Patent Application
20110223583
The present invention relates to the field of biosensing, and more particularly concerns biomolecule condensing devices and a method for providing a concentrated biomolecule condensate to any one of a plurality of biosensing devices.
Biological hazards are caused by minute life forms called microorganisms and include certain types of infective bacteria, viruses, protozoa, and substances derived from microorganisms, that invade and grow within other living organisms and cause disease. As the consumption of even a small amount of these pathogens can sicken or kill a living organism, biohazards are taking an enormous toll on humans, animals, the food chain, and the environment. The most effective way to prevent the spread of biohazards is to frequently test for the presence of pathogens in people, animals, insects, surfaces, water, air and the food chain, and then rapidly contain biohazards before transmission occurs. As there is no universal indicator for biohazards, each specific type of pathogenic bacteria, virus, protozoa and other species needs to be tested separately to determine its presence. This has created a demand for specific biotesting.
Detecting and identifying biological materials typically requires a capital-intensive laboratory, specialized equipment, costly materials, labor-intensive processing and highly-trained personnel. Biotesting can take several days or even weeks, as many steps are required, including the collection and transportation of samples to the biotesting laboratory. Because of the limited sensitivity of biotesting techniques, samples need to be processed before testing to increase the number of target biomolecules through time-intensive incubation and amplification techniques such as polymerase chain reaction (PCR). Any time delay is a problem, since pathogens and infectious diseases can spread before the test results are known. Furthermore, the high cost per test limits the number of tests that can be undertaken by government agencies, commercial organizations, and consumers due to budget constraints.
In addition to biotesting laboratories, there is a rapidly growing market focused on the identification of biological materials using biosensors, which are measuring devices that convert a biological interaction into a measurable electrical signal. Biosensors can operate independently of laboratories and be used in portable devices and wireless sensor networks. The lower infrastructure cost, reduced consumption of materials, and ease of use of biosensors can greatly reduce the cost per test when compared to laboratory testing, and will likely be in great demand in the future.
An example of an electrochemical biosensor is described in the Assignee's U.S. patent application Ser. No. 12/216,914 filed on Jul. 11, 2008, the contents of which are incorporated herein by reference. This biosensing device includes at least one working electrode having a systematic array of nano-electrode wires projecting vertically from an electrode pad. The nano-electrode wires all have a same shape and size, and are distributed non-randomly over the electrode pad. Biosensor probes are attached to the nano-electrode wires, each including a bioreceptor selected to bind with a complementary target biomolecule to create a binding event, and an electrochemical transducer transducing this binding event into an electrical signal conducted by the corresponding nano-electrode wire.
The strength of the signal obtained through a biosensing device depends on the concentration of the target biomolecules in the sample provided for biosensing. As even a small number of pathogens can pose a health risk, it is important to ensure that a sufficient quantity of the target biomolecules is included in the sample to which the biosensing device is exposed. This is not always the case for small amounts of fluid extracted directly from a potentially affected larger sample.
There is therefore a need for technology enabling the rapid preparation of target biomolecules for biosensor use that avoids time-intensive incubation and amplification techniques.
In accordance with a first aspect of the invention, there is provided a biomolecule condensing device for providing a concentrated biomolecule condensate to at least one biosensing device. The concentrated biomolecule condensate is obtained from a fluid sample potentially containing traces of at least one target biomolecule.
The biomolecule condensing device first includes a filtration module. The filtration module has at least one ultrafiltration assembly for separating the fluid sample into a filtered liquid and a retentate biomolecule condensate containing at least one of the target biomolecule, if present in the fluid sample.
A magnetic bead separation module is further provided for separating the retentate biomolecule condensate into a beaded biomolecule condensate, containing the target biomolecules, and waste materials. The magnetic bead separation module includes magnetic beads coated with antibodies of the at least one target biomolecule so that the target biomolecules in the retentate biomolecule condensate become attached to these magnetic beads.
Finally, a microfluidics module for processing the beaded biomolecule condensate to extract constituents of said target biomolecules therefrom is provided, thereby obtaining the concentrated biomolecule condensate. The microfluidics module enables the distribution of the concentrated biomolecule condensate to one of the at least one biosensing device.
In certain embodiments of the invention, microfluidics reagents and other processes can be placed on each module or, preferably, stored in central locations and then dispensed as required. One portion of the microfluidics module can typically be sanitized and reused with fresh reagents, whereas another portion of the microfluidics module can typically be used only once. A distribution capability is also provided to deliver the concentrated biomolecule condensate to an unused portion of the microfluidics module, and where applicable, unused biosensing device.
In accordance with another aspect of the present invention, there is provided a method for sanitizing the biomolecule condensing device described above. This method includes:
a) adding a sanitizing agent to the filtered liquid obtained through the filtering of the filtering module, thereby obtaining a sanitizing solution;
b) circulating the sanitizing solution through at least one of the filtration module, the magnetic bead separation module and the microfluidics module; and
c) leaving the sanitizing solution in the at least one of the filtration module, the magnetic bead separation module and the microfluidics module, for a soaking period.
