing chamber. In another embodiment, the collector element is not included within a processing chamber. When the collector element is not included within the processing chamber, an external collector element can be employed. Alternatively, the collector element can be included within the device, for example, within another chamber or within a valve. Exemplary collector elements include, but are not limited to, one or more collection magnets. Exemplary collection magnets can comprise or otherwise be arrayed in a number of configurations. Such collection magnets include a single magnetic sphere, a cylindrical stack of one or more magnets, or an open or closed chain of multiple magnetic spheres. In embodiments in which the collector element is contained within the device (e.g., contained within a chamber or a valve of the device), the invention contemplates that the collector element can be removable. Once removed from the remainder of the device, the collector element may be, for example, further analyzed, discarded, or reused as an external collector element or as a collector element within another device.
In one embodiment of any of the foregoing, the device may further comprise a valve which reversibly modulates the passage of material between an input chamber and a processing chamber. In another embodiment, the device may further comprise a valve which reversibly modulates the passage of material between the processing chamber and the eluate chamber. In still another embodiment, the device may further comprise the following two valves: a first valve which reversibly modulates the passage of material between an input chamber and a processing chamber and a second valve which reversibly modulates the passage of material between the processing chamber and the eluate chamber.
In another embodiment, the device may comprise a cap portion that reversibly seals an open end of the device. For example, the device may further comprise a first cap portion that reversibly seals an open end of the input chamber and/or a second cap portion that reversibly attaches to the eluate chamber.
For embodiments in which the device comprises one or more cap portions, the invention contemplates numerous configurations of cap portions. In one embodiment, the cap portion (the first cap portion and/or the second cap portion) comprises a plug portion and a handle portion. When the device contains two cap portions comprising plug and handle portions, the first and second plug and handle portions may be the same or different.
In a fifth aspect, the invention provides a filter device having a chamber with three sections and a filter passage through the sections with operable valves separating the chambers.
In one embodiment, the filter passage comprises a substrate that binds to a target to form a target-substrate complex. Exemplary substrates bind to the target with higher affinity than to non-target materials.
In another embodiment, the three sections comprise an input chamber, a processing chamber, and an eluate chamber.
In a sixth aspect, the invention provides a filter device having a chamber with two sections and a filter passage through the sections with an operable valve separating the chambers.
In one embodiment, the filter passage comprises a substrate that binds to a target to form a target-substrate complex. Exemplary substrates bind to the target with higher affinity than to non-target materials.
In another embodiment, the two sections comprise an input chamber and an eluate chamber. In another embodiment, the input chamber contains a valve which reversibly modulates the passage of material between an input chamber and a processing chamber.
In a seventh aspect, the invention provides a device for separating a target from a heterogeneous sample. The device comprises an input chamber. The input chamber can contain a substrate that binds to a target to form a target-substrate complex. Exemplary substrates bind to the target with higher affinity than to non-target materials. The device further comprises a first valve, a processing chamber, a second valve, and an eluate chamber. The first valve reversibly modulates the passage of material between the input chamber and the processing chamber and the second valve reversibly modulates the passage of material between the processing chamber and the eluate chamber.
In one embodiment, the processing chamber comprises a collector element that attracts or associates with the substrate. In another embodiment, the collector element is not contained within the processing chamber. In still another embodiment, the first or second valve comprises the collector element. In any embodiment in which the collector element is contained within a chamber or valve of the device, the invention contemplates that the collector element may be removable.
In an eighth aspect, the invention provides a device for separating a target from a heterogeneous sample. The device comprises an input chamber having a substrate that binds to a target to form a target-substrate complex. Exemplary substrates bind to the target with higher affinity than to non-target materials. The device further comprises a valve and an eluate chamber. The valve may be physically separate from the input chamber. Alternatively the valve may comprise or otherwise contain the input chamber. Accordingly, such devices represent two possible configurations for 2 chamber cartridges.
In one embodiment, the valve reversibly modulates the passage of material between the input chamber and the eluate chamber. In another embodiment, the valve comprises a collector element that attracts or associates with the substrate. In another embodiment, the collector element is not within the valve. In any embodiment in which the collector element is contained within a chamber or valve of the device, the invention contemplates that the collector element may be removable.
In another embodiment, the eluate chamber comprises an amount of an elution buffer sufficient to elute the target from the substrate, thereby separating the target from the substrate.
Certain embodiments of the invention are contemplated in combination with any of the foregoing aspects or embodiments of the invention. The substrate can be a magnetic or paramagnetic substrate. The substrate can be modified with one or more surface modifying agents. The one or more surface modifying agents can be appended to the substrate via a cleavable linker. The substrate can comprise one or more magnetic beads.
The device can be manufactured to include substrate within the input chamber. In another embodiment, the device can be manufactured without substrate in the input chamber. For such embodiments in which the device is manufactured without the substrate, substrate can be added prior to device use. Substrate added prior to device use can be supplied along with the device as a kit comprising the device and one or more substrates. Alternatively, substrate can be supplied separately by the end-user or a third party.
In embodiments including a collector element, the collector element can be included within the processing chamber, within a valve, or outside of (e.g., not contained within) the device. When the collector element is not included within the processing chamber, an external collector element can be employed. Furthermore, the collector element can comprise a collection magnet. Exemplary collector elements include, but are not limited to, one or more collection magnets. Exemplary collection magnets can comprise or otherwise be arrayed in a number of configurations. Such collection magnets include a single magnetic sphere, a cylindrical stack of one or more magnets, or a chain of multiple magnetic spheres.
In another embodiment, the device may comprise a cap portion that reversibly seals an open end of the device. For example, the device may comprise a first cap portion that reversibly seals an open end of the input chamber and/or a second cap portion that reversibly attaches to the eluate chamber.
For embodiments in which the device comprises one or more cap portions, the invention contemplates numerous configurations of cap portions. In another embodiment, the cap portion (the first cap portion and/or the second cap portion) comprises a plug portion and a handle portion. When the device contains two cap portions comprising plug and handle portions, the first and second plug and handle portions may be the same or different.
In a ninth aspect, the invention provides a method of separating a target from a heterogeneous sample using any of the foregoing aspects or embodiments of the devices or cartridges of the invention.
In one embodiment, the target is a eukaryotic cell, archaea, bacterial cell or spore, or viral particle. In another embodiment, the target is DNA, RNA, a protein, a small organic molecule, or a chemical compound.
In one embodiment, the heterogeneous sample is a liquid sample. In another embodiment, the heterogeneous sample is a dry sample. When the sample is a dry sample, the method may optionally comprise adding liquid to the sample (e.g., to form a slurry containing the sample) prior to addition of the sample to the input chamber. When liquid is added to the sample, the liquid may be a buffer, water, serum, or other suitable liquid. In a preferred embodiment, the liquid used to suspend the dry sample is otherwise free of target or other contaminants that may interfere with the analysis of the sample. Particularly preferred liquid includes, without limitation deionized water, purified water, treated water, autoclaved water, or buffer prepared using any of the foregoing.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.
a depicts a device with relatively small handles integrated into cap pieces that plug the input chamber and attach to the eluate chamber.
b depicts a device with relatively large handles integrated into cap pieces that plug the input chamber and attach to the eluate chamber.
a provides a close-up view of an input chamber.
b provides a close-up view of an input chamber with the cap removed.
a provides a close-up view of an eluate chamber.
b provides a close-up view of an eluate chamber removed from the cartridge body.
a depicts the following three configurations of collector elements: a single sphere, a multi-sphere chain, and a cylindrical stack. The collector elements shown in panel (a) are shown next to a penny to indicate their relative size.
b shows a close up view of a collector element in a multi-sphere chain configuration.
c shows a close up view of a collector element in a 2-cylinder stack configuration.
d shows a collector element submerged within an eluate chamber. The collector element depicted in panel (d) is in a 2-cylinder stack configuration.
a and the exploded view provided in
b depict a two-chambered device. Note that in this embodiment of a two-chambered device, the collector element is integrated into a valve. In this embodiment, the device comprises a single valve that reversibly modulates passage of materials between the input chamber and the eluate chamber.
a and the exploded view provided in
b depict another embodiment of a two-chambered device. Note that in this embodiment of a two-chambered device, the collector element is integrated into a valve. In this embodiment, the device comprises a single valve that reversibly modulates passage of materials between the input chamber and the eluate chamber.
c shows a close-up view of the collector element within a holder. The collector element depicted in the figure is a collection magnet in a single-cylinder stack configuration.
d shows the collector element within a holder and integrated into a valve.
(i) Overview
The biological, chemical, and environmental sciences often require the analysis of targets which must first be separated or otherwise detected from a heterogeneous population of materials. This process may be further complicated by the presence within a sample of contaminants that may degrade the target or otherwise inhibit the later analysis of the target. The present invention provides methods, compositions, and apparatuses for use in the purification of targets from heterogeneous populations of materials. These methods, compositions, and apparatuses can be used for a wide range of targets (e.g., DNA, RNA, protein, bacteria and bacterial spores (including gram+ and gram−), viruses (including DNA-based and RNA-based), small organic molecules, and chemical compounds) and have a variety of biological, chemical, and environmental applications.
The improved methods and compositions outlined in detail herein greatly enhance the ability to separate or otherwise detect targets from a wide range of gaseous, liquid, and solid samples. Additionally the present invention can be combined with previously described methods and apparatuses that help to maintain the integrity of the target during its separation and prior to further analysis. Such methods and compositions which help maintain the integrity of targets are described in detail in copending U.S. patent publication 2003/0129614, filed Jul. 10, 2003, which is hereby incorporated by reference in its entirety. Briefly, U.S. patent publication 2003/0129614 discloses methods and compositions designed to facilitate analysis of nucleic acids by processing the nucleic acids in the presence of compositions that inhibit degradative agents. By way of example, agents within a sample can degrade nucleic acids such as DNA. This degradation both decreases the concentration of DNA in a given sample and also decreases the quality of that DNA such that it may be difficult to process the DNA for further analysis in assays such as PCR.
Applications
There are many potential applications of the methods, compositions, and apparatuses of the present invention. For example, many assays used in forensic sciences require the purification of DNA, protein, or small organic molecules such as non-peptide hormones from amongst a complex sample. Such samples include human or animal fluid or tissues including, but not limited to, blood, saliva, sputum, urine, feces, skin cells, hair follicles, semen, vaginal fluid, bone fragments, bone marrow, brain matter, cerebro-spinal fluid, amniotic fluid, and the like. The purification and further analysis of target from these complex samples is hindered by (a) an often low concentration of target within the sample, (b) degradation of the sample by either environmental contaminants or by agents within the sample which degrade target over time, and (c) the presence of agents within these complex bodily fluids which interfere with techniques needed to analyze the target following its purification. Accordingly, the present invention has substantial application to the forensic sciences and would enhance the ability to analyze biological samples. Additionally we note that the methods and compositions of the present invention can be used effectively to separate target from mixtures of materials that may be present in a “dirty” environment such as soil or water. Accordingly, the present invention facilitates forensic and other studies performed not only on samples of fresh bodily fluids provided directly from individuals or found in a relatively undisturbed environment, but additionally can be used to analyze sample which must be recovered from soil, water (including fresh or salt water), or other sources which may contain a higher concentration of contaminants and other degradatory agents. Accordingly, the methods, compositions, and apparatuses of the present invention are broadly applicable to the analysis of biological materials in a laboratory, hospital, or doctor's office setting, as well to the analysis of biological materials in the field by police, medical examiners, emergency medical technicians, criminal investigators, Haz-mat personnel, and other field-based workers.
The application of the present invention in the biological sciences is not limited, however, to forensics. Advances in medical and genetic testing are already beginning to change the way in which we approach healthcare. A range of diagnostic tests are available or are currently being developed. Such tests rely upon the ability to analyze a particular target (DNA, protein, hormone) contained within a sample of human or animal fluid or tissue. Accordingly, the present invention can be used to further improve the ease and efficiency with which biological samples are analyzed. Additionally, given that the methods and compositions of the present invention allow the separation of smaller quantities of target, use of these methods and compositions in a diagnostic setting will help decrease the amount of sample that must be harvested from a particular patient. Additionally, the present invention provides methods that allow separation of targets from a wide range of samples at previously unattainable speeds and using minimal reagents. The ability to analyze samples quickly and at a reduced cost is advantageous in the health care and medical industry, as well as in many of the other applications of the invention outlined in detail herein.
By way of further example, the present invention can be used to screen blood, blood products, or other pre-packaged medical supplies to insure that these supplies are free from particular contaminants such as bacteria and viruses.
In addition to medical applications, the present invention has a variety of environmental uses. Water, soil, or air samples can be analyzed for the presence of particular targets. Such targets include DNA, RNA, protein, small organic molecules, chemical compounds, bacterial cells or spores (including gram+or gram-), and viruses (including DNA-based and RNA-based). DNA, RNA, and protein can be derived from humans, non-human animals, plants, bacteria, fungi, protozoa, and viruses. For example, samples of water collected from local ponds, lakes, and beaches can be analyzed to assess the presence and concentration of potentially harmful bacteria or chemical pollutants. Such analysis can be used to monitor the health of these water sources and to evaluate their safety for human recreation. Similarly, samples of soil can be collected and analyzed to assess levels of contamination from natural or industrial sources.
By way of further example, cartridges and filters containing the compositions of the present invention can be used to monitor air and water supplies. Such cartridges and filters can be used to assess air quality in buildings, airplanes, and other closed environments which rely on recirculating air. Furthermore, such cartridges can be used in fish tanks, aquariums, and the like to help monitor water quality and to help pinpoint the source of any changes to water quality.
A final non-limiting example of applications of the present invention can be widely classified in the field of home-land security. Given the threat of warfare employing biological and/or chemical weapons, methods and compositions which can be used to identify the presence of biological or chemical agents in food, water, soil, or air have tremendous possible applications. For example, samples of water and soil surrounding local reservoirs or other likely sources of attack could be collected and analyzed for the presence of biological or chemical contaminants. Furthermore, cartridges and filters can be used to monitor the air (either outside or within buildings, trains, airplanes, or other vehicles) for the presence of biological or chemical contaminants. The invention contemplates that biological contaminants can be identified by either the detection of DNA or RNA from a particular biological agent (such as a bacteria or virus) or by the detection of the bacteria or virus itself. Chemical contaminants may be identified by detection of the organic molecule itself, as well as by detection of its chemical by-products or metabolites. Exemplary biological and chemical agents which may be detected include anthrax, ricin, brucellosis, smallpox, plague, Q-fever, tularemia, botulism, staphylococcus, and viral hemorrhagic fevers including Ebola, mustard gas, Clostridium Perfringens, camelpox, sarin, soman, O-ethyl S-diisopropylaminomethyl methylphosphonothiolate, tabun, and hydrogen cyanide. Exemplary viruses of clinical and environmental relevance can be categorized based on their genome type and whether they are enveloped and include (i) single-stranded, positive sense strand, enveloped, RNA viruses; (ii) single-stranded, positive sense strand, non-enveloped, RNA viruses; (iii) single-stranded, negative sense stranded, enveloped, RNA viruses; (iv) double-stranded, non-enveloped, RNA viruses; and (v) double-stranded, enveloped, DNA viruses. Single-stranded, positive sense strand, enveloped, RNA viruses include, but are not limited to, Eastern equine encephalitis, Western equine encephalitis, Venezuelan equine encephalitis, St. Louis encephalitis, SARS, Hepatitis C, HIV, and West Nile virus. Single-stranded, positive sense stranded, non-enveloped, RNA viruses include, but are not limited to, Norwalk virus, Hepatitis A, and Rhinovirus. Single-stranded, negative sense stranded, enveloped, RNA viruses include, but are not limited to, Ebola, Marburg, and Influenza. Double-stranded, non-enveloped, RNA viruses include, but are not limited to, Rotavirus. Double-stranded, enveloped, DNA viruses include, but are not limited to, Hepatitis B and Variola major.
For each of the potential forensic, medical, diagnostic, environmental, industrial, and, safety applications of the invention outlined above, the invention contemplates the use of the methods, apparatuses, and compositions of the present invention to separate and/or identify target from the heterogeneous sample. Thus, these methods, compositions, and apparatuses are useful not only for further analysis of a particular target and sample, but also for removing a target (e.g., an unwanted target) from a sample. Exemplary uses of the invention for removing target include in decontamination of a sample. Following separation (e.g., removal; physical separation) of all or a portion of a target from a sample, the sample can be handled more safely than prior to removal of the target. The separated target can either be discarded (e.g., discarded appropriately in light of the nature of any hazard that may be associated with the target) or can be further studied using reagents and precautions appropriate in light of the nature of any hazard that may be associated with the target.
(ii) Definitions
For convenience, certain terms employed in the specification, examples, and appended claims are collected here. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.
The term “target” is used to refer to a particular molecule of interest. Exemplary targets include DNA, RNA, protein, gram+ bacteria, gram− bacteria, bacterial spores, DNA and RNA-based viruses (including retroviruses), small organic molecules (including non-peptide hormones), and chemical compounds. DNA, RNA, and protein can be derived from humans, non-human animals, plants, fungi, protozoa, bacteria, and viruses. For any of the foregoing targets, the invention contemplates the purification of the general class of target (e.g., all DNA in a sample), as well as the purification of a particular species of a class of target (e.g., a particular bacteria or an antibody against a given antigen). In the context of the present invention, the target is that molecule that is substantially purified from a heterogeneous sample using the methods, compositions, and apparatuses of the present invention.