In accordance with yet another aspect of the invention, there is also provided a microfluidics module packaged as a replacement cartridge that can be replaced when all of the one-time use microfluidics assemblies and biosensing devices have been used. The replaceable cartridge can include the entire microfluidics module or just the one-time use microfluidics and biosensing devices. For example, a set of one-time use components may be provided, including a filter component hosting a filter housing which contains a plurality of filter membranes, each having a corresponding outlet, and a sensor component hosting a plurality of biosensing devices in equal number to the filter membranes.
In accordance with yet another aspect of the invention, there is provided a condensing method for providing a concentrated biomolecule condensate to at least one biosensing device, the concentrated biomolecule condensate being obtained from a fluid sample potentially containing traces of at least one target biomolecule. The method includes:
a) separating the fluid sample into a filtered liquid and a retentate biomolecule condensate containing at least one of the target biomolecule, if present in the fluid sample;
b) attaching the target biomolecules in the retentate biomolecule condensate to magnetic beads coated with antibodies of the at least one target biomolecule, thereby obtaining a beaded biomolecule condensate, and separating the same from waste materials; and
c) processing the biomolecule condensate to extract constituents of the target biomolecules therefrom, thereby obtaining the concentrated biomolecule condensate, and distributing the same to one of the at least one biosensing device.
Advantageously, embodiments of the method according to the above aspect of the invention provide for detection of the target biomolecules without time-sensitive incubation and amplification techniques.
Furthermore, in accordance with another aspect of the invention, there is provided a filtration module for providing a retentate analyte condensate from a fluid sample potentially containing traces of at least one analyte. The filtration module includes at least one ultrafiltration assembly for separating the fluid sample into a filtered liquid and the retentate analyte condensate. Each ultrafiltration assembly includes:
In one embodiment, the filtration module includes two such ultrafiltration assemblies, a primary assembly and a secondary assembly, connected in a series to provide the retentate biomolecule concentrate extracted from the primary ultrafiltration assembly to the sample reservoir of the secondary ultrafiltration assembly, to define the fluid sample therein. Advantageously, the filtration module may be used both for biosensing applications and chemical sensing applications.
Other features and advantages of the present invention will be better understood upon a reading of the preferred embodiments thereof, with reference to the appended drawings.
Embodiments of the present invention will be described herein below in conjunction with the appended drawings, wherein like reference numerals refer to like elements throughout.
The present invention generally provides methods and devices allowing the processing of a fluid sample which potentially contains traces of at least one target biomolecule, in order to obtain a concentrated biomolecule condensate apt to be provided to one or more biosensing devices.
The starting fluid sample may be embodied by any fluid which may contain target biomolecules to be detected, such as water or other liquids, blood or other bodily fluids, liquefied solids or tissues, or liquefied materials from air or gases. Water samples may for example be obtained from a pressurized source connected to a municipal water network or the like, or an unpressurized source such as a lake. The target biomolecules may be any analyte which one may wish to detect and which is apt to bind with a bioreceptor, as described further below. The present invention may be particularly useful in the context of the detection of pathogens or biohazards such as specific strains of bacterium (e.g., E. coli, Salmonella, Vibrio cholerae), viruses (e.g., Hepatitis A, Norovirus), and protozoa (e.g., Cryptosporidium, Giardia). It is of course understood that the list above is given by way of example only, and is in no way limitative to the scope of the present invention.
The biosensing devices to which the concentrated biomolecule condensate is provided are preferably embodied by electrochemical sensors, a sensing approach commonly used for the detection of chemicals and in certain cases for the detection of biomolecules. Referring to the enclosed
The expression “electrochemical sensor” refers to an electrochemical system that determines the presence and concentration of a chemical material or biomaterial through measurements of electrical signal in a solution between a working electrode 202 and counter electrode 204, such as induced by a redox reaction or electrical potential from the release or absorption of ions. The redox reaction refers to the loss of electrons (oxidation) or gain of electrons (reduction) that a material undergoes during electrical stimulation such as applying a potential. Redox reactions take place at the working electrode 202, also referred to as the measuring electrode, and which, for chemical detection, is typically constructed from an inert material such as platinum or carbon. The potential of the working electrode 202 is measured against a reference electrode 206, which is typically a stable, well-behaved electrochemical half-cell such as silver/silver chloride. The electrochemical system can be used to support many different techniques for determining the presence and concentration of the target biomolecules including, but not limited to, various types of voltammetry, amperometry, potentiometry and conductimetry such as AC voltammetry, differential pulse voltammetry, square wave voltammetry, electrochemical impedance spectroscopy, cyclic voltammetry, and fast scan cyclic voltammetry.
The biosensing device 200 of
In the context of the present invention, one or more biosensing devices may be used, and selected to detect one or more types of target biomolecules. A single biosensing device may be use to detect more than one type of target biomolecule.
In addition to determining whether target biomolecules are present, a given biosensing device may be used to evaluate the concentration of these biomolecules in the solution, as well as the percentage of the cells in the biomolecules that are viable and therefore capable of dividing and increasing in number.
It will be readily understood by one skilled in the art that the condensing devices and methods of embodiments of the present invention may be used in combination with different types of biosensing devices than the one described above. For to example, these can include biosensing devices that measure changes in: temperature (calorimetric biosensors), light output or absorbance (optical biosensors), mass (piezo-electric biosensors), and size, shape and conductivity of a conductive channel in a field effect transistor (field effect biosensors), among others.
Referring to
It will be understood by one skilled in the art that the expression “solution” as used herein is meant to include suspensions or any other form taken by the mixture of the biomolecules and carrying fluid.
The condensing method may include a preliminary pre-treatment step 102 for the liquid solution, such as removing or neutralizing interfering materials and breaking clumps in the fluid sample. Many processes can be employed for this purpose depending on the liquid media; the type, concentration, size and properties of the materials in the liquid; and the environmental conditions such as temperature, pH, etc. In one embodiment, one or more dispensers are provided to add chemicals, such as sodium thiosulfate, to neutralize chlorine in drinking water. An adherent could also be employed to remove interfering materials. In the same embodiment, one or more disaggregation techniques such as surfactants, sonication or preferably hydrodynamic cavitation can also be employed to reduce clumping.
The method next includes separating 104 the fluid sample 100, after the pre-treatment step 102, into a filtered liquid 106 which is substantially free of biomolecules and a retentate biomolecule condensate 108 containing the target biomolecules, if present in the fluid sample. According to one embodiment, this separating involves passing the fluid sample successively though ultrafiltration assemblies, preferably including a primary filter 110 and a secondary filter 112, which may for example both be embodied by tangential flow filters. At each filtering substep, the fluid sample is received in a sample reservoir, circulated through an ultrafiltration filter for separating the filtered liquid and retentate condensate, and recirculated through the corresponding filter for multiple passes, defining filtering loops, additional portions of filtered liquid being extracted from the retentate biomolecule condensate at each pass. The retentate condensate is then extracted out of the corresponding ultrafiltration assembly. In one example, the primary filtering process may set aside 97% of the initial volume of the fluid sample (V) as the filtered liquid and retain 3% V as the retentate biomolecule condensate.
The secondary filtering may further extract another 2.95% V of filtered liquid, retaining about 0.05% of the initial sample volume. Optionally, additional clump-breaking processes and removing or neutralizing interfering materials steps 102 may be performed during the primary filtering, the secondary filtering or both, preferably as part of the corresponding filtering loops.
It will be noted from the above description of the separation in the filtration module 104 of the fluid sample that rather than discarding the biomolecule solution containing bacteria, viruses, protozoa, and other potentially disease causing biomaterials, as is typically done in industrial and medical applications, the biomolecule retentate is returned to the sample reservoir and repeatedly recirculated through the filter, while the filtered liquid, free of biomolecules, is removed. The filtered liquid is preferably passed to a filtered liquid reservoir and, as described below, can be used with a sanitizing agent to sanitize the device between uses.
As the fluid sample containing the biomolecules is repeatedly pumped though the filters, the biomolecules are returned to the input reservoir and the filtered liquid is removed. This greatly changes the concentration of biomolecules since the number of biomolecules stays the same but the volume of solution reduces over time. As a result, the biomolecules become greatly condensed. For example, 1,000 cells contained in 10 liters of solution can be condensed by the primary filter to 1,000 cells in 300 mL of solution, after 9,700 mL of filtered solution (or 97% of the liquid) is removed. As may be expected, some biomolecules may attach to the filter or related systems, and somewhat reduce the yield.
Multiple filtration circuits can be used depending on the volume of the input fluid sample and the volume of the condensate to be recovered after filtration. In one embodiment, 10 liters of potable water is used to condense down to about 1 to 10 mL of condensate for a reduction in volume of 99.9% to 99.99%. In this case, two filtration circuits are provided in a series sequence. A primary filtration circuit condenses the input volume to about 100 to 500 mL of retentate, which is fed into a secondary filtration circuit that further condenses the output to about 1 to 10 mL.
The method next involves a step 114 of mixing the retentate biomolecule condensate 108 resulting from the filtering step 104 with magnetic beads coated with antibodies of at least one target biomolecule and permitting the target biomolecules in the retentate biomolecule condensate to attach to the matching antibodies. One or more solutions can be added to the mix, such as filtered liquid extracted from the filtering step, a buffer solution, or a re-suspension. The mixture is then circulated to a condensation chamber and a beaded biomolecule condensate is obtained when a magnetic field is activated to retain the magnetic beads in the condensation chamber and the waste solution is removed. Once this step is done, the magnetic field is removed with a magnetic insulator, and a rinsing solution and/or compressed gas can bring the magnetic bead retentate to the microfluidics module. In one embodiment, the volume of the magnetic bead retentate is about 0.5-1 mL.