The term “sample” is used to refer to the heterogeneous mixture of biological, chemical, or environmental material. The methods, compositions, and apparatuses of the present invention allow the separation, detection, or substantial purification of a particular target from the sample. A sample can be gaseous, liquid or solid (e.g., either wet solid samples or dry solid sample), and can include biological, chemical, or environmental material. Exemplary biological samples include, but are not limited to, blood, saliva, sputum, urine, feces, skin cells, hair follicles, semen, vaginal fluid, bone fragments, bone marrow, brain matter, cerebro-spinal fluid, and amniotic fluid. Exemplary environmental samples include, but are not limited to, soil, water, non-laboratory-grade environmental water, sludge, air, plant and other vegetative matter, oil, liquid mineral deposits, and solid mineral deposits. The invention further contemplates the application of these methods and compositions in many commercial and industrial applications including the purification of contaminants during food processing or the production of other commercial products.
The term “substrate” is used to refer to any surface which can be modified or otherwise coated with a “surface modifying agent” in order to promote or enhance the interaction between the coated substrate and one or more targets. Substrates may vary widely in size and shape, and the particular substrate may be readily selected by one of skill in the art based on the modifying agent, the target, the sample, and other facts specific to the particular application of the invention. Exemplary substrates include, but are not limited to, magnetic beads, non-magnetic beads, tubes (e.g., polypropylene tubes, polyurethane tubes, etc.), glass slides or coverslips, chips, cassettes, filters, cartridges, and probes including fiber-optic probes.
The surface modifying agent may be coupled to the substrate covalently or non-covalently, and the surface modifying agent may optionally contain a cleavable linker such that the active region of the surface modifying agent can be released from the substrate. The term “active region” is used to refer to the portion of the modifying agent containing a region that interacts with the target. In embodiments in which the modifying agent contains a cleavable linker, cleavage of the linker releases target+the active region of the modifying agent while leaving some portion of the modifying agent attached to the substrate.
The term “Affinity Protocol” or “AP” is used to refer to the method by which a target is substantially purified or otherwise separated from a sample by contacting the sample with a substrate. The surface of the substrate may be coated with a modifying agent to promote or enhance the interaction between the substrate and a specific target.
The term “Affinity Magnet Protocol” or “AMP” is used to refer to embodiments of the AP method in which the substrate has magnetic characteristics. Similarly to substrates used in the AP method, substrates used for the AMP method may be coated with a modifying agent to promote or enhance the interaction between the substrate and a specific target.
The Affinity Protocol and Affinity Magnet Protocol includes a target capture phase where target and substrate interact to form a target-substrate complex. The time required for the binding of target and substrate to form a target-substrate complex is referred to herein as “capture time.” By “binding of target and substrate to form a target-substrate complex” is meant sufficient interaction between target and substrate such that greater than 50% (e.g., at least 51%) of the target in a sample binds to substrate to form a target-substrate complex. In certain embodiments, greater than 60%, 70%, 75%, 80%, 85%, 90%, or greater than 95% of target in a sample binds to substrate to form a target-substrate complex.
In certain applications of the AP and AMP, target-substrate complexes are disrupted and bound target is eluted from the substrate. The time required to elute target from substrate is referred to herein as “elution time.” By “eluting or removing of target from substrate to disrupt a target-substrate complex” is meant disruption of greater than 50% (e.g., at least 51%) of the target-substrate complexes. In certain embodiments, greater than 60%, 70%, 75%, 80%, 85%, 90%, or greater than 95% of target in a sample previously bound to target is eluted.
The term “coupling region” refers to the portion of the modifying agent that interacts with the substrate.
The term “MITLL protocol” and “SNAP/MITLL protocol” and SNAP/MITLL” will be used interchangeably throughout to refer to the methods outlined in detail in copending U.S. publication No. 2003/0129614 (U.S. application Ser. No. 10/193,742). Alternatively, the methods contained within U.S. publication No. 2003/0129614 are interchangeably referred to as “SNAP” or “SNAP method”, or “SNAP protocol.” As used herein, the use of these terms is not meant to be limited to the use of the particular devices and apparatuses presented in the copending application, but rather is meant to refer to the general method used to isolate a nucleic acid sample under conditions that inhibit degradation of the nucleic acid sample and/or inhibit agents within the sample that interfere with further processing and analysis of the sample (e.g., agents that inhibit analysis of the sample by PCR or RT-PCR).
Herein, the term “aliphatic group” refers to a straight-chain, branched-chain, or cyclic aliphatic hydrocarbon group and includes saturated and unsaturated aliphatic groups, such as an alkyl group, an alkenyl group, and an alkynyl group.
The terms “alkenyl” and “alkynyl” refer to unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond respectively.
The terms “alkoxyl” or “alkoxy” as used herein refers to an alkyl group, as defined above, having an oxygen radical attached thereto. Representative alkoxyl groups include methoxy, ethoxy, propyloxy, tert-butoxy and the like. An “ether” is two hydrocarbons covalently linked by an oxygen.
The term “alkyl” refers to the radical of saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. In preferred embodiments, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C1-C30 for straight chains, C3-C30 for branched chains), and more preferably 20 or fewer. Likewise, preferred cycloalkyls have from 3-10 carbon atoms in their ring structure, and more preferably have 5, 6 or 7 carbons in the ring structure.
Moreover, the term “alkyl” (or “lower alkyl”) as used throughout the specification, examples, and claims is intended to include both “unsubstituted alkyls” and “substituted alkyls”, the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents can include, for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety. It will be understood by those skilled in the art that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate. For instance, the substituents of a substituted alkyl may include substituted and unsubstituted forms of amino, azido, imino, amido, phosphoryl (including phosphonate and phosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl and sulfonate), and silyl groups, as well as ethers, alkylthios, carbonyls (including ketones, aldehydes, carboxylates, and esters), —CF3, —CN and the like. Exemplary substituted alkyls are described below. Cycloalkyls can be further substituted with alkyls, alkenyls, alkoxys, alkylthios, aminoalkyls, carbonyl-substituted alkyls, —CF3, —CN, and the like.
Unless the number of carbons is otherwise specified, “lower alkyl” as used herein means an alkyl group, as defined above, but having from one to ten carbons, more preferably from one to six carbon atoms in its backbone structure. Likewise, “lower alkenyl” and “lower alkynyl” have similar chain lengths. Throughout the application, preferred alkyl groups are lower alkyls. In preferred embodiments, a substituent designated herein as alkyl is a lower alkyl.
The term “heteroalkyl” as used throughout the specification, examples, and claims is intended to include both “unsubstituted alkyls” and “substituted alkyls,” (the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone) in which one or more carbons of the hydrocarbon backbone is replaced by an atom of any element other than carbon or hydrogen. Preferred heteroatoms are boron, nitrogen, oxygen, phosphorus, sulfur, and selenium.
The term “alkylthio” refers to an alkyl group, as defined above, having a sulfur radical attached thereto. Representative alkylthio groups include methylthio, ethylthio, and the like.
The terms “amine” and “amino” are art-recognized and refer to both unsubstituted and substituted amines, e.g., a moiety that can be represented by the general formula:
wherein R9, R10 and R′10 each independently represent a hydrogen, an alkyl, an alkenyl, —(CH2)m—R8, or R9 and R10 taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure; R8 represents an aryl, a cycloalkyl, a cycloalkenyl, a heterocycle or a polycycle; and m is zero or an integer in the range of 1 to 8. In preferred embodiments, only one of R9 or R10 can be a carbonyl, e.g., R9, R10 and the nitrogen together do not form an imide. In even more preferred embodiments, R9 and R10 (and optionally R′10) each independently represent a hydrogen, an alkyl, an alkenyl, or —(CH2)m—R8. Thus, the term “alkylamine” as used herein means an amine group, as defined above, having a substituted or unsubstituted alkyl attached thereto, i.e., at least one of R9 and R10 is an alkyl group.
The term “amido” is art-recognized as an amino-substituted carbonyl and includes a moiety that can be represented by the general formula:
wherein R9, R10 are as defined above. Preferred embodiments of the amide will not include imides, which may be unstable.
The term “aralkyl”, as used herein, refers to an alkyl group substituted with an aryl group (e.g., an aromatic or heteroaromatic group).
The term “aryl” as used herein includes 5-, 6-, and 7-membered single-ring aromatic groups that may include from zero to four heteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like. Those aryl groups having heteroatoms in the ring structure may also be referred to as “aryl heterocycles” or “heteroaromatics.” The aromatic ring can be substituted at one or more ring positions with such substituents as described above, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, —CF3, —CN, or the like. The term “aryl” also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings (the rings are “fused rings”) wherein at least one of the rings is aromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls.
The term “carbocycle”, as used herein, refers to an aromatic or non-aromatic ring in which each atom of the ring is carbon.
The term “carbonyl” is art-recognized and includes such moieties as can be represented by the general formula:
wherein X is a bond or represents an oxygen or a sulfur, and R11 represents a hydrogen, an alkyl, an alkenyl, —(CH2)m—R8 or a pharmaceutically acceptable salt, R′11 represents a hydrogen, an alkyl, an alkenyl or —(CH2)m—R8, where m and R8 are as defined above. Where X is an oxygen and R11 or R′11 is not hydrogen, the formula represents an “ester”. Where X is an oxygen, and R11 is as defined above, the moiety is referred to herein as a carboxyl group, and particularly when R11 is a hydrogen, the formula represents a “carboxylic acid”. Where X is an oxygen, and R′11 is hydrogen, the formula represents a “formate”. In general, where the oxygen atom of the above formula is replaced by sulfur, the formula represents a “thiocarbonyl” group. Where X is a sulfur and R11 or R′11 is not hydrogen, the formula represents a “thioester.” Where X is a sulfur and R11 is hydrogen, the formula represents a “thiocarboxylic acid.” Where X is a sulfur and R11′ is hydrogen, the formula represents a “thiolformate.” On the other hand, where X is a bond, and R11 is not hydrogen, the above formula represents a “ketone” group. Where X is a bond, and R11 is hydrogen, the above formula represents an “aldehyde” group.
The term “heteroatom” as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are boron, nitrogen, oxygen, phosphorus, sulfur and selenium.
The terms “heterocyclyl” or “heterocyclic group” refer to 3- to 10-membered ring structures, more preferably 3- to 7-membered rings, whose ring structures include one to four heteroatoms. Heterocycles can also be polycycles. Heterocyclyl groups include, for example, thiophene, thianthrene, furan, pyran, isobenzofuran, chromene, xanthene, phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine, phenanthroline, phenazine, phenarsazine, phenothiazine, furazan, phenoxazine, pyrrolidine, oxolane, thiolane, oxazole, piperidine, piperazine, morpholine, lactones, lactams such as azetidinones and pyrrolidinones, sultams, sultones, and the like. The heterocyclic ring can be substituted at one or more positions with such substituents as described above, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic moiety, —CF3, —CN, or the like.
As used herein, the term “nitro” means —NO2; the term “halogen” designates —F, —Cl, —Br or —I; the term “sulfhydryl” means —SH; the term “hydroxyl” means —OH; and the term “sulfonyl” means —SO2—.
The terms “polycyclyl” or “polycyclic group” refer to two or more rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls) in which two or more carbons are common to two adjoining rings, e.g., the rings are “fused rings”. Rings that are joined through non-adjacent atoms are termed “bridged” rings. Each of the rings of the polycycle can be substituted with such substituents as described above, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic moiety, —CF3, —CN, or the like.
The phrase “protecting group” as used herein means temporary substituents that protect a potentially reactive functional group from undesired chemical transformations. Examples of such protecting groups include esters of carboxylic acids, silyl ethers of alcohols, and acetals and ketals of aldehydes and ketones, respectively. The field of protecting group chemistry has been reviewed (Greene, T. W.; Wuts, P. G. M. Protective Groups in Organic Synthesis, 2nd ed.; Wiley: New York, 1991).
A “selenoalkyl” refers to an alkyl group having a substituted seleno group attached thereto. Exemplary “selenoethers” which may be substituted on the alkyl are selected from one of —Se-alkyl, —Se-alkenyl, —Se-alkynyl, and —Se—(CH2)m—R8, m and R8 being defined above.
As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described herein above. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this invention, the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. This invention is not intended to be limited in any manner by the permissible substituents of organic compounds.
It will be understood that “substitution” or “substituted with” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.
Analogous substitutions can be made to alkenyl and alkynyl groups to produce, for example, aminoalkenyls, aminoalkynyls, amidoalkenyls, amidoalkynyls, iminoalkenyls, iminoalkynyls, thioalkenyls, thioalkynyls, carbonyl-substituted alkenyls or alkynyls.
As used herein, the definition of each expression, e.g., alkyl, m, n, etc., when it occurs more than once in any structure, is intended to be independent of its definition elsewhere in the same structure.
The terms triflyl, tosyl, mesyl, and nonaflyl are art-recognized and refer to trifluoromethanesulfonyl, p-toluenesulfonyl, methanesulfonyl, and nonafluorobutanesulfonyl groups, respectively. The terms triflate, tosylate, mesylate, and nonaflate are art-recognized and refer to trifluoromethanesulfonate ester, p-toluenesulfonate ester, methanesulfonate ester, and nonafluorobutanesulfonate ester functional groups and molecules that contain said groups, respectively.
The abbreviations Me, Et, Ph, Tf, Nf, Ts, Ms represent methyl, ethyl, phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl, p-toluenesulfonyl and methanesulfonyl, respectively. A more comprehensive list of the abbreviations utilized by organic chemists of ordinary skill in the art appears in the first issue of each volume of the Journal of Organic Chemistry; this list is typically presented in a table entitled Standard List of Abbreviations. The abbreviations contained in said list, and all abbreviations utilized by organic chemists of ordinary skill in the art are hereby incorporated by reference.
Certain compounds of the present invention may exist in particular geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis- and trans-isomers, R— and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the invention. Additional asymmetric carbon atoms may be present in a substituent such as an alkyl group. All such isomers, as well as mixtures thereof, are intended to be included in this invention.
If, for instance, a particular enantiomer of a compound of the present invention is desired, it may be prepared by asymmetric synthesis, or by derivation with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomers. Alternatively, where the molecule contains a basic functional group, such as amino, or an acidic functional group, such as carboxyl, diastereomeric salts may be formed with an appropriate optically active acid or base, followed by resolution of the diastereomers thus formed by fractional crystallization or chromatographic means well known in the art, and subsequent recovery of the pure enantiomers.
For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover. Also for purposes of this invention, the term “hydrocarbon” is contemplated to include all permissible compounds having at least one hydrogen and one carbon atom. In a broad aspect, the permissible hydrocarbons include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic organic compounds which can be substituted or unsubstituted.
“amino acid”—a monomeric unit of a peptide, polypeptide, or protein. There are about eighty amino acids found in naturally occurring peptides, polypeptides and proteins, all of which are L-isomers. The term also includes analogs of the amino acids and D-isomers of the protein amino acids and their analogs.
The term “hydrophobic” refers to the tendency of chemical moieties with nonpolar atoms to interact with each other rather than water or other polar atoms. Materials that are “hydrophobic” are, for the most part, insoluble in water. Natural products with hydrophobic properties include lipids, fatty acids, phospholipids, sphingolipids, acylglycerols, waxes, sterols, steroids, terpenes, prostaglandins, thromboxanes, leukotrienes, isoprenoids, retenoids, biotin, and hydrophobic amino acids such as tryptophan, phenylalanine, isoleucine, leucine, valine, methionine, alanine, proline, and tyrosine. A chemical moiety is also hydrophobic or has hydrophobic properties if its physical properties are determined by the presence of nonpolar atoms.
The term “hydrophilic” refers to chemical moieties with a high affinity for water. Materials that are “hydrophilic” are, for the most part, soluble in water.
As used herein, “protein” is a polymer consisting essentially of any of the about 80 amino acids. Although “polypeptide” is often used in reference to relatively large polypeptides, and “peptide” is often used in reference to small polypeptides, usage of these terms in the art overlaps and is varied.
The terms “peptide(s)”, “protein(s)” and “polypeptide(s)” are used interchangeably herein.
The terms “polynucleotide sequence” and “nucleotide sequence” are also used interchangeably herein.
As used herein, the term “nucleic acid” refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). The term should be understood to include single (sense or antisense) and double-stranded polynucleotides.
The term “small molecule” refers to a compound having a molecular weight less than about 2500 amu, preferably less than about 2000 amu, even more preferably less than about 1500 amu, still more preferably less than about 1000 amu, or most preferably less than about 750 amu.
(iii) Exemplary Methods
The present invention provides an improved method for separating target from a sample so that the target can be further analyzed. This method will be referred to herein as the “Affinity Protocol”, “AP” or the “Affinity Method”. Certain embodiments of this methodology will utilize magnetic substrates and may also be referred to as the “Affinity Magnet Protocol” or “AMP”.
The Affinity Protocol uses substrates to help identify one or more targets from a sample. AP may be used for any of a wide range of targets including, but not limited to, nucleic acids (e.g., DNA and RNA), proteins, bacterial cells or spores (e.g., gram+ and gram−), viruses (e.g., DNA- or RNA-based), small organic molecules (e.g., toxins, hormones, etc), and large chemical compounds. AP may be used to identify target from any of a wide range of samples including gaseous samples (e.g., filtered or unfiltered air), environmental liquid samples (e.g., fresh water, sea water, sludge, mud, re-hydrated soil, gasoline, oil), biological liquid and semi-solid samples (e.g., blood, urine, sputum, saliva, feces, cerebro-spinal fluid, bone marrow, semen, vaginal fluid, brain matter, bone fragments), and environmental solid samples (e.g., dry soil or clay). Additionally, AP may be used to analyze the presence of target on solid surfaces which are not amenable to whole processing. For example, the presence of a target on a desktop, computer keyboard, doorknob, and the like. In such cases, the presence of target can be assessed by first taking a surface wipe of the solid surface, and then processing the surface wipe for the presence of a target. Furthermore, AP may be used to identify target in any of a number of industrial applications such as food processing, chemical processing, or any large scale production effort which would be hindered by the presence of certain contaminating targets within a preparation.
The present invention contemplates that the Affinity Protocol can be used alone to identify target in a sample, and to facilitate the further analysis of that target. For example, the Affinity Protocol can be used to identify the presence of particular bacterial cells in a water sample. These bacterial cells can then be further analyzed cytologically or molecularly.
The Affinity Protocol has many significant advantageous over other methods of isolating or separating targets from heterogeneous samples. Substrates for use in the Affinity Protocol and the Affinity Magnet Protocol are either uncoated (e.g., underivatized) or are derivatized with relatively simple chemical moieties (e.g., non-antibody moieties). This is in contrast to many previously available separation techniques which require substrate coated with antibodies immunoreactive with particular targets. Antibodies are more expensive to produce and append to substrates, their use requires tremendous a priori knowledge of the target of interest, and each antibody likely has a narrow spectrum of immunoreactivity. Furthermore, antibodies are prone to denaturing and degradation when exposed to chemicals and components present in environmental samples such as soils, whereas the simple modifying agents disclosed herein are more robust than antibodies against such degradation. Additionally, the Affinity Protocol and Affinity Magnet Protocol allow rapid separation of target from a heterogeneous sample, and the method requires the use of minimal reagents. These features decrease the cost of the Protocol, and allow its use in the field (e.g., non-laboratory conditions) as well as in the laboratory.
However, the invention further contemplates that the Affinity Protocol can be used in combination with the previously disclosed SNAP method or with other methodologies for further analyzing nucleic acids. The SNAP method, which is outlined in detail in U.S. publication No. 2003/0129614 and is hereby incorporated by reference in its entirety, allows for the isolation of nucleic acids from samples in a manner that prevents their degradation and/or inhibits agents in the sample that interfere with the further analysis of the nucleic acid. An exemplary commercially available product that typifies SNAP-like methodology is IsoCode™ paper. By coupling the Affinity Protocol with SNAP methodology, the present invention provides a vastly improved method for identifying targets from complex, heterogeneous samples. As the examples provided herein illustrate, the use of both the Affinity Protocol and SNAP methodology, improves the quality of the target identified in a sample and thus facilitates the further analysis of the target. Additionally, the combined methods are more sensitive than the SNAP methodology alone, and thus allow the identification of lower concentrations of target within a sample.
The Affinity Protocol uses substrates that interact with target present in a sample. The substrate may be of virtually any size and shape, and exemplary substrates include beads, tubes, probes, fiber-optics, plates, filters, cartridges, coverslips, chips, films, dishes, swabs, paper or other wipes, and the like. Furthermore, the substrate may be composed of any of a number of materials including, but not limited to, glass, plastic, and silica. The substrate may be magnetized (e.g., possess magnetic characteristics). The substrate may be porous or non-porous, and porous substrates may have any of a range of porosities.
Substrates for use in the Affinity Protocol should have an increased affinity for target in comparison with non-target materials in the sample. As will be detailed herein, some substrates have a higher affinity for certain targets in comparison to certain other targets, and one of skill in the art can readily select a particular substrate depending on factors including the target, the sample, etc. The invention additionally contemplates that the surface of the substrate can be modified to further promote the interaction of the substrate with one or more targets. Moieties that are attached to the surface of a substrate to influence the interaction of the substrate with target are referred to as surface modifying agents. The invention contemplates that one or more surface modifying agents can be appended to the surface of a substrate to promote the interaction of the substrate with a particular target. Exemplary surface modifying agents are provided herein, and in one embodiment of the present invention, a substrate modified with one or more of the surface modifying agents disclosed herein is used in the Affinity Protocol to identify and/or separate a target from a sample.
The invention further contemplates Affinity Protocols which employ a cocktail of substrates. For example, the method may use two or more substrates modified with different surface modifying agents to identify more than one target, and/or the method may use substrates which vary in size, shape, or composition, but are modified with the same surface modifying agent.
To further illustrate the Affinity Protocol,
In the hypothetical example outlined in
Identification and/or separation of a target from a sample using a substrate has numerous applications. One of skill in the art will recognize that the term “separation” can have two meanings in the context of the present invention. The term separation can refer to the association of a target with the substrate (e.g., the formation of a target-substrate complex) such that the target is now separated from the remainder of the sample by virtue of its association with the substrate. The term separation can additionally refer to the physical removal of the target and/or target-substrate complex from the remainder of the sample. The invention contemplates embodiments in which either of these are preferred.
The present application provides an improved method (the Affinity Protocol) for identifying and/or separating a target from amongst a heterogeneous liquid, solid, or gaseous sample. As will be appreciated from the examples provided herein, the Affinity Protocol provides an improved method that can be used in a controlled setting such as a laboratory, hospital, or food processing plant, as well as in a less-controlled field setting. The Affinity Protocol is amenable to rapid identification and/or separation, and is amenable to use with any of a large number of substrates which can be chosen based on the specific requirements of the application, sample, and target.
(iv) Exemplary Compositions
As outlined in detail above, in one embodiment of the Affinity Protocol, the surface of the substrate can be modified with a surface modifying agent. Exemplary surface modifying agents can be used to promote the interaction of the coated substrate with target. Preferred surface modifying agents provide an increased affinity between the coated substrate and the target in comparison to either other coated substrates or uncoated substrates.
The invention contemplates that substrates can be coated with any of a number of surface modifying agents, and furthermore that a substrate can be coated with a single surface modifying agent or with more than one surface modifying agents. It is anticipated that some surface modifying agents will have an affinity for a particular class of target (e.g., all DNA or all RNA or all bacterial cells) while other surface modifying agents will have an affinity for a specific target (e.g., a particular bacterial species or the spore versus the cellular form of a particular bacteria or class of bacteria). One of skill in the art can readily test various surface modifying agents and select agents which have the desired affinity for the desired target.
Following the identification of a desired surface modifying agent or agents, any of a number of substrates can be coated or otherwise derivatized such that the surface of the substrate is coated with the surface modifying agent. The invention contemplates that certain surface modifying agents may more readily coat or covalently interact with particular substrates, and thus every surface modifying agent may not be suitable for coating every possible substrate. However, the selection of a suitable substrate for coating with a surface modifying agent can be readily made by one of skill in the art given the particular application, target, sample, etc.
One aspect of the invention is to take a silicon containing surface modifying agent and modify the surface of a substrate to give the surface-modified substrate represented in
The left panel of
R1=F, Cl, Br, I, OH, OM, OR, R, NR2, SiR3, NCO, CN, O(CO)R
R2=F, Cl, Br, I, OH, OM, OR, R, NR2, SiR3, NCO, CN, O(CO)R
R3=F, Cl, Br, I, OH, OM, OR, R, NR2, SiR3, NCO, CN, O(CO)R
M=metal
X═NR, O
R=substituted or unsubstituted alkyl, alkenyl, heteroalkyl, aryl or heteroaryl, hydrogen
Y =a linker/spacer=substituted or unsubstituted alkyl, alkenyl, aryl or heteroaryl, silanyl, siloxanyl, heteroalkyl
Z=F, Cl, Br, I, OH, OM, OR, R, NR2, SiR3, NCO, CN, O(CO)R, N(CO)R, PR2, PR(OR), P(OR)2, SR, SSR, SO2R, SO3R
The example in
The surface modifying agent typically contains a coupling region containing a silicon atom bonded to at least one hydrolyzable moiety, optionally a spacer/linker region shown as Y, and an active region shown as Z. The silicon atom is typically substituted with a spacer region shown as Y but this group is optional and Z may be directly attached to the silicon. The silicon is also typically substituted with three groups designated as R1, R2, and R3 which can be identical or different provided that one group is hydrolyzable. Hydrolyzable groups can be, but are not limited to H, F, Cl, Br, I, OH, OM, OR, NR2, SiR3, NCO, and OCOR.
The spacer region is typically an alkyl (substituted or unsubstituted), alkenyl, aromatic silane, or siloxane based organic moiety which may be substituted with other organic moieties such as acyl halide, alcohol, aldehyde, alkane, alkene, alkyne, amide, amine, arene, heteroarene, azide, carboxylic acid, disulfide, epoxide, ester, ether, halide, ketone, nitrile, nitro, phenol, sulfide, sulfone, sulfonic acid, sulfoxide, silane, siloxane or thiol. The alkyl, alkenyl, or aromatic based organic moiety may contain up to 50 carbon atoms and contains more preferably up to 20 carbon atoms and contains most preferably up to 10 carbon atoms. The silane or siloxane based silicon moiety may contain up to 50 silicon or carbon atoms and contains more preferably up to 20 silicon or carbon atoms and contains most preferably up to 10 silicon or carbon atoms. Attached to the Y spacer region, or optionally directly to the silicon, is the active region shown as Z. The active region is employed to attract and bind the organism or biological molecule of interest (the target). The binding of target to the active region can occur via any of a number of interactions. Without being bound by theory, the binding between the active region and target can occur via van der Waals interactions, hydrogen bonding, covalent bonding, and/or ionic bonding.
Additionally, we note that the active region can also contain an alkyl, alkenyl, or aromatic based organic moiety which may be substituted with other organic moieties such as acyl halide, alcohol, aldehyde, alkane, alkene, alkyne, amide, amine, arene, heteroarene, azide, carboxylic acid, disulfide, epoxide, ester, ether, halide, ketone, nitrile, nitro, phenol, sulfide, sulfone, sulfonic acid, sulfoxide, silane, siloxane or thiol. The alkyl, vinyl, or aromatic based organic moiety may contain up to 50 carbon atoms and contains more preferably up to 20 carbon atoms and contains most preferably up to 10 carbon atoms.
A second aspect of the invention is to take a silicon containing surface modifying agent and modify the surface of a substrate to give the material shown in
The active regions on the surface modifying agent can be the same or different and the spacer regions on the surface modifying agent can be the same or different. The substrate can be modified with any number of surface modifying agents with the degree of surface modification typically expressed as the amount of surface coverage in moles per gram. The substrate can also be modified with more then one type of surface modifying agent by attaching the agents either sequentially or concurrently.
The left panel of
R1=F, Cl, Br, I, OH, OM, OR, R, NR2, SiR3, NCO, CN, O(CO)R
R2=F, Cl, Br, I, OH, OM, OR, R, NR2, SiR3, NCO, CN, O(CO)R
R3=F, Cl, Br, I, OH, OM, OR, R, NR2, SiR3, NCO, CN, O(CO)R
M=metal
X═NR, O
R=substituted or unsubstituted alkyl, alkenyl, heteroalkyl, aryl or heteroaryl, hydrogen
Y=substituted or unsubstituted alkyl, alkenyl, aryl or heteroaryl, silanyl, siloxanyl, heteroalkyl
Z=F, Cl, Br, I, OH, OM, OR, R, NR2, SiR3, NCO, CN, O(CO)R, N(CO)R, PR2, PR(OR), P(OR)2, SR, SSR, SO2R, SO3R
For substrates modified with either the modifying agents represented in
The substrate can be made of any material. Preferred substrates have a surface composed in whole or in part of a metal oxide, a nucleophile, a hydroxide, amine, thiol, or a halide. Those skilled in the art will recognize that any metal oxide surface can contain hydroxide functionality either innately or through a treatment to partially hydrolyze the metal oxide. Furthermore, any metal halide can also contain hydroxide functionality either innately or through a treatment to partially hydrolyze the metal halide. Organic surfaces can also be employed in this invention provided the surface has a nucleophile present such as a hydroxide moiety either present or in latent form. A preferred material is a material that contains silicon oxides or silicon hydroxide either with or without the presence of other metals or metal oxides or metal halide. Additional substrates for use in the methods of the present invention include glass and plastic
In some aspects of the invention, the substrate will contain material in sufficient quantity to make the substrate paramagnetic (herein referred to as possessing magnetic character) in that the substrate is attracted to magnetic fields. In a preferred form of the invention, the substrate will contain iron, nickel, or cobalt, and in a more preferred form the substrate will contain iron or an iron oxide. In this aspect the use of a paramagnetic substrate is advantageous in that a magnetic field can be used to separate the magnetic substrate from other non-magnetic materials.
In some other aspects of the invention the substrate will contain a perforation such that a string that can be passed through the substrate. Such a string, tether or other linking means can connect substrates together and can be used to facilitate later recover of either the substrate or of the substrate-target complexes.
There are aspects of this invention in which it would be advantageous to detach the active region of the surface modifying agent from the substrate. Accordingly, the invention contemplates modifying agents that contain a cleavable linker. The presence of a cleavable linker allows the release of the active region of the modifying agent+target from the remainder of the substrate. The ability to release the target in this way may greatly facilitate the further analysis of the target. For example, the ability to release the target may be especially important in scenarios in which the association between the substrate and the target is very strong.
The method of detachment can include treatment of the surface modified substrate with any process or chemical that disrupts or reverses the binding forces that attract the target and the active region. These include altering the pH or salt concentration, exposing the complex to heat, and exposing the complex to light. We note that the use of such methods does not disrupt or cleave the modifying agent itself, but rather releases the target from the active agent while leaving the modifying agent intact.
In other aspects, the invention contemplates that the release of target involves cleavage within a site in the modifying agent (e.g., cleavage of the linker and release of the active region+target). This can be accomplished by cleaving a covalent bond in the spacer region thereby separating the active region of the surface modifying agent from the substrate. This may also be accomplished by cleaving covalent bonds in the coupling region thereby separating the active region of the surface modifying agent from the substrate. Particular specific examples of methods that can be used to induce a cleavage event within the modifying agent can be found in the Examples.
(v) Exemplary Screening Assays
The invention provides an Affinity Protocol for identifying and/or separating target from a sample. The substrate can be modified in any of a variety of ways to further promote the interaction of the substrate with a particular target. For example, the surface of the substrate can be modified with one or more surface modifying agents such as the amine-containing agents provided herein.
Given the identification of a number of surface modifying agents that promote interaction of a target with the modified substrate, the present invention contemplates screens to identify further agents that can be used as modifying agents. Armed with an appropriate assay or assays to allow the relatively efficient evaluation of substrate coatings, one of skill in the art can readily screen any of a number of coatings and identify coatings that may be useful for promoting the interaction of substrate with a particular target. For example, one could specifically screen for coatings that promote the interaction of substrate with DNA, RNA, bacterial cells and spores generally, or a particular bacterial cell or spore.
We provide several screening assays that can be used to efficiently identify surface modifying agents for use in the Affinity Protocol. Substrates modified with candidate surface modifying agents can be screened using any of these assays, and the ability of substrates coated with one or more of the candidate surface modifying agents to interact with a target can be assessed. Substrates coated with candidate agents that interact with a particular target with a greater affinity than that of the uncoated substrate may be further analyzed to determine their target specificity, ease of manufacture, etc.
Assay 1—Flow Cytometry Screening Assay. The following protocol, represented schematically in
Using this type of assay, a large number of substrate coatings can be rapidly assessed and compared. Candidate coatings worth further analysis are those that bind bacterial cells more readily (e.g., promote the interaction between target and substrate) than uncoated substrate.
Counting bacteria by flow cytometry was found to be reproducible between samples, and cell densities calculated by flow cytometry agreed with expected cell densities as determined by light microscopy within two standard deviations.
Assay 2—Fluorescence Screening Assay. The following protocol, represented schematically in
Place a suitable volume of an appropriate mixing buffer in a centrifuge tube. The buffer can be selected based on the particular sample and target. Measure the amount of dsDNA prior to the addition of any substrate. For an in vitro screening assay, a starting concentration of dsDNA in the range of 50 μg/ml-1 μg/ml is appropriate. Add Pico-green dsDNA intercalating dye to the dsDNA. Pico-green has an excitation wavelength of 488 nm and an emission wavelength of 522 nm. Other fluorescent intercalating dyes can also be used and one of skill in the art can select a dye that has appropriate excitation and emission characteristics for easy laboratory analysis. Other commonly used, fluorescent intercalating dyes include, but are not limited to, Acridine Orange, Propidium Iodine, DAPI, SYBR Green 1, and ethidium bromide. Following addition of dye, allow dye and DNA to mix, and measure the fluorescence. This provides a baseline for the analysis.
Add coated substrate to the labeled DNA sample and allow substrate and sample to mix. Shake and vortex for approximately 30 seconds to allow adhesion to occur. Separate substrate from free DNA by centrifugation or settling, and measure the fluorescence of DNA remaining in solution.
By comparing the fluorescence of the DNA mixture before and after the addition of the coated substrate, one can quantify the capture efficiency of each coated substrate. This allows the evaluation of any of a number of substrate coatings.
Assay 3—PCR Screening Assay. PCR can also be used to determine adhesion by determining the cycle number of a sample before and after the addition of coated substrate. The steps are similar to those outlined above for the fluorescence assay, except staining of the DNA with an intercalating agent is not required. A sample of the initial stock solution of DNA and a sample of the supernatant removed following substrate addition and mixing are compared by PCR. An increase in the cycle number required to amplify DNA from a sample following addition of substrate indicates that DNA adhered to the substrate.
(vi) Exemplary Apparatuses
The present invention provides several classes of apparatuses. The first class of devices is designed to facilitate the efficient interaction of modified substrate with large amounts of sample. Such devices are useful for applications of the Affinity Protocol in large-scale industrial settings in which it may be difficult to readily contact a substrate with a sample containing a particular target, and is especially important when the target may not be evenly distributed throughout the entire sample.