The purpose of the magnetic beads is to separate target biomolecules that will be detected by the biosensing device from non target biomolecules that can interfere with the detection, and need to be removed from the biomolecule retentate and discarded. In the example given above, the separation step condenses a 10 L fluid sample to approximately 5 mL after two filtration circuits. When analyzing potable water, the number of biomolecules per 10 L can be in the thousands or millions of cells. Up to 100% of the cells can be non-pathogenic heterotrophic species that do not need to be detected, and should not be sent to the biosensing device. In this case, the magnetic bead separation is provided to extract the target biomolecules using magnetic beads with antibodies selected to match a suite of target biomolecules commonly identified as waterborne pathogens. These can include E. coli (Indicator), E. coli O157:H7, Campylobacter, Cryptosporidium, Giardia, Enterovirus, etc. The remaining liquid incorporating the non-target biomolecules is preferably discarded 116. Other suites of target biomolecules can be used for different applications such as in testing pathogens for Listeria bacteria in meat or Hepatitis A virus in blood.
The method finally includes a step of processing 118 the beaded biomolecule condensate to extract constituents of the target biomolecules, thereby obtaining a retentate biomolecule condensate which is distributed to an associated biosensing device. Referring to the enclosed
Preferably, processing step 118 is performed in a microfluidics module. Preferably, the processing first includes lysing cells 120 of the biomolecule condensate to release the biomolecule constituents. In one example, the beaded condensate is pumped to a microfluidics chamber and a cell lysis reagent is added. The lysis reagent and sample are mixed in a mixing chamber and the temperature is maintained at a proper value in a range of 25 to 37° C., to open the cell walls and release the cell constituents. In some lysis methods, the temperature could be as high as 90° C. Optionally, additional processing could be performed to separate the biomolecule constituents from the cell walls. In one embodiment, sonication is used for this purpose; that is, ultrasonic energy is projected through the beaded biomolecule condensate. In another embodiment, the beaded biomolecule condensate is submitted to an alternating low and high temperature cycle, to break open cell walls. This technique may for example be employed for cryptosporidium oocysts that may be more resistant to conventional cell lysis. A binding buffer and washing buffer may be added to improve the extraction and collection of target constituents.
The beaded biomolecule condensate is then filtered 122 to separate the biomolecule constituents from the magnetic beads and waste materials left over from the cell lysis. For this purpose, the mixture is preferably pumped through a membrane and waste material is discarded. An elution buffer is pumped through the membrane and carries off the target constituents.
In one embodiment, for example for 16S ribosomal RNA detection, the method next includes a step of preparing 123 the biomolecule constituents for detection. This may for example involve mixing the biomolecule constituents for DNA digestion with a RW buffer before DNase, DNase and RDD, RPE buffer (trademarks of the Qiagen company) and Ethanol. The temperature may be raised to approximately 70° C. to provide denaturing and unfolding the RNA strands in the biomolecule constituents. The biomolecule constituent may further be mixed with a wash buffer immediately prior to distribution 124 to one of the biosensing devices.
The microfluidics module can further support the biosensing device for temperature control of 25 to 65° C. that may be required for hybridization, and adding other materials such as chemical mediators and positive control target biomolecules. At this stage, the biosensing device is activated to measure the cell concentrations of target biomolecules.
In a preferred embodiment, the beaded biomolecule condensate is divided 128 into two equivalent portions by a metering system in the microfluidics module. One of the portions of the sample is immediately processed as above, and then provided to an available biosensing derive. The other portion of the sample is pumped to a culture chamber and cultured 130 with growth medium, heat and other requirements to encourage any viable cells to reproduce in number. Once a predetermined period of time or condition is attained, the second sample is then sent to an available microfluidics chamber and the above process is repeated so that the second biosensing device can calibrate the difference between the first and second readings, to determine the viability of the target biomaterials in the sample as described in the co-assigned U.S. patent application Ser. No. 12/216,914, filed on Jul. 11, 2008.
With reference to
The biomolecule condensing device 300 further includes a microfluidics module 307, which processes the beaded biomolecule condensate to extract constituents of the target biomolecules, thereby obtaining the concentrated biomolecule condensate. Preferably, the microfluidics module 307 includes a first microfluidics assembly 312 which hosts cell lysing means for lysing cells of the biomolecules attached to the magnetic beads of the beaded biomolecule condensate. Various components and processes which may be used for this purpose will be described further below. Cell lysis opens the cell walls and releases the biomolecule constituents of the target biomolecules, i.e. the strands of nucleic acid of the biomolecules. In order to separate these constituents from the magnetic bead, cell walls and other waste material from the cell lysis, the beaded biomolecule condensate from the first microfluidics assembly is then receive in a filter housing 308 which contains one or more filter membranes for retaining waste material from the cell lysis and allowing the biomolecule constituents therethrough. The biomolecule constituents are then received in a second microfluidics assembly 309 which hosts preparation means for the preparation of the biomolecule constituents for detection. This may involve several processes which will also be described further below. The biomolecule constituents are then ready for distribution to an unused biosensor 200, for detection.
In some embodiments, the biomolecule condensing device's pumps, pipes, valves and other components can be configured to support a sanitizing method as described further below, operated either automatically with Programmable Logic Controllers (PLCs) or manually by an operator to direct and control the flow of the liquids, additives and processes.
Each module of condensing device 300 according to embodiments of the invention will now be described in more detail.
As mentioned above, the condensing device 300 includes a filtration module 302 which separates the fluid sample into a retentate biomolecule condensate and filtered liquid, the retentate condensate being extracted for further processing and eventual detection of the biomolecules it contains. Although the filtration module 302 is described hereinbelow in the context of biosensing, one skilled in the art will understand that a similar module could be use for the condensation of any soluble analyte, whether biological or chemical. The analyte could for example be embodied by pathogens, drugs, pesticides, industrial chemicals, metals and natural toxic compounds. All the components of the filtration module 312 described below could therefore be adapted for chemical sensing without departing from the scope of this aspect of the present invention.
In the embodiments of
With reference to
Throughout the present description, the expression “in fluid communication” is understood to signify that one or more pipe, conduit or any other fluid path connects two components to allow fluid to flow from one to the other, at least in one direction. The communication may be direct or indirect, that is, the fluid may traverse intermediate components during its travel from one component to the other.
In one embodiment, the ultrafiltration filter is a hollow fiber tangential flow filter or membrane filter. Tangential flow filters generally permit a sample solution to flow through a feed channel along the surface of a membrane (tangentially thereto).
Liquid is extracted through the membrane with applied pressure, whereas the particles in the solution remain in the feed channel and are carried along to the retentate outlet. The cross flow prevents build up of molecules at the surface of the membrane, which could cause fouling. This process prevents the rapid decline in flux rate often seen in direct flow filtration, allowing a greater volume to be processed per unit area of membrane surface.
A concentration loop 326 circulates the retentate condensate from the retentate outlet 324 of the filter housing 316 back to the reservoir 314, and further circulates the retentate condensate through the filter housing 316 for multiple passes, additional portions of filtered liquid such as water, liquid food products, beverages, chemicals, urine or blood being removed therefrom at each pass. An extraction line 328 allows the extraction of the retentate biomolecule condensate out of the ultrafiltration assembly 304 after a sufficient number of passes through the ultrafiltration filter 318. This is preferably done when the total volume of solution passing through the sample reservoir 314 is reduced to the desired Output Volume as measured by a sensor 315, flow meter or other suitable devices.
In the illustrated embodiment of
In this embodiment, once the fluid sample enters the sample reservoir 314, the pump 334 is used to propel the fluid sample though the ultrafiltration filter 318. For example, the fluid sample may be pumped at about 20 to 30 psi into the ultrafiltration filter 318, producing a filtrate of filtered liquid and a retentate containing target and non-target biomolecules. Rather than discarding the retentate, as is typically done in industrial and medical applications, the retentate is returned to the sample reservoir 314 and repeatedly recirculated through the filter 318. Preferably, the filtered liquid is passed to a filtered liquid reservoir 329. As the sample is repeatedly pumped though the ultrafiltration filter 318, the biomolecules are returned to the sample reservoir 314 and the filtered liquid is removed. This greatly changes the concentration of biomolecules since the number of biomolecules stays the same but the volume of solution reduces over time. As a result the biomolecules become greatly condensed. For example, 1,000 cells contained in 10 liters of solution can be condensed to 1,000 cells in 300 mL of solution after 9,700 mL of filtered liquid (or 97% of the liquid) is removed.
Multiple filtration circuits can be used depending on the volume of the fluid in the input sample and the volume of the condensate to be used after filtration. In one embodiment, 10 liters of potable water is used to condense down to about 1 to 10 mL of condensate. In this case, a primary ultrafiltration assembly condenses the input volume to about 100 to 500 mL of retentate, which is fed into a secondary ultrafiltration assembly that further condenses the output to about 1 to 10 mL. Additional rinses with filtered water, buffers and/or air can be employed to release biomolecules attached to the filters or other components of the filtration module.
As mentioned above,
The fluid sample is received in the first sample reservoir 314a. A vent may be to provided to evacuate any air pressure created therein during its fill-up process. Preferably, a sensor 315a is provided in the sample reservoir 314a for sensing a level of the fluid sample therein as the reservoir is being filled. The sensor 315a is operationally connected to both the pump 334a and the valve 336a of the first ultrafiltration assembly 304a. The sensor 315a measures the level of solution against an upper and a lower threshold. When the upper threshold is reached, a valve closes the input loop 344a and the pump 334a is activated to start circulating the fluid sample in the concentration loop of the primary filtration assembly 304a. When a lower threshold level is reached, the valve 336a is set to direct the retentate from the second outlet 342a out to the secondary filtration assembly 304b. The sensor 315a may also be used to control the circulation of the fluid sample in the concentration loop before the sample reservoir 314a is filled with the incoming fluid sample, allowing the ultrafiltration assembly 304 to process an initial volume of fluid greater than the total capacity of the sample reservoir 314a. In this case a flow meter in the input loop 344 can initiate the pump 334a to start while the input sample in still filling sample reservoir 314a and provide the added benefit of reduced processing time.