The Affinity Protocol and Affinity Magnet Protocol described in detail herein use substrates such as beads to capture target from materials such as liquids, slurries, and air. Large quantities of sample material require effective mixing to maximize substrate-target interaction and capture efficiency on the bead surfaces. The first class of device of the present invention was designed based on modifications of known techniques for mixing viscous slurries. These techniques use the principle of chaotic mixing, and are known as journal bearing flow (which refers to the flow of fluids in a journal bearing—a hollow cylinder enclosing a solid shaft that rotates about its axis). Journal bearing flow is typically used to mix viscous fluids such as oils and cement, in large (multi-gallon) quantities. The principle is to place the material in a cylindrical container with an annulus, formed by placing a second cylinder inside the first. The two cylinders are aligned eccentric to each other, and are co- or counter-rotated about their longitudinal axes at slow speeds (typically less than 20 revolutions per minute). The slow rotation causes the material inside the annulus to stretch and fold, thereby decreasing the interaction distance between any two particles in the material. Over the course of many rotations, efficient mixing can be achieved.
A particular apparatus designed to facilitate mixing of substrate and sample is described in detail in the examples section of this application. Furthermore, the examples provide data demonstrating the performance of this device in a representative scenario. The invention contemplates multiple variations on this class of devices which are referred to herein as “Class I apparatuses”, “Class I devices”, “Chaotic Mixing apparatus”, or “Chaotic Mixing device”. The device can be of virtually any size, and the size of the device can be scaled up or down depending on the total volume of sample which must be accommodated. The key aspect of the device is not its overall size, but rather (a) the presence of two eccentrically placed cylinders, (b) an outer cylinder which is larger than an inner cylinder, and (c) the rotation of the cylinders at relatively low speeds. The cylinders may vary in size and shape, and the two cylinders need not have the same shape. Additionally, one or both cylinders can be altered to increase its surface area by, for example, the addition of fins, vanes, or ribs to the outer surface of the inner cylinder and/or to the inner surface of the outer cylinder. Such fins or vanes not only increase the surface area but can also increase vertical circulation of the sample during mixing, thereby increasing substrate-target interaction.
The invention contemplates that the cylinders can be either solid or hollow, and whether the cylinder should be solid or hollow can be determined based on the size of the cylinders and based on the material used to construct the cylinder. These factors will influence the weight and strength of the cylinders, as well as the cost of their construction. The cylinders can be constructed from any of a number of materials, and the two cylinders need not be constructed of the same materials. The materials can be selected based on the size and shape of the cylinders, as well as the particular type of sample, substrate and target. Exemplary materials include, but are not limited to, Teflon, stainless steel, iron or other metal, and plastic. Additionally, the invention contemplates that the cylinders can be plated with a material such as gold, platinum, iron, Teflon, and the like, to improve particular characteristics of the cylinders.
The rotation of the cylinders can be in the same direction or in opposite directions (e.g., both cylinders can be rotated clockwise, both cylinders can be rotated counter-clockwise, or one cylinder can be rotated clockwise while the other is rotated counter-clockwise). The rotation of the cylinders should occur at relatively slow speeds ranging from 5-50 rpm, preferably from 10-20 rpm. The rotation of the cylinders in exemplary devices should occur at 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 rpm, however, the invention contemplates that the optimal rotation can be selected based on the particular sample, the total volume being mixed, and the particular target.
The invention further contemplates that the dynamics of the beads as they are circulated through the mixture can be influenced by using a varying external magnetic field, such as a rotating magnetic field external to the outer cylinder. This may be especially useful when the substrate has a magnetic character (e.g., coated or uncoated magnetic beads). In a further application of the use of magnetic fields in these devices, the inner cylinder can serve a dual purpose by being constructed as an electromagnet, with a coil of wire wrapped around an iron-based core. When the electromagnet is activated, the inner cylinder can serve as a collection rod for the substrate in embodiments which use a substrate with a magnetic character. In this way, the inner cylinder can serve two functions as both an instrument to facilitate mixing of substrate and target and as a means for collecting substrate-target complexes following mixing.
The invention further contemplates a second class of devices. These devices comprise filters or cartridges that contain one or more substrates. The design of filters and cartridges containing one or more substrates capable of interacting with targets will facilitate the monitoring and analysis of a variety of air and liquid samples. For example, such filters and cartridges will allow a more detailed analysis of air that circulates in buildings, airplanes, and public transportation vehicles, as well as the analysis of water in reservoirs and streams.
The invention contemplates that Affinity Protocol-adapted filters and cartridges can be used alone, in combination with previously disclosed devices that facilitate the analysis of DNA (see, U.S. publication No. 2003/0129614, hereby incorporated by reference in its entirety), and in combination with other commercially available filters used to analyze air and water (e.g., HVAC air filters, HEPA filters, charcoal-based water filter, and the like). U.S. publication No. 2003/0129614 discloses exemplary devices used to facilitate further PCR analysis of targets. Such devices can be used to carry out the MITLL protocol, and several such exemplary designs are reproduced herein for illustrative purposes (See,
Of particular note, as with all of the substrates and modified substrates of the present invention, the Affinity Protocol adapted filters and cartridges are amenable to use under a range of conditions, can be readily changed or processed for analysis, and can be used at the bench (e.g., in a doctor's office, hospital, laboratory, processing plant) or in the field (e.g., at a site of suspected contamination, on the runway of an airport, at a crime scene).
AP Devices
These devices can be relatively small. Exemplary devices are approximately 8-18 inches in length. Specifically, an exemplary device can be 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 inches in length when configured linearly, as depicted in
In addition to their relatively small size, these devices can be self contained. Stated another way, the device can contain the substrate necessary to separate particular targets from a heterogeneous sample. Such substrates comprise coated or uncoated substrates, particularly coated or uncoated magnetic substrates. The substrates can be packaged along with and inside of the device, or the substrates can be packaged separately and added to the device prior to sample processing. Furthermore, the device may optionally contain the necessary elution buffers and other reagents that might otherwise need to be separately transported and maintained. Integration of all of the necessary components of the Affinity Protocol into a single device reduces the need to separately transport various reagents, as well as the need to transport disposables required to manipulate reagents.
Devices 10a and 10b are exemplary of a multi-chambered device. In one embodiment of a multi-chamber device, these exemplary devices comprise the following components: an input chamber (15 or 15′), a first valve (16 or 16′), a processing chamber (20 or 20′), a second valve (22 or 22′), and an eluate chamber (24 or 24′). These exemplary devices may further comprise: one or more connector elements (18 or 18′), a first cap portion (14 or 14′), a first plug portion (13 or 13′), a first handle portion (12 or 12′), a second cap portion (27 or 27′), a second plug portion (26 or 26′) and a second handle portion (25 or 25′). These components are described in more detail below.
Device 10a and 10b comprise an input chamber (15 or 15′). The input chamber serves as the initial collection chamber for a particular sample. The input chamber can be constructed of any of a number of materials, for example plastic, polystyrene, polypropylene, and the like. For example, the input chamber can be constructed from a commercially available polystyrene test tube. The interior surface of an exemplary input chamber is substantially inert. In other words, the chamber itself does not substantially react biochemically with the sample. Furthermore the interior surface of the input chamber can be coated with one or more agents that help prevent degradation of sample or of constituents of the sample. For example, the interior surface of the input chamber can be coated with one or more of a DNase inhibitor, RNase inhibitor, protease inhibitor, or anti-coagulent.
The input chamber serves both as the initial collection vessel and may also serve as the location of target capture. Sample, for example a heterogeneous, liquid or solid sample containing one or more targets, can be added to the input chamber. The input chamber may comprise one or more substrates that capture particular target from amongst constituents contained in the sample. Exemplary substrates include coated or uncoated substrates, particularly coated or uncoated magnetic substrates.
The input chamber can be manufactured to include substrate within the input chamber. Alternatively, the input chamber can be manufactured without substrate. In such instances, substrate can be sold along with the device as a kit. The end-user could then add substrate to the input chamber at some appropriate point prior to addition of sample. Alternatively, the end-user could separately purchase or make a suitable substrate, and add the substrate to the input chamber at some appropriate point prior to addition of sample.
Heterogeneous sample added into the input chamber can contact the substrate. Exemplary substrates bind to target within a heterogeneous sample to form a target-substrate complex. Such exemplary substrates bind to target with a higher affinity than to non-target materials within the heterogeneous sample.
In certain embodiments, the input chamber can be reversibly sealed using a cap portion (14 or 14′). In one embodiment, the cap contains a plug portion (13 or 13′) and a first handle portion (12 or 12′) integrated into a single unit. The plug portion interacts directly with the open end of the input chamber. Exemplary plug portions interact with the open end of the input chamber via, for example, a screw-cap, snap-cap, or magnetic cap mechanism. The handle portion provides a means for holding, moving, and otherwise manipulating the device. In another embodiment, the cap contains a plug portion and a first handle portion, but the plug portion and the first handle portion are two separate units. In another embodiment, the device is reversibly sealed with a plug portion but does not contain a handle portion. In such embodiments, the cap comprises a plug portion but does not comprise a handle portion.
For any of the foregoing, the invention contemplates devices with first handle portions of varying sizes and shapes.
The device further comprises a processing chamber (20 or 20′). The processing chamber can be constructed of any of a number of materials, for example plastic, polystyrene, polypropylene, and the like. For example, the processing chamber can be constructed from a commercially available polystyrene test tube. The interior surface of an exemplary processing chamber is substantially inert. In other words, the chamber itself does not substantially react biochemically with the sample. Furthermore the interior surface of the processing chamber can be coated with one or more agents that help prevent degradation of sample or of constituents of the sample. For example, the interior surface of the processing chamber can be coated with one or more of a DNase inhibitor, RNase inhibitor, protease inhibitor, or anti-coagulent.
The processing chamber may optionally contain a collector element that associates with or otherwise attracts a substrate-target complex. Regardless of whether the processing chamber contains a collector element, the processing chamber is the place where target-substrate complexes are separated from the remainder of the heterogeneous sample.
As used herein, a collector element refers to any implement used to associate with, bind to, or otherwise attract a substrate-target complex. By way of example, a collector element may comprise a collection magnet. Collection magnets are particularly useful for embodiments of the invention where the substrate is a magnetic substrate. In such embodiments, a collection magnet can be used within the device to physically bind to the substrate-target complex, thereby helping to separate the substrate-target complex from the remainder of the sample. Alternatively, the collection magnet can be used within the device to magnetically attract (e.g., with or without actually binding to) the substrate-target complex, thereby helping to separate the substrate-target complex from the remainder of the sample. In still another example, the collection magnet can be used outside of the device to magnetically attract (e.g., without actually binding to) the substrate-target complex, thereby helping to separate the substrate-target complex from the remainder of the sample. However, the collector elements according to the invention may also include non-magnetic collector elements. Such non-magnetic collector elements can be used with either non-magnetic substrates or with magnetic substrates. By way of example, a non-magnetic collector element may include a hook that engages or otherwise associates with the substrate (e.g., the substrate contains a notch or other point of engagement for the collector element hook). Thus, following formation of substrate-target complex, the collector element hook can associate with the engagement point on the substrate, thereby separating the substrate-target complex from the remainder of the sample.
The device further comprises a first valve (16 or 16′) which can reversibly modulate the flow of materials between the input chamber and the processing chamber. Appropriate valves can be readily selected by one of skill in the art depending on the volume of sample, as well as the size of particles or other matter that the user wishes to prevent from passing from one chamber to the next chamber.
The device further comprises an eluate chamber (24 or 24′). The eluate chamber contains elution buffer needed to elute target from substrate-target complex, thereby separating target for further analysis. The eluate chamber can be constructed of any of a number of materials, for example plastic, polystyrene, polypropylene, and the like. For example, the eluate chamber can be constructed from a commercially available polypropylene vial. The interior surface of an exemplary eluate chamber is substantially inert. In other words, the chamber itself does not substantially react biochemically with the sample. Furthermore the interior surface of the eluate chamber can be coated with one or more agents that help prevent degradation of sample or of constituents of the sample. For example, the interior surface of the eluate chamber can be coated with one or more of a DNase inhibitor, RNase inhibitor, protease inhibitor, or anti-coagulent.
The eluate chamber can be reversibly closed using a stopper ((29) in
The device further comprises a second valve (22 or 22′) which can reversibly modulate the flow of materials between the processing chamber and the eluate chamber. Appropriate valves can be readily selected by one of skill in the art depending on the volume of sample, as well as the size of particles or other matter that the user wishes to prevent from passing from one chamber to the next chamber.
The eluate chamber can be reversibly sealed using a stopper. A stopper that reversibly closes the eluate chamber is not depicted in
Exemplary plug portions and/or stoppers interact with the end (e.g., the open end or the closed end) of the eluate chamber via, for example, a screw-cap, snap-cap, a magnetic cap mechanism, or simply fit over the top of the eluate chamber. The handle portion provides a means for holding, moving, and otherwise manipulating the device. In another embodiment, the cap contains a plug portion and a handle portion, but the plug portion and the handle portion are two separate units.
For any of the foregoing, the invention contemplates devices with handle portions of varying sizes and shapes.
The device may further comprise linker elements. Linker elements, referred to interchangeably as connector elements (18 or 18′), is the term used to describe segments of the device that join together the functional components of the device. By way of example, connector elements (18 or 18′) can be used to link together one or more of the input chamber (15 or 15′), the first valve (16 or 16′), the processing chamber (20 or 20′), the second valve (22 or 22′), and the eluate chamber (24 or 24′). Exemplary connector elements are constructed from durable materials, for example, plastic, polystyrene, or polypropylene. The interior surface of an exemplary connector element is substantially inert. In other words, the element itself does not substantially react biochemically with the sample. Furthermore the interior surface of the connector element can be coated with one or more agents that help prevent degradation of sample or of constituents of the sample. For example, the interior surface of the connector element can be coated with one or more of a DNase inhibitor, RNase inhibitor, protease inhibitor, or anti-coagulent. Alternatively or in addition to, the interior surface of the connector element can be coated with one or more agents that decrease adherence between the interior surface of the connector element and the sample. Connector elements of various sizes and shapes can be readily selected to construct a device of the appropriate size and shape. Alternatively, connector elements need not be used, and all or a portion of the chambers or valves can be attached directly to the preceding chambers or valves. Regardless of whether the chambers or valves are attached directly or via connector elements, the devices are constructed such that the various chambers and valves are in fluid contact with each other.
As outlined above, the interior surface of one or more components of the device can be coated with materials that prevent degradation of sample or of target during sample processing. Furthermore, the interior surface of one or more components of the device can be coated with materials that prevent adherence between the interior surface and either sample or target.
Note that the device depicted in
a and 40b show close-up views of certain components of a multi-chambered device.
In embodiments where the device further includes elements that reversibly attach to the eluate chamber, cap portion (14) is understood to comprise a first cap portion, handle portion (12) is understood to comprise a first handle portion, and plug portion (13) is understood to comprise a first plug portion.
a and 40b also depict a connector element (18). In the view depicted in
a and 41b show close-up views of certain components of a multi-chambered device.
Given that plug portion (26) does not reversibly close the open end of the eluate chamber, the invention contemplates that an additional mechanism can be used to reversibly close the eluate chamber.
Collector elements can be arrayed in any of a variety of shapes.
b provides a close-up view of a collector element having a multi-sphere chain (51) configuration.
c provides a close-up view of a collector element having a cylindrical stack (52) configuration.
d depicts a collector element (52) located within an eluate chamber (24). The collector element depicted in
As outlined above, one class of substrates are magnetic substrates. Accordingly, one important class of collector elements comprises collection magnets. Thus, the invention contemplates that any of the foregoing exemplary configurations of collector elements can be configurations of collection magnets. Exemplary collection magnets can be composed of, for example, neodymium-boron-rare-earth magnetic material. Further exemplary collection magnets can be composed of any magnetic or paramagnetic material. Collection magnets for use in the devices and methods of the invention can be nickel plated.
Although one important class of collector elements capable of binding to, attracting, or otherwise associating with a substrate are collection magnets, the invention also contemplates non-magnetic collection elements that can be used to attract or otherwise associate with magnetic or non-magnetic substrates. By way of example, a non-magnetic collection element can comprise a hook that associates with a notch or one or more other engagement points on a substrate (e.g., a Velcro-type mechanism, a single hook and loop, etc.).
Regardless of whether a particular collector element is magnetic or non-magnetic, collector elements for use in the devices and methods of the invention bind, attract, or otherwise associate with substrate. In this way, the collector element can be used to separate substrate-target complexes from the remainder of the sample. Furthermore, regardless of whether a particular collector element is magnetic or non-magnetic, the invention contemplates embodiments in which the collector element is within the device (e.g., within the processing chamber, within a valve, etc.), as well as embodiments in which the collector element is not within the device.
The cartridges depicted in the foregoing figures can be more explicitly illustrated by an explanation of a method of using the device to separate a target from a sample. The following is merely exemplary of methods using the devices depicted in, for example,
Following addition of sample to the input chamber, the input chamber is reversibly sealed using a cap portion. For example, a cap portion comprising a plug portion and a handle portion is used to reversibly seal the open end of the input chamber. The cartridge is shaken for approximately 10 seconds (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more than 15 seconds) to mix the sample with the substrate contained in the input chamber. The cartridge is then allowed to sit to allow target capture. In other words, the device is allowed to sit for a time (target capture time) sufficient to allow substrate within the input chamber to associate with target present in the heterogeneous sample to form target-substrate complexes. Exemplary target capture times include, for example, approximately 1-5 minutes.
Following target capture, a first valve is opened and the cartridge is gently tapped to facilitate movement of the sample plus the target-substrate complexes into the processing chamber. After the contents of the input chamber have emptied into the processing chamber, the first valve is closed. At this point, the cartridge may optionally be briefly inverted so that any debris can fall toward the input chamber.
The contents of the processing chamber (sample plus substrate-target complexes) are allowed to sit for approximately 1-5 minutes. This capture time allows attraction or association between the substrate-target complex and a collection element. The collection element may be located within the processing chamber or within the valve. Alternatively, the collection element may be outside of the device, but can be used to attract target-substrate complexes to a particularly place within the processing chamber by placing the collection element in close proximity to that location. Regardless of the particular configuration and location of the collection element, attraction to or association of the target-substrate complexes with the collection element helps facilitate separation of the substrate-target complexes from the remainder of the sample.
Following capture, the cartridge is inverted and the first valve is opened. Sample which was not previously bound to substrate (and thus, material that was not attracted to or associated with the collection element) passes back into the input chamber. The first valve is then closed leaving target-substrate complexes in the processing vessel. In device configurations in which the collection element is also contained within the processing vessel or first valve, the collection element can remain within the device. For example, at this point in the methodology, the processing chamber can contain substrate-target complexes and the collection element. Alternatively, if the collection element was an external collection element (e.g., the collection element was not contained within the device), the collection element would remain external to the device and could be used to facilitate target separation in other devices. In still another alternative embodiment, the collection element could be contained within the device, but could be removable after its use for use in another device.
Regardless of the particular configuration of collection element, the processing vessel know contains substrate-target complexes. The device is then placed upright and the second valve is opened. The target-substrate complexes and, optionally, the collection element pass into the eluate chamber which contains elution buffer. The second valve is closed. The cartridge is shaken for approximately 10 seconds (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more than 15 seconds) and left for approximately 1 minute. This period is the elution time. The elution time is the time sufficient to allow separation of the target-substrate complexes, thereby allowing separation of the target from the heterogeneous sample.
Note that whether or not the collection element will also be present in the elution chamber depends on the particular configuration of the device and the particular substrate and collection element employed. For example, when external collection elements are used, the collection element is outside of the device, and thus is not present in the elution chamber. Even if the collection element is present within a chamber or valve of the device, the collection element need not be passed into the elution chamber. If, for example, the collection element attracts the substrate-target complexes, but does not bind to these complexes, the substrate-target complexes can be passed into the elution chamber without the need to also bring the collection element into the elution chamber. If, on the other hand, the collection element binds to the target-substrate complexes, the collection element will enter the elution chamber along with the target-substrate complexes.
Following elution of the target from the target-substrate complex, the cartridge is inverted. The substrate-collector element complex moves toward the valve. In one embodiment, the end of the second valve contains a metal collar that attracts the collector element (in this case the collection magnet), thereby separating the substrate-collector element complex from the eluted target. The eluate chamber can be detached from the remainder of the cartridge, thus removing eluted target from the remainder of the device. The eluate chamber can comprise a separate plug or reversibly sealing means so that target can be contained in the eluate chamber.
One example of such a plug (29) was depicted in
Like other configurations of the devices of the invention, the device depicted in
The input chamber can be manufactured to include substrate within the input chamber. Alternatively, the input chamber can be manufactured without substrate. In such instances, substrate can be sold along with the device as a kit. The end-user could then add substrate to the input chamber at some appropriate point prior to addition of sample. Alternatively, the end-user could separately purchase or make a suitable substrate, and add the substrate to the input chamber at some appropriate point prior to, addition of sample.
The input chamber (75) is incorporated into the body of a valve. The valve stem (96), valve body (76), and valve lever (86) are depicted in
The device further comprises an eluate chamber (74). The eluate chamber contains elution buffer needed to elute target from substrate-target complex, thereby separating target for further analysis. The eluate chamber can be constructed of any of a number of materials, for example plastic, polystyrene, polypropylene, and the like. For example, the eluate chamber can be constructed from a commercially available polypropylene vial. The interior surface of an exemplary eluate chamber is substantially inert. In other words, the chamber itself does not substantially react biochemically with the sample. Furthermore the interior surface of the eluate chamber can be coated with one or more agents that help prevent degradation of sample or of constituents of the sample. For example, the interior surface of the eluate chamber can be coated with one or more of a DNase inhibitor, RNase inhibitor, protease inhibitor, or anti-coagulent.
The eluate chamber can be reversibly closed using a stopper (79). For example, a stopper can be tethered to the eluate chamber, and the stopper can be used to reversibly close the eluate chamber using, for example, a screw-cap, snap-cap, or magnetic-cap mechanism. Furthermore, the eluate chamber can be removed from the rest of the device, and target contained within the eluate chamber can be stored or further analyzed within the eluate chamber. Alternatively, the eluate chamber can be removed from the rest of the device, and target contained within the eluate chamber can be transferred to another container or vessel for subsequent analysis.
The following methodology more clearly exemplifies the device depicted in
Like other configurations of the devices of the invention, the device depicted in
The input chamber can be manufactured to include substrate within the input chamber. Alternatively, the input chamber can be manufactured without substrate. In such instances, substrate can be sold along with the device as a kit. The end-user could then add substrate to the input chamber at some appropriate point prior to addition of sample. Alternatively, the end-user could separately purchase or make a suitable substrate, and add the substrate to the input chamber at some appropriate point prior to addition of sample.
The valve modulates the passage of materials from the input chamber (75) into the eluate chamber (74). In the particular design depicted in
The device further comprises an eluate chamber (74). The eluate chamber contains elution buffer needed to elute target from substrate-target complex, thereby separating target for further analysis. The eluate chamber can be constructed of any of a number of materials, for example plastic, polystyrene, polypropylene, and the like. For example, the eluate chamber can be constructed from a commercially available polypropylene vial. The interior surface of an exemplary eluate chamber is substantially inert. In other words, the chamber itself does not substantially react biochemically with the sample. Furthermore the interior surface of the eluate chamber can be coated with one or more agents that help prevent degradation of sample or of constituents of the sample. For example, the interior surface of the eluate chamber can be coated with one or more of a DNase inhibitor, RNase inhibitor, protease inhibitor, or anti-coagulent.
The eluate chamber can be reversibly closed using a stopper (79). For example, a stopper can be tethered to the eluate chamber, and the stopper can be used to reversibly close the eluate chamber using, for example, a screw-cap, snap-cap, or magnetic-cap mechanism. Furthermore, the eluate chamber can be removed from the rest of the device, and target contained within the eluate chamber can be stored or further analyzed within the eluate chamber. Alternatively, the eluate chamber can be removed from the rest of the device, and target contained within the eluate chamber can be transferred to another container or vessel for subsequent analysis.
The following methodology more clearly exemplifies the device depicted in.
When sample is added to the input chamber, the valve is oriented so that the collector element is not exposed to the input chamber (e.g., is not exposed to substrate prior to formation of target-substrate complexes). Sample is mixed to facilitate interaction between substrate and target within the heterogeneous sample. Following formation of target-substrate complexes (e.g., target capture), the valve is rotated 90 degrees. This exposes the collector element to the interior of the input chamber (e.g., exposes the collector element to substrate-target complexes occurring within the input chamber). The collector element attracts target-substrate complexes. This collection of target-substrate complexes occurs rapidly (e.g., less than 1 minute, 1, 2, 3, 4, or 5 minutes).
Following attraction of the target-substrate complexes to the collector element, the valve is further rotated to expose the target-substrate complexes to the eluate chamber. Elution buffer present in the eluate chamber separates the target from target-substrate complexes. The substrate remains associated with the collector element which is integrated into the valve. Thus, the target is separated from the heterogeneous sample, as well as from the substrate used to facilitate the separation.
Exemplification
The invention now being generally described, it will be more readily understood by reference to the following examples which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention.
As outlined in detail above, the Affinity Protocol provides an improved method for identifying targets in a sample. The protocol can be used either alone or in combination with SNAP/MITLL methodology, can be used to identify a wide range of targets from a diverse array of samples, and can be used with a variety of substrates. One substrate that can be useful for identifying particular targets is commercially available magnetic beads. Such beads are available from a number of manufacturers, come in a range of sizes and shapes, and are composed of any of a number of materials. Each of these factors can be optimized based upon the particular target, sample, and other factors.
The following methodologies briefly summarize methods employed to use commercially available magnetic beads as a substrate in the Affinity Protocol. Commercially available magnetic beads are shipped in a buffer. Prior to use, the beads were washed as follows: place 1 mL magnetic beads in a microcentrifuge tube, pellet beads at maximum (14,000 rpm) microcentrifuge speed, remove all liquid from above the bead pellet, resuspend in distilled water, and repeat as necessary to wash beads.
To perform the Affinity protocol on liquid samples as outlined schematically in
Following initial preparation of sample, add prepared magnetic beads to the tube containing the sample and close the tube. Place the tube with sample and beads in a rotating mixer for 10-20 minutes. Use the collection magnet to draw the beads to the side of the tube, taking enough time to ensure all beads have migrated. Collection time should be 10-20 seconds. Using a pipettor with a filter tip, remove all but a small volume of liquid from the tube, taking care not to disturb the pellet of magnetic beads collected at the side of the tube. Gently resuspend the substrate (which should be bound to target) using the small volume of liquid left behind in the previous step. After the target-substrate complex is resuspended, remove all of the liquid (containing target-substrate complex) and apply to commercially available medium such as IsoCode™ paper (this allows the performance of SNAP/MITLL methodology on your sample).
Following the Affinity Protocol steps outlined in detail above, nucleic acid from the sample can be processed using the IsoCode™ paper or other SNAP/MITLL methodology, and then the nucleic acids can be analyzed via PCR or other commonly employed technique for analyzing nucleic acids. Briefly, dry the IsoCode™ paper triangles in dishes, using one of four methods: place dishes (uncovered) with triangles in a vacuum oven at 60°±5° C. for 15 minutes, place dishes (uncovered) with triangles in an incubator at 60°±5° C. for 15 minutes (ensure that there is no water in the humidity tray), place dishes (uncovered) with triangles in a biosafety hood at room temperature until completely dry, or place each dish with triangle in a sealed pouch with a desiccant packet at room temperature until completely dry. After the sample has been dried, continue processing with SNAP/MITLL protocol for elution of target from IsoCode™ and analyze nucleic acid by PCR or other commonly used molecular biological approach.
We conducted an initial screening of 19 commercially available magnetic beads of varied coatings and sizes (Table 1) to ascertain their usefulness in the Affinity Protocol. The goal was to determine which commercially available beads provided the best overall efficiency in increasing signal (decreasing cycle number using PCR) in comparison to that achieved by the use of the SNAP/MITLL protocol alone. The identification of the characteristics of commercially available substrates and coatings that provide increased efficiency in the separation and identification of nucleic acid from various samples can be used to develop a rationale strategy for designing additional substrates and coatings. In these experiments using commercially available beads, the efficacy of each bead was assessed in comparison to the analysis of target with SNAP/MITLL alone. Binding efficiency of each bead was evaluated using the flourescence and flow cytometry assays described above.
Briefly,
We also examined several commercially available non-magnetic beads. We note that although a large number of beads were initially screened, only those of 50 μm size were directly compared and data reported.
The efficacy of these beads was assessed by measuring the percentage of DNA that adhered to the bead following incubation of the bead with a sample, and these results are summarized in
We note that although the interaction of substrate with DNA was directly tested in this experiment, the interaction of substrate with other nucleic acids such as RNA can also be evaluated. Based on the chemical structure of RNA, substrates that interact with DNA are likely to interact with RNA, and may be used to separate target RNA from a sample. Methodologies in which RNA is the target may be further modified to prevent the degradation of RNA which is generally less stable than DNA.
Following our analysis of commercially available beads (e.g., substrates) containing various commercially available coatings, we prepared a variety of novel coated substrates to assess the usefulness of these coated substrates in the Affinity Protocol. Specifically, we focused on amine containing surface modifying agents, however, similar experiments can be readily performed using other classes of surface modifying agents. As detailed herein, we prepared a number of surface modifying agents and used these agents to modify substrates of various sizes, shapes, and materials.
A. Preparation of 50-Micrometer Surface Modified Silica Gel
A slurry was prepared from 2.0 grams of 50-μm particle size silica gel purchased from Waters Corporation (YMC-gel silica) and 20 ml of isopropyl alcohol. To the slurry was added 10 mmole of the surface modifying agent. The slurry was gently stirred for 16 hours and then filtered. The silica gel was resuspended in 20 ml of isopropyl alcohol and filtered two additional times to remove unreacted surface modifying agent. The surface modified silica gel was dried overnight in a vacuum oven at 50° C. The amount of surface modification was determined by thermogravimetric analysis. Table 3 lists the surface modifying agents employed and the resulting surface coverage determined for modified 50-μm particle size silica gel. The W designation indicates that the resultant substrate is modified Waters Corporation silica gel, and the letters are used to indicate the surface modifying agent employed.
B. Preparation of 1-Millimeter Surface Modified Soda Lime Glass Beads
A suspension was prepared from 2.0 grams of 1-mm soda lime glass beads from PGC Scientific and 2 ml of 10% aqueous nitric acid and allowed to reflux with gentle stirring for 30 minutes. The nitric acid solution was decanted off and the beads were filtered and washed with deionized water. The beads were then added to 2 ml of 10 N sodium hydroxide and allowed to reflux with gentle stirring for 120 minutes. The sodium hydroxide solution was decanted off and the beads were filtered and extensively washed with deionized water. The beads were dried under vacuum for 4 hours at 100° C.
A suspension was prepared from the dried beads, 1 ml of the surface modifying agent, and 19 ml of dry toluene. The suspension was gently stirred for 45 minutes and filtered. The beads were washed with toluene, washed with ethanol, and vacuum dried for 3 hours at room temperature and 30 minutes at 100° C. The amount of surface modification was determined by performing a Kaiser test and following the change in absorbance at 575-nm. Table 4 lists the surface modifying agents employed and the resulting surface coverage determined for modified 1-mm soda lime glass beads. The PS designation indicates that the resultant substrate is modified PGC soda lime glass beads, and the letters are used to indicate the surface modifying agent employed.
C1. Preparation of 1-Millimeter Surface Modified Borosilicate Glass Beads
A suspension was prepared from 2.0 grams of 1-mm borosilicate glass beads from PGC Scientific and 2 ml of 10% aqueous nitric acid and allowed to reflux with gentle stirring for 30 minutes. The nitric acid solution was decanted off and the beads were filtered and washed with deionized water. The beads were then added to 2 ml of 10 N sodium hydroxide and allowed to reflux with gentle stirring for 120 minutes. The sodium hydroxide solution was decanted off and the beads were filtered and extensively washed with deionized water. The beads were dried under vacuum for 4 hours at 100° C.
A suspension was prepared from the dried beads, 1 ml of the surface modifying agent, and 19 ml of dry toluene. The suspension was gently stirred for 5 hours and filtered. The beads were washed with toluene, washed with ethanol, and vacuum dried for 3 hours at room temperature and 30 minutes at 100° C. The amount of surface modification was determined by performing a Kaiser test and following the change in absorbance at 575-nm. Table 5A lists the surface modifying agents employed and the resulting surface coverage determined for modified 1-mm borosilicate glass beads. The P designation indicates that the resultant substrate is modified PGC borosilicate glass beads, and the letters are used to indicate the surface modifying agent employed.
C2. Preparation of 1-Millimeter Surface Modified Borosilicate Glass Beads
A suspension was prepared from 2.0 grams of 1-mm borosilicate glass beads from PGC Scientific and 2 ml of 10% aqueous nitric acid and allowed to reflux with gentle stirring for 30 minutes. The nitric acid solution was decanted off and the beads were filtered and washed with deionized water. The beads were then added to 2 ml of 10 N potassium hydroxide and allowed to reflux with gentle stirring for 120 minutes. The potassium hydroxide solution was decanted off and the beads were filtered and extensively washed with deionized water. The beads were dried under vacuum for 4 hours at 100° C.
A suspension was prepared from the dried beads, 1 ml of the surface modifying agent, and 19 ml of deionized water. The suspension was gently stirred for 18 hours and filtered. The beads were extensively washed with deionized water, and vacuum dried for 3 hours at room temperature and 30 minutes at 100° C. The amount of surface modification was determined by performing a Kaiser test and following the change in absorbance at 575-nm. Table 5B lists the surface modifying agents employed and the resulting surface coverage determined for modified 1-mm borosilicate glass beads. The P designation indicates that the resultant substrate is modified PGC borosilicate glass beads, and the letters are used to indicate the surface modifying agent employed.
D. Preparation of 6.0 Micrometer Surface Modified Magnetic Particles
A suspension was prepared from 0.1 grams of 6.0-μm magnetic particles suspended in 1.9 ml of water purchased from Micromod Partikeltechnologie (Sicastar-M-CT), 0.5 mmole of the surface modifying agent, and 1.25 ml of isopropyl alcohol. The slurry was gently stirred for 16 hours. The particles were allowed to settle on a magnet and the liquid decanted. The following step was performed twice. An additional 4 ml of isopropyl alcohol was added to the particles, the new suspension was vigorously stirred for one minute, the particles were allowed to settle on a magnet, and the liquid decanted. The surface modified silica gel was dried in a vacuum oven at 50° C. overnight. The amount of surface modification was determined by thermogravimetric analysis. Table 6 lists the surface modifying agents employed and the resulting surface coverage determined for modified 6.0-μm magnetic particles. The S6 designation indicates that the resultant substrate is modified 6 μm magnetic beads from Sicastar, and the letters are used to indicate the surface modifying agent employed.