The fluid sample from the sample reservoir 314a circulates through the primary ultrafiltration assembly 304a for multiple passes through its ultrafiltration filter, as explained above. Preferably, a filtered liquid reservoir 329 is provided and connected to the filtered liquid outlet 322a of the filter housing 316a to receive the filtered liquid that is removed from the liquid solution by the filtration module. Of course, the primary ultrafiltration assembly 304a may include any additional devices typically included in liquid treatment apparatuses, such as valves, pressure gauges and the like.
In the embodiment of
The filtration module 302 and pre-filtration module 350 may also include one or more processes to break up clumps of biomolecules 354, 354a and 354b. For example, bacteria tend to form aggregates or clumps of materials since cells can naturally attach to other cells as well as to different materials. When a traditional cell culture is done, the output of a Colony Forming Unit (CFU) can actually be 1 cell, or 10 cells, or 100 cells or a clump with an even bigger amount of cells. As a result, traditional cell cultures can understate the true bacteria count because these clumps are not broken up and appear as lower number of CFUs than if the clumps were broken up. In other cases, biomolecules can be trapped in biofilms formed in the incoming fluid sample. Disaggregation techniques such as surfactants, sonication or hydrodynamic cavitation can be added to the prefiltration module and/or each ultrafiltration assembly to reduce clumping.
In one embodiment, hydrodynamic cavitation is used to work like a garden hose to increase the speed of the solution flow and subsequently mechanically erode and decompose the surface of the clumps or biofilms. This will allow the device to break up the clumps when the retentate is recirculated though one or more re-circulation loops and/or pre-filtration module, and ultimately provide a more realistic cell count which will be higher and more accurate.
Referring back to
With reference to
The magnetic bead module 306 is preferably provided between the filtration module 302 and the microfluidics module 307. The purpose of the magnetic bead module is to separate target biomolecules that will be detected on a biosensing device 200 from non target biomolecules that can interfere with the detection and need to be discarded. In an embodiment described above, the filtration module condenses a 10 L water sample to approximately 5-7 mL. For example, when sampling potable water, the number of biomolecules per 10 L can be in the thousands or millions of cells. Up to 100% of these biomolecules can be non-pathogenic heterotrophic species that should not be sent to the biosensing device. The magnetic bead module 306 extracts the target biomolecules using magnetic beads 362 with antibodies selected to match a suite of target biomolecules commonly identified as waterborne pathogens. These can include E.coli (Indicator), E.coli O157:H7, Campylobacter, Cryptosporidium, Giardia, Enterovirus, etc. All the magnetic beads 362 mixed with the retentate biomolecule condensate is may be of a same type, or of multiple types depending on the biomolecules to be detected.
In one embodiment, shown in
The magnetic bead separation is preferably performed at room temperature. It may be necessary to provide refrigeration capabilities for storing the magnetic beads onboard for a finite time between replacement cartridges, such as up to 6 months.
The magnetic bead module may be refined as needed to support other types of solutions such as water drippings from washed fruits; liquefied particles from air; liquefied tissues from plants, animals and humans; blood and other body fluids. Variants to the magnetic beads module described above can include the types of antibodies, the number and size of beads, contact time, magnet type, and mediator depending on the target biomolecules to be detected, the solution type and properties, and the interfering materials in the solution.
The magnetic beads module can be configured to flow the retentate back and forth with pump 376a as in
With reference to
The microfluidics module generally includes a first microfluidics assembly 312, a filter housing 316 and a second microfluidics assembly 309.
Referring to
In both branches of the first microfluidics assembly 312, or in a single branch, as the case may be, cell lysing means are provided for lysing cells attached to the magnetic beads of the beaded biomolecule condensate. The lysing releases the biomolecule constituents of the target biomolecules. Preferably, a lysis mixing chamber 404a, 404b is provided, in which the beaded biomolecule condensate is mixed with cell lysis reagents, such as a Bacterial Protect Reagent (BPR) solution or a lysozyme lysing solution. Appropriate solutions may for example be obtained from the company Qiagen suh as a RLT (trademark) buffer solution. Each solution reagent may be provided from a suitable dispenser 418b in fluid communication with the corresponding mixing chamber 404a, 404b. The solution containing the mixed beaded biomolecule condensate and cell lysis reagents is agitated back and forth in the lysis mixing chamber and then sent to extraction chamber 406a. A heater 410 preferably collaborates with the lysis mixing chamber 404a, 404b, to heat this chamber to an optimum temperature in the range of 25 to 37° C., preferably for 5 to 10 minutes, should it be necessary to heat the sample to facilitate cell lysis. Of course, other temperature ranges or heating times may be considered depending on the particular application. For example, temperatures as high as 90° C. can be reached depending on the lysis method used. This process opens the cell walls of the target biomolecules, exposing the RNA strands therein. Optionally, the beaded biomolecule is further processed to help separate the biomolecule constituents from their cells. In one embodiment, a sonication device (not shown) projects ultrasonic energy through the mixing chamber. Other methods can also be employed to open the cell walls and extract the target biomolecule constituents used for biodetection. For example, the beaded biomolecule condensate can be submitted to an alternating low and high temperature cycle to break open cell walls. This technique may for example be employed for cryptosporidium oocysts that may be more resistant to conventional cell lysis. This process may take place in an additional mixing chamber and the same heater as mentioned above or a different one may be provided for this purpose.
Still referring to the embodiment of
The filtered biomolecule constituents are then processed through a second microfluidics assembly, which includes preparation means for the preparation of the filtered biomolecule constituents for detection. For this purpose, one or more mixing chamber 412 is provided. Each mixing chamber 412 mixes the biomolecule constituents with an appropriate additive, such as an elution buffer, binding buffer or washing buffer, Appropriate solutions may for example be as provided from the company Qiagen such as a RW buffer, a DNase buffer, a DNase and RDD solution, a RPE buffer (all trademarks?) and an Ethanol solution. Preferably, the mixture in each mixing chamber 412 is agitated back and forth, preferably through alternative pumping means (not shown). Heating means such as an additional heater 411 may be provided for heating the biomolecule constituents at an appropriate temperature and for a length of time sufficient to denature and unfold the RNA strands therein. In one embodiment, the biomolecule constituents are heated to about 70° C. to permit denaturing and unfolding of target constituents such as rRNA.
The biomolecule constituents are then delivered to an unused one-time use biosensing device 200 for measuring the total number of target cells. A second distribution manifold 311b, in fluid communication with the second microfluidics assembly 309, receives the biomolecule constituents therefrom, and distributes them to one of a plurality of outlets, each connected to a corresponding biosensing device 200. Any waste output is sent to a waste container. In another embodiment, the RNA output is passed to an unused biosensing device and is agitated back and forth and heated to 25-65° C. for hybridization. A mediator and positive control target are added to the solution and a detection process commences at about 25° C.
Although the first microfluidics assembly 312, first manifold 311a, filter housing 316, second microfluidics assembly 309, second manifold 311b and biosensing devices 200 are shown in
Referring to
In one embodiment, a chemical mediator for amplifying the electrochemical signal from the biosensing device and target biomolecules for the positive control electrodes, is also available for adding to the biosensing device. The different steps of this process are shown in
Alternatively, the components of the second microfluidics assembly may be integrated at the level of the filter membranes or the manifold.
A delivery system for moving liquid though the microfluidics module can include a pump 416 to provide compressed gas to push fluids through the module, and a vacuum 419 that can pull fluids through the microfluidics module. The pump 416 can also be used to control pneumatic switches 414 in the manifold.
In another embodiment, a cartridge houses a plurality of biochips, or a plurality of one-time use microfluidics assemblies and one-time use biosensing devices, also referred to herein as an insert. For example, the cartridge could have a vertical stack of inserts similar to a PEZ (trademark) candy dispenser, with an unused insert placed from one end into a housing to receive the magnetic bead condensate and then replaced after use. In another embodiment, inserts are loaded into a circular carousel resembling a 35 mm slide projector, where an unused insert is placed into a housing from the carousel to receive the microfluidics condensate and then replaced after use.
In the illustrated embodiment, in accordance with one aspect of the invention, a set of one-time-use components for the microfluidics module of a condensing device as described herein may be provided. This set may include a filter component holding the filter housing which includes a plurality of filter membranes, each having a corresponding outlet. A separate sensor component may host a plurality of said biosensing devices in equal number to the filter membranes. The two components may, for example, take the form of a disk and be provided individually, or held in a fixed arrangement through appropriate holding means. In one embodiment, each biosensor and each membrane is provided on a biochip manually inserted into a structure to receive the processed biomolecule constituents. The filter component and sensor component are preferably fabricated on separate undiced substrates, such as wafers. This embodiment has the advantage of minimal moving parts that may cause electrical interference or additional maintenance. Furthermore, the ability to fabricate and deliver multiple inserts or biochips of microfluidics and biosensing device duplexes on undiced wafers is enabled by a novel fabrication technique for the volume production of biochips that provides cost efficiencies in the volume production of semiconductors, shared circuitry to reduce the number of connectors and simplified packaging, which as one skilled in the art will recognize, provides significant cost reductions over the fabrication and packaging of individual biochips. This will be further described below.