E. Preparation of 5.0 to 10.0 Micrometer Surface Modified Magnetic Particles
A suspension was prepared from 0.1 grams of 5.0- to 10.0-μm magnetic particles suspended in 3.2 ml of water purchased from CPG, Inc (MPG Uncoated), 0.5 mmole of the surface modifying agent, and 1.25 ml of isopropyl alcohol. The slurry was gently stirred for 16 hours. The particles were allowed to settle on a magnet and the liquid decanted. The following step was performed twice. An additional 4 ml of isopropyl alcohol was added to the particles, the new suspension was vigorously stirred for one minute, the particles were allowed to settle on a magnet, and the liquid decanted. The surface modified silica gel was dried in a vacuum oven at 50° C. overnight. The amount of surface modification was determined by thermogravimetric analysis. Table 7 lists the surface modifying agents employed and the resulting surface coverage determined for modified 5.0- to 10.0-μm magnetic particles. The M designation indicates that the resultant substrate is modified MPG beads, and the letters are used to indicate the surface modifying agent employed.
Table 8 provides the chemical names for the surface modifying agents analyzed in more detail herein. The invention contemplates the coating of any substrate with one or more of these surface modifying agents, the use of coated substrates in the Affinity protocol (either alone or in combination with SNAP/MITLL methodology), and the design of devices such as filters and cartridges with a layer containing a substrate modified with one or more of these surface modifying agents.
Furthermore, the chemical structures for each of surface modifying agents A-Y are provided in
F. Peptide-Based Surface Modifying Agents
In addition to the foregoing amine-based chemical functionalities, the present invention contemplates surface modifying agents composed in whole or in part of peptides. Such peptides can be attached to the surface of a substrate directly, via a cleavable linker, or via a chemical functionality which is itself directly appended to the surface of the substrate.
Exemplary peptides for use as surface modifying agents include any peptide that interacts with a target such that it increases the affinity of a coated substrate for that target. Specific examples of peptides suitable as surface modifying agents include the family of anti-microbial peptides, aptamers, and PNA. As with other types of substrates and substrate coatings, peptide-based surface modifying agents can be used to bind to any of a wide range of targets including DNA, RNA, protein, bacterial cells or spores (gram+ or gram−), viruses (DNA- or RNA-based), small organic molecules, and chemical compounds. Preferred peptide-based surface modifying agents will be relatively stable under the particular conditions required to promote interaction of the peptide-based coated substrate with the target.
The following are non-limiting examples of methods that can be used to release active region-target complexes from the remainder of the surface modifying agent and substrate.
A. Fluoride Labile Alkylsilyl Linker in Coupling Reaction
An alkylsilyl moiety can be used in the coupling region to attach the surface modifying agent to the substrate. Following binding of target to the active region of the surface modifying agent, hydrofluoric acid can be employed to cleave the silicon-oxygen bond and detach the active region from the substrate.
B. Fluoride Labile Alkylsilyl Linker in Spacer Region
An alkylsilyl moiety can be used in the backbone of the spacer region that is used to attach the active region to the substrate. Following binding of target to the active region of the surface modifying agent, hydrofluoric acid can be employed to cleave the silicon-oxygen bond and detach the active region from the remainder of the surface modifying agent and substrate.
C. Acid Labile Carbonyl Linker in Spacer Region
An acid labile carbonyl moiety can be used in the backbone of the spacer region that is used to attach the active region to the substrate. Examples of acid labile carbonyl moieties are amides, esters, carbonates, urathanes, and ureas. Following binding of target to the active region of the surface modifying agent, acids such as trifluoracetic acid, hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, and sulfuric acid can be employed to cleave the acid labile carbonyl moiety.
D. Base Labile Carbonyl Linker in Spacer Region
A base labile carbonyl moiety can be used in the backbone of the spacer region that is used to attach the active region to the substrate. Examples of base labile carbonyl moieties are amides, esters, carbonates, urathanes, and ureas. Following binding of target to the active region of the surface modifying agent, bases such as ammonium hydroxide, sodium hydroxide, and potassium hydroxide can be employed to cleave the base labile carbonyl moiety.
E. Nucleophile Labile Linker in Spacer Region
A nucleophile labile moiety can be used in the backbone of the spacer region that is used to attach the active region to the particle. An example of a nucleophile labile moiety is an oxime or a sulfonamide. Following binding of target to the active region of the surface modifying agent, any organic based amine can be employed as a nucleophile to affect cleavage.
F. Photo Labile Linker in Spacer Region
A photo labile moiety can be in the backbone of the spacer region which is used to attach the active region to the particle. Examples of photo labile moieties are esters, nitro substituted arylhydroxymethyl esters and arylsubstituted diazo derivatives. Following binding of target to the active region of the surface modifying agent, light can be employed to induce cleavage of the photo labile moiety. The wavelength of light employed is not critical, however the light will preferably have a wavelength of between 800 and 100 nm, with a more preferred wavelength between 465 and 190 nm, and a most preferred wavelength between 365 and 240 nm.
G. Base Labile Silyl Linker in Coupling Reaction
An alkylsilyl moiety can be used in the coupling region to attach the surface modifying agent to the substrate. Following binding of target to the active region of the surface modifying agent, base can be employed to cleave the silicon-oxygen bond and detach the active region from the substrate. Bases such as ammonium hydroxide, sodium hydroxide, and potassium hydroxide can be employed to cleave the surface modifying agent from the substrate.
As described in detail above, we synthesized a variety of bead-shaped substrates modified with various amine-functionalized surface modifying agents. Coated beads were assessed for their interaction with doubled-stranded DNA, as well as for their interaction with bacterial cells and spores. The beads are referred to using letters A-P, and A-P refer to the same modification as presented in Table 8 above, except where otherwise noted (bead P corresponds to bead W-U). Specifically, the beads are the 50 μm silica gel beads described in Table 3 and indicated with a W.
The conditions used to examine the adhesion efficiency of cells and spores to the beads were largely the same as that used to measure interaction with DNA. Briefly, 5 mg of beads were mixed with a sample of ˜109cells/mL in 1.5 mL water at pH 5 for 5 min. Samples with beads were mixed by slow rotation and the solution tested for fluorescence or using flow cytometry before and after the addition of beads. A decrease in the amount of target in the sample indicates better adhesion and thus more efficient capture. For the measurements of cell adhesion, absorbance measurements were also run to confirm results.
An important goal of the methods of the present invention is the identification of parameters which will allow Affinity Protocol technology to be used under conditions that (a) can be easily employed in the field (e.g., at a crime scene, environmental site, accident scene, etc) and (b) are adaptable to a wide range of samples, substrates, and targets. Accordingly, we performed a series of experiments designed to understand the factors that influence DNA adhesion to substrates.
We examined the impact of a range of pH and salt concentrations on the interaction of beads coated with coating B (a triamine coating). Briefly, the experiments involved adjusting the pH and ionic strength of the sample solutions and measuring the corresponding effects on target capture and subsequent release from the beads. Both pH and ionic strength have a profound effect on the % efficiency of DNA adhesion to the beads.
In a next set of experiments, we analyzed the interaction of beads coated with coating D with DNA seeded into samples of either water, bacterial culture supernatant, or non-laboratory-grade environmental water.
Although the first step in evaluating the utility of a particular coated or uncoated substrate is determining the ability of that substrate to interact with a target, further analysis of the target likely requires the ability to recover the target from the substrate. Given the high level of sensitivity of many modern techniques for analyzing targets, it is not necessary for all of the target to be readily released from the substrate. However, the ability to recover an amount of target sufficient for further analysis is important.
As our previous analysis of the factors which influence DNA adhesion to a substrate indicated, adhesion (e.g., both adhesion and release of target) between substrate and target DNA is greatly influenced by pH and salt concentrations. Accordingly, methods which can be used to release target from a substrate include the manipulation of pH and salt concentration. Additionally, we found that temperature influences the adhesion of target DNA to a substrate (
The invention contemplates that manipulation of any of a number of variables can be used to release target (DNA, RNA, protein, bacterial cells, etc) from a substrate. One of skill in the art can readily select from amongst these variables, and the optimal elution (e.g., release) conditions will vary based on the specific substrate employed, the specific target, the concentration of the target, and the initial adhesion conditions. Exemplary variables which can be manipulated include, without limitation: salt concentration (e.g., NaCl, CaCl2, NaOH, KOH, LiBr, HCl), pH, the presence of spermidine, the presence of SDS, the type of buffer (e.g., carbonate buffer, Tris buffer, MOPS buffer, phosphate bugger), the presence of serum, the presence of detergents, the presence of alcohols, the time of adhesion, the temperature, and the application of mechanical agitation. Exemplary mechanical manipulations include sonication, use of a French press, electrical shock, microwaves, dehydration, vortexing, or application of a laser.
The invention further contemplates that the release of the target can be achieved by cleavage of a moiety that links the surface modifying agent to the substrate.
In still another embodiment, the invention contemplates the use of electroelution to recover target nucleic acid from a substrate.
Amine surface-functionalized beads have been developed and have been shown to exhibit a high affinity for DNA. The DETAP modified beads captured nucleic acids exceedingly well in a variety of liquid environments. However, although the high affinity for this substrate to DNA is desirable, it is equally desirable to be able to efficiently release target from the substrate so that the target can be further analyzed.
In addition to other methods for promoting release of targets from substrates, we have used an electric field to improve the efficiency of recovery of DETAP bead-bound DNA. Although the protocol currently being tested has not been efficient in recovering trace amounts of DNA from a substrate, this methodology has proved successful in releasing DNA when larger initial concentrations were adhered to the substrate.
Agarose and Calf Thymus DNA were purchased from Invitrogen (Carlsbad, Calif.). Agarose was melted in 0.5×TBE Electrophoresis Buffer (45 mM Tris-Borate, 1 mM EDTA). DETAP beads were synthesized, and the batch label PB-7 will be used to denote the amine-functionalized beads. GeneCapsule™ devices were obtained from Geno Technology (St. Louis, Mo.). Other standard reagents were of molecular biology grade purity.
Twenty PB-7 beads were loaded overnight in 1 mL water containing 50 μg/mL Calf Thymus DNA. Beads were loaded in a normal-mode 0.5% Agarose-TBE gel with 0.2 μg/mL Ethidium Bromide for visualization and covered with a top agarose containing 1N NaOH. Beads were also loaded in the GeneCapsule™ device using 0.5% Agarose-TBE containing various concentrations of NaOH. A 100 μL bed of agarose was set in the GelPICK™. Loaded beads were layered above this support bed, and an overlay of agarose was set. The GelTRAP™ was equilibrated in TBE for 15 minutes before the addition of 150 μL of fresh TBE and the insertion of the GelPICK™ to the level of the trap TBE as depicted in
All low DNA load experiments were conducted with the GeneCapsule™ device with 0.5% Agarose-TBE containing either 0.1N NaOH or 0.1N NaOH plus 100 μg/mL Calf Thymus DNA. Sets of twenty PB-7 beads were loaded for 30 minutes in 1 mL water containing 5, 50, or 500 μg/mL pCR2.1Topo-BtkCryIA Bacillus thuringiensis subspecies kurstaki gene copy standard plasmid. As above, loaded beads were layered above a 100 μL support gel in the GelPICK™, and an approximately 450 μL agarose overlay was set to fill the remaining volume. Pre-equilibrated GeneTRAPs™ were filled with 150 μL fresh TBE, the loaded GelPICK™ was inserted. Electrophoresis of the loaded GeneCapsules™ was conducted at 200V for either 15 minutes or 45 minutes. Eluates were removed through the pierced Collection Port via pipette. Control samples were eluted by incubation in 150 μL of 0.01N NaOH plus 100 μg/mL Calf Thymus DNA for 15 minutes at room temperature. Samples were assayed by TaqMan® real-time PCR.
As indicated by the gel presented in
Initially we note that our experiments indicate that DNA could be separated from the amine beads with relatively low voltages (˜10 V/cm within 15 minutes). The table below summarizes the results obtained using several low voltage electroelution to release DNA from a substrate. We note that under conditions of varying salt concentrations, the yield of DNA is good, however, the highest recovery was observed under higher NaOH concentration (e.g., a more alkaline environment).
These experiments indicate that electroelution is another mechanism that can be used to release target from a substrate. The present conditions have not been optimized for very low concentrations of DNA, however, the results indicate that electroelution represents a quick, safe, and cost-effective mechanism for releasing target from substrate.
As outlined in detail above, an important aspect of the invention is the ability to release target from the substrate so that the target can be further analyzed. One mechanism that can facilitate the release of target from substrate is the use of surface modifying agents containing cleavable linker that can be specifically cleaved to release target from substrate. The invention contemplates the use of any of a number of cleavable linkers.
One possible concern with the use of cleavable linkers is that the agents needed to induce cleavage of the linker may either degrade the target or may otherwise inhibit the further analysis of the target. To address this possible concern, we analyzed target DNA in the presence of DETAP or the cleavage product DETA to evaluate a possible inhibitory role for these moieties in further molecular analysis of the DNA by PCR. Based on our analysis, we concluded the presence of DETAP, and the cleavage product DETA, does not prevent further analysis of DNA by real-time PCR.
Briefly, Diethylenetriamine and (3-trimethoxysilyl-propyl)-diethylenetriamine were obtained from Sigma-Aldrich (DETA 103.2 g/mol, 0.95 g/mL; DETAP 265.4 g/mol, 1.031 g/mL). Serial dilutions of each were made in autoclaved diethylpyrocarbonate-treated water from Ambion.
Target DNA was either crude plasmid DNA from Bacillus thuriengensis subspecies kurstaki or the gene copy standard pCR2.1Topo-BtkCryIA. TaqMan® real-time PCR chemistry was used to assay samples on the ABI 7700 Sequence Detection System.
TaqMan® real-time PCR assays were performed in a standard 50 μL volume. Except for negative controls, assay reagent was spiked with 50 pg/mL of target DNA. Samples were spiked with varying concentrations of either DETAP or DETA, and water was added to the positive controls.
Inhibition of PCR was measured as a change in threshold cycle relative to the threshold cycle of the positive control containing no amine additive. Percent inhibition was taken as the ratio of the change in threshold cycle to the threshold cycle of the positive control. Our result indicated that DETAP can be inhibitory to PCR at higher concentrations. However, at concentration relevant to the application of bead-based DNA capture and release (˜25 nmol amine functionality), the level of inhibition drops significantly. The addition of 20 nmol of DETAP to a 50 ˜L PCR reaction results in a threshold cycle shift of approximately 2 (˜9% inhibition of signal).
In contrast, our results indicated that DETA alone does not significantly inhibit PCR. At both quantities relevant to the bead-based assay and at quantities that are several orders of magnitude greater, there is no apparent shift in threshold cycles due to the DETA additive relative to positive controls.
These results indicate that the use of surface modifying agents containing cleavable linkers is a feasible approach for facilitating the substrate based capture of targets, the release of those targets, and the further molecular analysis of those targets.
A second class of cleavable linkers that can be used to reversibly attach surface modifying agents to substrate is ammonia labile linkers. Accordingly, in a second set of experiments, we analyzed whether ammonia inhibits the further analysis of target DNA by PCR.
Two experiments were performed. The target was supernatant from vegetative Ba grown in BHI (culture medium) overnight, and centrifuged for 5 minutes at 3000 rpm to pellet the cells. Supernatant dilutions were prepared in BHI.
Various concentrations of ammonia were mixed with various dilutions of Ba supernatant, and allowed to incubate at room temperature. The resulting mixture was used as the eluate in a standard TaqMan reaction in the ABI7700. 5 μL of each eluate (out of a total of 50 μL) was added to the PCR reaction well, with the Ba primer-probe set. All samples were prepared in duplicate. Controls consisted of supernatant dilution (in the absence of ammonia) placed directly into the PCR well.
The results of two independent sets of experiments demonstrated that the addition of ammonia can be sustained up to a level of 0.005M concentration in the PCR reaction without any loss of PCR efficiency. Even at an ammonia concentration of 0.05M, a loss of PCR efficiency of only approximately 1-2 orders of magnitude was observed. Additionally, our observations indicated that low levels of ammonia may actually improve the efficiency of the PCR reaction—perhaps due to a favorable change in the pH of the PCR reaction mix.
The Affinity Protocol is broadly applicable to identifying and/or separating any of a number of targets from amongst heterogeneous liquid and solid samples. Even in a relatively unoptimized form, the Affinity Protocol provides increased sensitivity for detecting small concentrations of target from a heterogeneous sample, and thus even an unoptimized form of the protocol has substantial benefits in a variety of settings. However, further optimization of the Affinity Protocol has a variety of additional benefits including, but not limited to (i) the ability to detect a smaller concentration of target, (ii) the ability to identify and/or separate target in less time, (iii) the ability to detect capture upon the substrate of a higher percentage of the available target within a sample, (iv) the ability to release/elute from the substrate (e.g., for further analysis or separation) a higher percentage of the bound target, and (v) the ability to perform the Affinity Protocol using fewer starting materials (e.g., fewer consumables, less substrate).
The following examples detail experiments conducted to optimize the Affinity Protocol, and to thus achieve some of the benefits outlined above.
(a) Capture and Elution Efficiencies of Coated Substrates.
We tested several commercially available and laboratory-synthesized coated substrates to access the efficiency with which each coated substrate captured and released target. In this particular example, the target was DNA and the substrates were various magnetic beads modified with a surface modifying agent.