Referring to
All or some of the layers of
With reference to
In the preferred embodiment, the middle layer defines one or more substrates containing a plurality of one-time use microfluidics assemblies at predetermined coordinates on the substrate. The middle layer makes use of a multitude of dies of one-time use microfluidics with microscale features and can be fabricated on multiple types of water-proof materials, preferably a thermal polymer or machinable polymer, which is low cost, easy to align and bond with the bottom layer, and readily mass produced, preferably using NanoImprint Lithography or photolithography, depending on the critical dimensions of the nano-electrodes, or micromachining or injection molding, and/or other processes.
The top layer defines one or more substrates containing a single reusable microfluidics assembly and distribution manifold with microscale features, and can be fabricated on multiple types of water-proof materials, preferably a thermal polymer or machinable polymer, which is low cost, easy to align and bond with the bottom layer, and readily mass produced using Lithography, micromachining, injection molding, and/or other processes.
The method then includes aligning the bottom and middle layers and then attaching 452 them to each other so that the one-time use biosensing devices and microfluidics assemblies are provided as duplexed biochips. The top layer, containing the reusable portion of the microfluidics assembly, is then also aligned and attached.
The method preferably next includes providing for connections 454 associated with electrical connections for each biosensing device, fluid input and output, compressed gas and control mechanism for the manifold, as well as packaging 456 the resulting cartridge in view of its intended use.
In accordance with another aspect of the invention, a sanitizing method is provided that allows for a fully automated or semi automated process to sanitize the reusable modules of a condensation device according to at least some of the embodiments above, between uses.
Referring to
In one embodiment, the sanitizing solution comprises filtered water that has been filtered by the primary and secondary filtration systems, and the sanitizing agent is 30 mL of 35% hydrogen peroxide per 10 L of filtered water. However, other filtered liquids, sanitizing agents and concentrations can be used depending on the input media, target biomolecules and other materials in the liquid solution. The sanitizing method's pumps, pipes, valves and other components can be configured to support the sanitizing method automatically with Programmable Logic Controllers (PLCs) or manually by an operator to direct and control the flow of the sanitizing liquid and sanitizing agent. A dispenser releases the sanitizing agent into the filtered solution as the solution is being pumped from its reservoir.
Once the condensing device is filled with the sanitizing solution, a predetermined contact time is preferably measured to permit the sanitizing solution to adequately disinfect the reusable components before the next biodetection test. In the above embodiment, the minimum contact time is 60 minutes. After the minimum contact time is reached, the pumping system 442 is used to pump the sanitizing solution out of the modules and empty the systems. The discharge can be sent to a drainage discharge or to a carboy.
The system is then prepared for the next detection test, as per appropriate maintenance procedures. In one embodiment, the sanitizing solution can be left in the ultrafiltration filters for a storage period, as such filters can degrade when in contact with air. Alternatively, the filters can be filled with a different liquid soaking solution and flowmeters can be reset to zero.
It should be noted that the condensing device according to embodiments of the present invention may advantageously, although not limitatively, be used with the abovementioned biosensing device in a biohazard early warning system for the fully-automated or semi-automated sampling, condensing, and detection of pathogenic bacteria, viruses and protozoa, and other target biomolecules in water, food, air, surfaces, insects, animals, and humans in a sensor network or portable device. Without the need for time-intensive sampling, incubation and amplification techniques, less time is needed to identify a potential pathogen outbreak and provide the appropriate response to stop the transmission.
However, many other configurations and related applications can also be devised without departing from the scope of the invention. For example, the condensation device can replace PCR or other incubation and amplification techniques in screening specific genes for unknown mutations and in genotyping using known sequence-tagged site (STS) markers for medical testing, drug discovery and other biological applications. As well, the condensing device can be used for chemical sensors and other sensing applications in addition to biosensing devices
The condensing device can be used with all of its modules as described in the embodiments and figures, or alternatively can be effective in certain applications when some of the modules are streamlined or removed. For example, when detecting target biomolecules known to be in very high concentrations in a solution, such as in sewage waste water or recreational beach water, then a much smaller sample may be sufficient for detection accuracy. In these cases the filtration module and/or the magnetic bead separation module may not be needed, and can be omitted from the configuration to reduce the processing time. Furthermore, it is anticipated that the various new sensing technologies and enhancements will improve sensing sensitivity and specificity, making condensing less required.
In other cases, the biochip may be used in a rapid screening mode, which tolerates a much greater range of false positive and false negative results than the diagnostic mode described in the embodiments above, in order to have preliminary test results in minutes rather than hours. When the biochips are used in a screening mode in a handheld device or wearable sensor or in front of the diagnostic device with a biomolecule condensing device, then the condensing device modules may be further streamlined or omitted.
Finally, other types of modules can be employed in combination with the condensing device to collect and liquefy solids in air, or from insects, food, tissues, feces, and other solid materials.
Of course, numerous other modifications could be made to the embodiments above without departing from the scope of the present invention.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/CA2009/001709 | 11/24/2009 | WO | 00 | 5/17/2011 |
| Number | Date | Country | |
|---|---|---|---|
| 61117337 | Nov 2008 | US |