The following commercially available beads were used: Cortex-Biochem polystyrene-amine beads, Dynal M-270 polystyrene-amine beads, Polysciences polystyrene beads, Biosource silanized FeO-amine beads, and streptavidin functionalized beads. Additionally, the following laboratory-synthesized beads were used: M-B-1, M-B-2, and M-B-3. The laboratory synthesized beads were made as follows: 5-10 μm of uncoated magnetic particles (aka—beads of 5-10 μm particle size or beads of 5-10 μm in diameter; obtained from CPG, Inc.) were suspended in a combination of water, the surface modifying agent, and isopropyl alcohol. This slurry was gently stirred for 16 hours. The particles were allowed to settle on a magnet; and the liquid was decanted. The following was repeated two times. Additional isopropyl alcohol was added to the particles, the suspension was stirred vigorously for one minute, the particles were allowed to settle on a magnet, and the liquid was decanted. The surface-modified silica beads were dried in a vacuum overnight at 50° C., and following drying, the amount of surface modification was determined by thermogravimetric analysis.
In certain embodiments, the invention contemplates capture efficiencies of greater than 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or greater than 99%. In certain other embodiments, the invention contemplates capture efficiencies of 100%.
In certain embodiments, the invention contemplates elution efficiencies of greater than 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or greater than 99%. In certain other embodiments, the invention contemplates elution efficiencies of 100%.
In any of the foregoing, the invention contemplates an overall efficiency of greater than 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or greater than 99%. In certain other embodiments, the invention contemplates an overall efficiency of 100%.
(b) Substrate Quantity and Capture Time
The Affinity Protocol is suitable for a number of applications. Many of these applications are sensitive to cost, time, and the amount of consumable supplies required to conduct the method. Accordingly, we performed a number of experiments to examine capture efficiency as a function of the amount of substrate and the capture time (e.g., the amount of time allotted for substrate-sample interaction). The results of these experiments are summarized graphically in
We note that as little as 1 mg of substrate (e.g., beads) for 1 minute is sufficient to capture greater than 90% of the target in this sample. Increasing the substrate concentration, the capture time, or both increased the capture efficiency to greater than 99.99%. One can manipulate these parameters depending on the requirements of the particular application of the Affinity Protocol to arrive at the appropriate combination of efficiency and cost.
(c) Substrate Quantity and Elution Time
As outlined in detail above, for many of the possible applications of the Affinity Protocol, the total amount of time required to perform the method is an important factor. Accordingly, we examined the elution efficiency as a function of both substrate quantity and elution time. The results of these experiments are summarized graphically in
(d) Elution Volume
As outlined in detail above, for many of the possible applications of the Affinity Protocol, the amount of reagents required to perform the method is an important factor. The need for reagents not only increases the cost of the method, but also increases the amount of materials that must be transported and maintained in the field for applications of the invention that are not conducted in a traditional laboratory setting. One of the possible reagents required for the Affinity Protocol is the elution buffer needed to recover captured target from the substrate. Accordingly, we examined the effect of elution buffer volume on elution efficiency.
The results of these experiments are summarized in
In certain embodiments, the method of eluting target from substrate is performed in a volume of elution buffer less than ⅕th the volume of the initial sample from which the target was captured. In certain other embodiments, the method of eluting target from substrate is performed in a volume of elution buffer less than ⅙th, 1/7th, ⅛th, 1/9th, 1/10th, 1/15th, 1/20th, or 1/25th the volume of the initial sample from which the target was captured. In certain other embodiments, the method of eluting target from substrate is performed in a volume of elution buffer less than 1/30th, 1/40th, or 1/50th the volume of the initial sample from which the target was captured.
(e) Elution pH
The standard elution buffer used in these experiments (100 μg/mL of calf thymus DNA in 0.01N NaOH) has a pH of 11.8. We examined the effect on elution efficiency of small changes in the pH of the elution buffer. The results of these experiments are summarized in
(f) Elution Buffer Optimization
As outlined in detail above, calf thymus DNA was included in the elution buffer. Accordingly, we conducted experiments to assess whether elution efficiency was sensitive to the concentration of calf thymus DNA included in the buffer. Briefly, we varied the concentration of calf thymus DNA in the elution buffer between 50 μg/mL and 500 μg/mL. We observed no significant increase in elution efficiency with concentrations of calf thymus DNA greater than 100 μg/mL. Thus, we selected a standard concentration of 100 μg/mL of calf thymus DNA for use in the elution buffer given that the use of additional reagent (e.g., with the concomitant expense) produced no significant benefit with respect to elution efficiency.
(g) Washing
One or more wash steps are typically employed in many isolation or separation protocols. Accordingly, one embodiment of the Affinity Protocol could involve a wash step following target capture but prior to target release. Such a wash step could be used to remove low affinity materials from the substrate, and to thus increase the specific capture and elution of target that binds with increased affinity to the substrate. However, the need for one or more wash steps increases the time, cost, and amount of reagents necessary to perform the Affinity Protocol. Accordingly, we conducted a series of experiments to assess the need for one or more wash steps following target capture but prior to target elution.
Briefly, we performed the Affinity Protocol in the presence or absence of two 1 mL wash steps. The results of these experiments indicated that the wash steps were not required and, in fact, did not significantly altered the efficiency of DNA recovery. Additional experiments performed using DNA suspended in other, more heterogeneous sample such as growth media or non-laboratory water indicated that wash steps were not necessary. We note that the presence of two wash steps did not significantly decrease the efficiency of DNA recovery, and thus wash steps could be employed if necessary or desired in certain applications. For example, if the sample is extremely heterogeneous, hazardous, or contains a high concentration of inhibitory materials that may effect further analysis of isolated target, then wash steps can be employed without a significant negative effect on recovery efficiency. If, on the other hand, speed or cost is a significant issues, the post-capture wash step can be omitted.
The Affinity Protocol provides an improved method for separating and/or identifying a target from a heterogeneous sample using a substrate. The substrates can be of virtually any size or shape, can be magnetic or non-magnetic, and can be modified with one or more surface modifying agents that preferentially increase the affinity for the modified substrate to a particular target in comparison to the affinity of the modified agent for other material in the sample.
The Affinity Protocol is suitable for any of a large number of laboratory or field applications. Furthermore, as outlined in detail in Example 9, aspects of the Affinity Protocol can be manipulated to (i) decrease the time required to perform the method, (ii) decrease the cost of the materials required to perform the method, and (iii) decrease the number of materials required to perform the method. For example, the Affinity Protocol can be performed in a range of sample volumes, for example, 1 mL-5 mL. The Affinity Protocol can be performed using a range of substrate concentration, for example, 1 mg/mL-5 mg/mL of a substrate such as beads. The Affinity Protocol can be performed with a capture time of 5 minutes, or even less than 5 minutes, and with an elution time of 1 minute, less than one minute, or thirty seconds. Of course, one of skill in the art will readily appreciate that the present invention contemplates the use of any of a number of parameters, and the foregoing are merely indicative of parameters that can be advantageously used to decrease time and cost of carrying out this method.
We provide in detail herein a rapid application of the Affinity Protocol that was used to separate target from a heterogeneous sample. In this example, the total time required to separate target is less than 5 minutes. In this example, the substrate was 2.7 μm, amine derivatized, magnetic beads (Dynal), the target was DNA, and the sample was bacterial supernatant diluted in deionized, laboratory water. Below we have provided an exemplary, rapid protocol. Beside each step both the time required to conduct each step of the protocol and the total time elapsed is provided.
One application of the methods, compositions, and apparatuses of the present invention is for long term storage of targets separated from a sample. Such long term storage is useful in a variety of contexts. For example, efficient and reliable long term storage is useful in a forensic context for cataloging biological evidence. Furthermore, long term storage is useful in a medical context for preservation of samples for educational purposes, as well as preservation of samples for analysis that cannot be performed immediately upon target collection. Furthermore, long term storage is useful in a variety of environmental contexts where target collection may take place in the field but where target analysis will occur in a laboratory that may be geographically separated from the field site.
One example of long term storage involves the use of the substrate itself as a vehicle for the target. For example, following target capture on the substrate, the target-substrate complex can be separated from the sample, vacuum dried, and stored. This can be done extremely rapidly. In the rapid protocol summarized above, this drying and storage step may be optionally inserted following step 5 (e.g., following approximately 2 minutes of handling time). By way of specific example, the tube containing target-bead complex can be placed in a vacuum oven at 80° C. for approximately 30 minutes or until the bead pellet is dry. The dried pellet can be stored, for example, in a dark container with dessicant.
As outlined in detail above, the Affinity Protocol can be effectively used to separate target from a sample. We have additionally tested the particular bead, capture, and elution conditions described in detail in Example 9 to assess the efficiency of target recovery from more complex samples. These more complex samples may more accurately mimic the types of medical and environmental samples to which this technology applies. Exemplary complex samples include solid samples such as soil, mud, clay, and sand or other high humic soils. Further exemplary complex samples include biological samples such as blood, urine, feces, semen, vaginal fluid, bone marrow, and cerebrospinal fluid. Still further exemplary complex samples include sea water, pond water, oil, liquid or solid mineral deposits, and dry or wet food ingredients.
Briefly, we separated target DNA from a number of complex samples using the Affinity Protocol. Separated target DNA was amplified using PCR. Our results indicated that target DNA could be separated from a complex sample using the Affinity Protocol, and that the separation was sufficient to remove agents that might inhibit PCR. Target DNA from both B. anthracis (Ba) and B. thuringiensis (Btk) culture supernatant was efficiently separated from non-laboratory grade, environmental water containing any of a number of complex contaminants not found in laboratory-grade water. Not only was the DNA efficiently captured and eluted, but it was also separated from inhibitory contaminants sufficiently to allow amplification of the DNA in a PCR reaction.
In a second set of experiments, target DNA from both B. anthracis (Ba) and B. thuringiensis (Btk) culture supernatant was efficiently separated from concentrated growth media (BHI) which contains any of a number of complex additives not found in laboratory or non-laboratory grade water. Not only was the DNA efficiently captured and eluted, but it was also separated from inhibitory contaminants sufficiently to allow amplification of the DNA in a PCR reaction.
In a third set of experiments, we separated target bacterial cells from complex samples using the Affinity Protocol. Briefly, we separated target DNA from a number of complex samples using the Affinity Protocol. DNA from separated target cells was amplified using PCR. Our results indicated that bacterial cells could be efficiently separated from complex samples, and furthermore that DNA from these bacterial cells could then be amplified by PCR. Ba, Btk, and Yp vegetative cells were used as target bacterial cells, and these targets were separated from non-laboratory grade, environmental water containing any of a number of complex contaminants not found in laboratory-grade water.
As detailed herein, the affinity protocol can be used to separate a wide range of targets from various samples including gaseous, liquid, and solid samples. We now demonstrate that the separation of targets from various types of samples does not require that the samples first be rehydrated in water or otherwise processed to form a slurry. Although the rehydration of certain types of samples may be useful, certain materials such as clay soils are either difficult to rehydrate or become difficult to process further following their rehydration.
Dry biological particles typically carry a charge, and this charge can be used to help facilitate the separation of targets from dry samples such as soil samples or air. To more particularly illustrate, a magnetic substrate or a magnetic substrate coated with a surface modifying agent would be added to a sample and the sample and substrate would then be mixed so that the substrate contacts the sample. Following mixing, a target-substrate complex forms, and this can be processed using any of a number of methods detailed herein for examining targets separated by the Affinity Protocol.
Application of the Affinity Protocol to non-liquid samples has a variety of important environmental, medical, industrial, and safety applications. As outlined above, separation of target from dry sample can be accomplished by first rehydrating the dry sample to create a slurry which is then contacted with substrate to form target-substrate complexes that can be separated, and optionally analyzed further. Alternatively, separation of target from dry sample can be accomplished without the need to first rehydrate the dry sample.
We conducted additional experiments to separate and optionally analyze target from samples that were originally in a dry state. In these experiments, cartridges comprising surface modified, magnetic substrates were used to perform the Affinity Protocol on dry samples. Briefly, Ba spores (target) were seeded at varying dilutions (0-106 spores/mL of sand) into samples of sand. Each cartridge was loaded with 1 gram of sand wetted with 5 mL of distilled water. 15 mg (3 mg/mL) of magnetic beads (substrate) were used in the cartridge to capture the target. Capture time in this application of the Affinity Protocol was 5 minutes, and elution time was 1 minute.
Following elution of the target spores, DNA from the target was analyzed by PCR to assess the limit of detection of target in sand using the Affinity Protocol prior to PCR analysis, in comparison to the limits of detection using PCR alone.
We note that this cartridge containing magnetic beads (the substrate) was similarly used effectively to perform the Affinity Protocol on other samples containing target. For example, this cartridge was used to separate bacterial cells or bacterial spores from non-laboratory grade, environmental water. Using substrate concentrations of 3 mg substrate/mL of sample, target capture times of 5 minutes, and target elution times of 1 minute, we observed one order of magnitude or greater improvements in detection in comparison to PCR alone. Specifically, we detected concentrations of bacterial cells and bacterial spores as low as 10 cells/mL of sample.
As outlined in detail above, the large-scale application of the Affinity Protocol and the Affinity Magnet Protocol may be facilitated by the development of devices which promote the efficient mixing of substrate and target within a large sample. We have constructed an apparatus to achieve journal bearing flow based on the principles outlined in
We have used the Chaotic mixing device with the Affinity Protocol to extract bacterial targets from various types of soil, in quantities of 2 grams per sample. The large scale application of the affinity protocol demonstrates that these methods and devices are suitable for not only small sample sizes, but can also be scaled-up for industrial applications. The ability to scale-up the Affinity Protocol has implications not only for industrial applications of this technology. The results provided herein also demonstrate that certain target-substrate interactions may be more readily detected in larger volumes.
As outlined in detail herein, the present invention contemplates that a wide range of substrates can be used in the Affinity Protocol. Such substrates may be further coated with one or more surface modifying agents. One example of an alternative substrate that can be coated with one or more surface modifying agents is provided in
The use of functionalized tubes and culture vessels would help eliminate sample transfer—which would reduce both possible error and contamination, and reduce the need for additional supplies. Additionally, the use of such substrates would allow the target adhesion and further analysis to occur in a single vessel, and is thus readily adaptable to field applications or other settings where supplies and time may be limiting.
Other specific devices that can be designed based on the Affinity Protocol described herein are devices which facilitate gaseous or liquid sample collection and analysis. These devices will be broadly referred to as Class 2 devices. The invention contemplates the construction of both wet and dry filters. The filters can contain one or more layers of substrate (e.g., beads, paper, etc). Dry or wet samples that pass over/through the filter will pass through the substrate, and target within the sample will adhere to the substrate.
By way of further example of a dry format filter, one or more layers of substrate such as beads can be packed. The invention contemplates filters containing multiple layers of either the same substrate or of different substrates, as well as filters containing a single layer. In embodiments where the filter contains a single layer, the layer may contain a single substrate, a single substrate derivatized with multiple surface modifying agents, or multiple substrates. Air flows through the filter, and targets in the air sample are adsorbed onto the beads.
The invention contemplates the use of these filters alone, or in combination with other air filters commonly used in buildings and vehicles. For example, an Affinity Protocol-based filter can be added to a buildings HVAC system to provide a means for further analyzing the quality of the air circulating in the building.
Similarly, wet-filters can be used to assess the presence of targets in water samples. Such filters can be used to monitor reservoirs and thus assess the quality of drinking water, to monitor lakes or ponds and thus assess the health of these environments. These filters can be modified for use in aquariums, and thus help to both evaluate the quality of the water and to diagnose any water-related problems. Furthermore, these filters can be used in the home in combination with commercially available water purification devices. The invention contemplates the use of these filters alone, or in combination with other water filters commonly used in home, environmental or industrial applications.
The invention further contemplates the construction of another class 2 device: Affinity Protocol cartridges. These particular cartridges were designed based on cartridges previously designed and disclosed in U.S. publication No. 2003/0129614 (U.S. patent application Ser. No. 10/193,742, hereby incorporated by reference in its entirety), however, the present invention contemplates cartridges that contain only a means for performing the Affinity Protocol on a sample, as well as cartridges that contain both a mean for performing the Affinity Protocol and a means for performing the SNAP protocol.
The following device, used for the collection and purification of an environmental, clinical, bioagent, or forensic sample containing DNA, was described in U.S. publication No. 2003/0129614. This device can be further modified to include a means for performing the Affinity Protocol on a sample.
The outer container can be attached to the inner housing by means of a tether and screw or snap fastener on the bottom of the outer container. The outer container can also have a flange integrated into the bottom surface, to provide stability and prevent tipping when the cylinder is resting on a surface.
In one modification of this device, an additional layer is introduced such that sample is brought into contact with a means for performing the Affinity Protocol (e.g., a substrate that binds to target) prior to being brought into contact with the SNAP filter.
Another possible modification of the device involves the addition of processing steps after the purification and inhibitor binding steps described earlier. It is well-known that under the appropriate salt and pH conditions, nucleic acid will bind strongly to silica and glass, while other classes of compounds will not be as strongly bound (for example, see Tian et al. 2000 Analytical Biochemistry, 283:175-191). By changing the pH and/or salt conditions, the nucleic acid can be eluted from the silica/glass material, thus allowing selective binding and subsequent release of nucleic acid from a mixed sample. This effect, described in the “Boom” U.S. Pat. No. 5,234,809, is the basis of several existing commercial nucleic acid purification technologies, produced by companies such as Qiagen and Promega. We provide a novel implementation of this “Boom” effect that is mechanically and chemically compatible with our devices and can further facilitate the detection and analysis of target within a sample.
The processing of the sample with the device proceeds as described earlier up to the point at which it is brought into contact with a chaotropic salt on a solid matrix and eluted from that matrix. At this point in the process, the sample contains high concentrations of chaotropic salt, which promotes binding of nucleic acid to silica or glass. The sample is next brought into contact with a silica or fused glass substrate. In a preferred embodiment, the sample is eluted through a silica column by applying positive pressure with a plunger (see
This method and device can be coupled to numerous variants of existing sample capture and cell lysis techniques already described in this and earlier patent applications. This method could also be coupled to other sample capture and cell lysis techniques, so long as the composition of the sample immediately prior to beginning this process include high concentrations of salt and was in a practical pH range (for example, pH 3-12).
As described previously, the preferred embodiment of the device includes applying the sample to a porous support that contains a high concentration of chaotropic salt, which, among other functions, inactivates or kills agent in the sample. This effect renders the cartridge safe for subsequent handling and transport. For some applications, however, the user may want to culture any organisms present in the sample while still gaining the other advantages of processing the sample with chaotropic salt. Two alternate configurations of the sample cartridge address these conflicting goals are provided (see
In a second design, the inner chamber of a device is divided into two sub-chambers that have no fluidic communication. The porous support is also divided into two sections, with one section containing chaotropic salt while the other does not but instead may contain chemicals that enhance culture. This design is better suited for archival purposes, because both halves must be processed simultaneously. Although it is expected that it will be possible to culture from eluate taken from the chaotropic salt-free side of the inner cylinder, culturing from the porous support prior to elution will yield a higher concentration of organism.
As outlined in detail above, the similar characteristics and structure of DNA and RNA suggests that substrates that interact with DNA will also interact with RNA. The invention contemplates that the compositions and methods for the separation and/or identification of DNA from a sample can also be used for the identification and/or separation of RNA. However, given that RNA is typically less stable and more susceptible to degradation than DNA, the invention further contemplates that the separation and/or identification of RNA may require additional modifications to the present methods.
The ability to rapidly isolate and purify RNA from a sample of interest requires isolating the RNA under conditions that preserves the RNA. RNA is present in all organisms, so the methods described herein could be applied to RNA isolation from eukaryotes, prokaryotes, archaea, or viruses. In particular, we have explored isolation of RNA from viruses.
RNA isolation is complicated by the susceptibility of RNA to rapid degradation by nucleases in the environment. Viral RNA must be isolated from the virion particles in a way that inactivates these ribonucleases (RNases). Agents that inhibit or otherwise inactivate RNases are incorporated into many of the currently available laboratory procedures and commercial kits used to isolate RNA, however many of these methods are slow, labor intensive, and expensive.
We have previously reported the use of the SNAP/MITLL method and the use of reagents such as IsoCode™ paper to help efficiently isolate DNA under conditions that inhibit the degradation of the DNA. Furthermore, we have previously reported the development of devices referred to as LiNK which incorporate SNAP/MITLL methodology into a cartridge format for easier handling, portable, and field-related use. The present invention contemplates that SNAP/MITLL and LiNK technologies can be adapted to further enhance ability to separate and analysis target RNA from a sample. Such RNA-focused modifications of SNAP/MITLL and LiNK could be used alone, or could further enhance the efficacy of the Affinity Protocol described in the present application.
RNA-specific modifications of SNAP/MITLL and LiNK technologies would be based on the following principles. Preservation of RNA should involve both the prevention of degradation of RNA by RNases, and the prevention of nonenzymatic hydrolysis of the phosphodiester bonds in RNA. This hydrolysis is mediated by high temperature or pH extremes and divalent cations. RNA purification, therefore, must take place in appropriately buffered solutions.
Identification of an RNA virus by reverse transcription PCR (RT-PCR) can be broken down into four steps: extraction and isolation of RNA, prevention of degradation of RNA by RNases and hydrolysis, conversion of RNA to cDNA via RT-PCR, and amplification of DNA via PCR. These steps are discussed in more detail below.
a) Extraction and Isolation of RNA
RNA isolation from viruses requires the dissociation of the external viral coatings without degradation of the RNA. Commonly used RNA-extraction methods include SDS, phenol, or high-molarity chaotropic salt. IsoCode™ paper, used in the SNAP/MITLL protocol, also has the capability of releasing RNA from sample applied to the paper.
b) Prevention of RNA Degradation by RNases
Numerous RNase inhibitors exist. Many of these inhibitors could be used singly, or in combination for a rapid, simple RNA isolation protocol. Useful inhibitors must have a wide specificity (some RNase inhibitors act only against one class of RNases) and must not themselves inhibit downstream RT-PCR reactions (some RNase inhibitors are general enzyme inhibitors), or they need to be easily and completely removed from the extracted RNA.
The invention contemplates the following inhibitors for use in the separation and/or identification of RNA target: clays (bentonite, macaloid); aurintricarboxylic acid (ATA); chaotropic salts, including guanidinium thiocyanate (GT) and guanidinium hydrochloride (GH); diethylpyrocarbonate (DEPC); SDS; urea; and vanadyl-ribonucleoside complexes (VRCs).
The invention further contemplates that inhibition of hydrolysis by pH and temperature extremes can be mediated by eluting RNA in pH-buffered solutions such as Tris-EDTA.
The following RNase inhibitors have characteristics that make them preferred agents for use in the methods of the present invention: macaloid, bentonite, ATA, SDS, urea, DEPC, and the chaotropic salts. These agents are stable at room temperature, and either do not inhibit downstream RT and PCR reactions or are easily removed or diluted without organic extraction. The following paragraphs provide brief descriptions of each of these inhibitors.
Overview of RNase Inhibitors
Two of the RNase inhibitors, macaloid and bentonite, are types of clay. Their inhibitory properties are thought to be caused by their overall negative charge, which allows them to bind RNases and other basic proteins. Macaloid is a purified hectorite (a clay consisting of sodium magnesium lithofluorosilicate). Bentonite is a montmorillonite clay (Al2O3.5SiO2.7H2O). A fraction prepared from each of the clays is stable at room temperature and appears to be compatible with incorporation into a cartridge format. They have different pH optima for RNase inhibition and so could be used separately or together.
Aurintricarboxylic acid (ATA) is a general inhibitor of nucleases (DNases and RNases, included) in in vitro assays, and has been used in bacterial RNA isolation. ATA is the primary constituent of a commercial RNase inhibitor, RNase block (Innogenex, Inc.). It is a highly water soluble, dark red solution that can be removed from purified nucleic acids by gel filtration (through Sephadex G-100). RNA isolated with ATA can be used for RT-PCR. ATA does not appear to inhibit DNA isolation, however trace amounts may inhibit the action of reverse transcriptases. If such inhibition of reverse transcriptases is observed, an extraction step to eliminate the ATA prior to reverse transcription may be readily employed.
Chaotropic salts such as the guanidinium compounds (GT and GH) are strong protein denaturants that inhibit the action of RNases and are the basis of many RNA extraction procedures. These compounds are the basis of the IsoCode™ paper that is used in the SNAP/MITLL protocol.
Vanadyl-ribonucleoside complexes (VRCs) are competitive inhibitors of RNases. They are superior to DEPC, polyvinyl sulfate, heparin, bentonite, macaloid, SDS, and proteinase K. Unfortunately, they have significant drawbacks in that trace amounts inhibit RT and PCR polymerase activity, requiring removal by organic extraction. Additionally, VRCs do not inhibit all RNases, and specifically do not inhibit the activity of RNase H. A further, although not insurmountable, limitation is that VRC require storage at <−20° C. We note however, that the physical attachment of VRCs to a particular surface (for example, a cartridge over which a sample is passed or a bead which can be added and removed from a sample) would enable binding of RNases by mixing the sample in the presence of the modified surface and subsequent physical separation of VRCs from the sample prior to subsequent molecular analysis.
SDS is a detergent that denatures proteins, including RNases.
For any of the foregoing, as with all currently employed RNA-isolation procedures, relevant solutions will be pretreated with DEPC. DEPC is not useful as a standalone RNase inhibitor for environmental samples as it reacts with amines and becomes inactivated.
c) Reverse Transcription and PCR
The extracted RNA must be compatible with downstream analysis, i.e. free of reverse-transcriptase and PCR inhibitors. As reviewed in Wilson, 1997, materials to remove inhibitors include 5% DMSO, BSA, and the T4 Gene 32, among others. In addition, RT-PCR reaction conditions are available for the detection of many viruses of interest (De Paula, 2002; Drosten, 2002; Leroy, 2000; Pfeffer, 2002; Warrilow, 2002).
One application of the above outlined methodologies for separating and further analyzing target RNA is in the construction of devices which incorporate reagents which help prevent the degradation of target RNA and/or prevent the action of compounds which inhibit the later molecular analysis of an RNA target. Such devices and methodologies can be used alone or in combination with methods and devices based on the Affinity Protocol described herein.
The following provides a detailed description of an exemplary layered device. However, the invention contemplates the construction of devices that utilize the same or similar reagents but are not organized in a layered configuration. Construction of a device or development of a cartridge approach into which a sample is placed could be done in a layered approach as follows:
a) Lysis of the Organism of Interest
The part of the device which first contacts the sample could contain reagents to lyse viruses, bacteria, eukaryotic, or archaeal organisms. This lysis will split the organism open and allow DNA or RNA to be extracted. Reagents to do this could consist of chaotropic salts, SDS, or urea. Additionally, heat or cold could be used to lyse samples. Temperature changes could be provided by a battery-powered resistor-based heating circuit built into the support structure for a cartridge or by means of a chemical reaction.
Possible implementations of the lysis mechanism could include addition of solutions containing the aforementioned reagents; addition of the sample to a dry filter or matrix containing those reagents, which upon the addition of water (for a dry sample) or the sample itself (for a liquid sample), the reagents would re-dissolve to the correct concentration.
b) Inhibition of RNases
Intermixed with the reagents to lyse the sample, reagents to inhibit the action of RNases, to physically trap the RNases, or to bind the RNases should be present. These reagents include GT, GH, urea, SDS, bentonite; macaloid, ATA, VRCs, and cellulose-based papers like IsoCode™. GT, GH, urea, and SDS can be present in solution and can be removed by the addition of a desalting step or dilution to a concentration that doesn't inhibit the action of downstream detection steps. The clays bentonite and macaloid can be layered on top of IsoCode™ or other cellulose-based papers. Incorporation of ATA or VRCs can be done by chemically linking the ATA or VRCs to a solid support, so that they are not present in the eluate that contains RNA, or by addition of a filtration step.
c) Filtration to Remove ATA
In the event that the device incorporates ATA as an RNase inhibitor, it is necessary to remove the ATA from the eluate. This can be done by filtration through a size exclusion column (e.g., a Sephadex G-100 column). Such a column could be included as a layer in a cartridge-based device.
d) Binding of Nucleic Acid and Removal of RNases
A layer of size-fractionated silica, chemically-treated beads, or a chemically treated membrane or surface can be used to bind nucleic acids (DNA or RNA) to allow subsequent purification by rinsing the lysed sample to remove metals, salts, or other materials that have not been specifically bound in the previous layers. Nucleic acids can then be eluted from the silica, beads, or surface with appropriate conditions and analyzed using standard methods in molecular biology.
For many applications of the present invention, the ability to simultaneously assess the presence of multiple target is advantageous. For example, the ability to separate two different bacterial cell types would enable medical diagnostics that assess the presence of multiple, potentially infectious agents in a single test. Similarly, the ability to separate both DNA and RNA from the same sample would allow simultaneous assessment of bacterial and viral organisms, or of DNA and RNA-based viruses.
We evaluated the ability to isolate DNA and RNA using a commercially available glass fiber filter, and a standard protocol for the use of this filter. Our results indicated that DNA and RNA can be simultaneously isolated from the same sample using standard protocols and indicated that simultaneous isolation of multiple targets using the Affinity Protocol is also possible. The use of the Affinity Protocol would greatly simplify separation of multiple agents in comparison to currently available techniques which are more time, labor, and reagent intensive.
Briefly, samples containing bacteria (bacillus thuringiensis-Btk), MS2 bacteriophage (a bacteriophage that infects E. coli and serves as a model for single-stranded, RNA viruses), or both Btk and MS2 were analyzed. Samples were diluted in L6 buffer (buffer containing: guanidine isothiocyanate; 0.1M Tris-HCl (pH 6.5); 0.2M EDTA (pH 8.0); Triton-X 100) and passed over a commercially available, glass fiber filter in a volume of 1 mL. 60 mL of air was passed through the filter using a 60 mL syringe. 2 mL of L2 buffer (buffer containing: guanidine isothiocyanate; 0.1M Tris-HCl (pH 6.5); 0.2M EDTA (pH 8.0); Triton-X 100) was applied to the filter. Application of L2 buffer was followed by 60 mL of forced air, 3 mL of 70% EtOH, and then another 60 mL of forced air (repeated 2×). The filter was then dried, and target was eluted with TE (Tris, 1.0 mM EDTA—final pH=7.0).
RT-PCR and PCR were performed on aliquots of the eluate to detect viral RNA and bacterial DNA, respectively. RT-PCR was performed in a reaction volume of 25 μl. A One-Step RT-PCR Reaction (TaMan One-Step, Applied Biosystems) was prepared using an MS2 specific primer and probe set and run in an ABI7700 real-time PCR machine (Applied Biosystems). Each 25 μl reaction contained 2.5 μl of sample eluate. The following RT-PCR conditions were used: 30 minutes at 48° C., 10 minutes at 95° C., 50 cycles of 15 seconds each at 95° C., and 1 minute at 60° C. PCR was similarly performed, however, Btk specific primers were used.
The presence of MS2 was detected by RT-PCR in samples containing either MS2 alone or a combination of MS2 and Btk. Detection of MS2 by RT-PCR in samples containing only MS2 occurred with a cycle threshold of 20.65 (standard deviation=0.33). Detection of MS2 by RT-PCR in samples containing both MS2 and Btk occurred with a cycle threshold of 21.75 (standard deviation=2.04).
The presence of Btk was detected by PCR in samples containing either Btk alone or a combination of Btk and MS2. Detection of Btk by PCR in samples containing only Btk occurred with a cycle threshold of 23.65 (standard deviation=0.23). Detection of Btk by PCR in samples containing both Btk and MS2 occurred with a cycle threshold of 23.81 (standard deviation=0.39).
Although commercially available glass-fiber filters, and the accompanying methodologies, can be used to separate DNA and RNA targets. These methods are time and reagent intensive, and thus present limitations to (i) their use in the field; (ii) their use for time-sensitive applications; (iii) their use for cost-sensitive applications. As outlined in detail in the present application, the Affinity Protocol overcomes many of the limitations of other analytical methods known in the art and allows separation and, optionally, further analysis of a variety of targets with minimal reagents and time.
We have demonstrated that the Affinity Protocol can be effectively used to separate a variety of targets including bacterial cells and bacterial spores, and additionally that DNA from bacterial cells and spores separated by the Affinity Protocol can be further analyzed by methods such as PCR. We now show that the Affinity Protocol can be effectively used to separate viral targets, and additionally that RNA from viral targets separated by the Affinity Protocol can be further analyzed by methods such as RT-PCR.
MS2 was separated from a sample of water using either a commercially available, glass fiber filter and the manufacturers instructions (as outlined in Example 18), or using the Affinity Magnet Protocol (amine derivatized magnetic beads for target capture and elution in buffer containing 100 ug/ml of calf thymus DNA in 0.01N NaOH). Following separation of MS2 using either method, eluate was processed by RT-PCR to identify MS2 RNA. Briefly, we successfully separated and further analyzed by RT-PCR MS2 using either methodology. Detection of MS2 by RT-PCR following separation of MS2 using the glass fiber filter occurred with a cycle threshold of 29.83 (standard deviation=0.19). Detection of MS2 by RT-PCR following separation of MS2 using the Affinity Protocol occurred with a cycle threshold of 33.02 (standard deviation=0.72). Although sensitivity of detection appears slightly higher following separation using the glass fiber filter, significant improvements with respect to time, cost, and ease of operation are achieved using the Affinity Protocol.
Further experiments indicated that the differences in sensitivity in the detection of RNA following separation using the glass fiber filter method versus the Affinity Protocol were due to an inhibitory effect on RT-PCR analysis, and not due to inefficient capture or elution of target using the Affinity Protocol. Briefly, prior to RT-PCR analysis, MS2 containing eluate was diluted in either water or in AP-elution buffer and incubated for 0, 30, or 60 minutes prior to RT-PCR analysis of MS2. Detection of MS2 by RT-PCR following incubation of the sample in water for 0, 30, or 60 minutes occurred with a cycle threshold of 20.57, 20.65, and 21.02, respectively (standard deviation=NA). Detection of MS2 by RT-PCR following incubation of the sample in elution buffer for 0, 30, or 60 minutes occurred with a cycle threshold of 24.15, 24.05, and 24.14, respectively (standard deviation=0.03, 0.93, and 0.04, respectively).
All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein.
This application is a continuation-in-part of and claims priority to U.S. application Ser. No. 10/916,784, filed Aug. 12, 2004, which claims priority to U.S. application Ser. No. 60/494,702, filed Aug. 12, 2003. The disclosures of each of the foregoing are hereby incorporated by reference in their entirety.
This invention was supported, in whole or in part, by Lincoln Contract Number F19628-95-C-0002 from Defense Directorate of Research and Engineering and by Lincoln Contract Number F19628-00-C-0002 from the U.S. Air Force. The Government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 60494702 | Aug 2003 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10916784 | Aug 2004 | US |
| Child | 11056518 | Feb 2005 | US |
