Patent Grant
7083920
The present invention relates to methods and systems for the detection of specific sequences of nucleic acids or oligonucleotides, including deoxyribonucleic acids (DNA) and ribonucleic acids (RNA). It relates more particularly to a conjugated enzyme based assay system utilizing reflective and/or transmissive optical discs for detection of specific sequences of nucleic acids.
Assay systems utilizing optical discs have been described. See, for example, Virtanen, U.S. Pat. No. 6,030,581 entitled “Laboratory in a Disk”. Such systems have enormous potential in the field of medicine, for diagnostic and other clinical assays, as well as in fields such as environmental testing and the like. Nonetheless, there remains a continuing need to develop assays that are faster, more efficient, and more economical.
Commonly assigned U.S. application Ser. No. 10/035,836 discloses a bead based DNA assay developed for the optical disc platform. Although such assays are qualitatively reproducible, quantitation of bead binding through different methods showed relatively high variations.
Enzyme assays have been widely used in a microtiter plate format. Commonly assigned U.S. Provisional Application Ser. No. 60/353,017, entitled “Data Capture and Signal Processing for Colorimetric and Fluorescent Assays as Implemented on Reflective Optical Analysis Discs”, filed on Jan. 29, 2002, discloses an enzyme assay implemented in an optical bio-disc system. In this assay, the reactants are not immobilized on the disc surface, and the reporter is not localized. This format is very useful for the detection of small molecules, but is not easily adapted to macromolecules, such as antibodies and DNA.
Assays that detect the presence of specific sequences of nucleic acids have a number of applications. For example, nucleic acid detection systems are used to test for the presence of specific disease causing agents, such as viruses or bacteria, in biological samples taken from patients. Nucleic acid detection systems are also used to test water and soil samples for specific microorganisms. Indeed, nucleic acid testing can be used to identify particular strains or types of a microorganism, which may have important implications for the appropriate response or treatment. Nucleic acid testing is also helpful in monitoring agricultural products as, for example, in testing for the presence of genetically modified crop products. As is well known, nucleic acid testing has important forensic applications as well.
What is needed, therefore, is a rapid, efficient, and economical assay system for testing various samples for specific nucleic acid sequences that also provides reproducible quantitation of the target nucleic acid.
This invention relates to identification of at least one target DNA or RNA that may exist in a sample and test methods relating thereto. The invention is further directed to an optical bio-disc used to test a sample of DNA or RNA for a target DNA or RNA of a prescribed sequence. The bio-disc includes a flow channel having target or capture zones, a return channel in fluid communication therewith, and in some embodiments a mixing chamber in fluid communication with the flow channel. The bio-disc may be implemented on an optical disc including an information encoding format such as CD, CD-R, DVD, DVD-R, any standard optical disc format, or a modified version thereof. Methods of manufacturing the optical bio-disc according to the present invention are also provided.
A bio-disc drive assembly is employed to rotate the disc, read and process any encoded information stored on the disc, and analyze the DNA samples in the flow channel of the bio-disc. The bio-disc drive is thus provided with a motor for rotating the bio-disc, a controller for controlling the rate of rotation of the disc, a processor for processing return signals form the disc, and an analyzer for analyzing the processed signals. The rotation rate is variable and may be closely controlled both as to speed, direction, and time of rotation.
The bio-disc drive assembly may also be utilized to write information to the bio-disc either before, during, or after the test material in the flow channel and target zones is interrogated by a read beam of the drive and analyzed by the analyzer. The bio-disc may include encoded information for controlling the rotation rate of the disc, providing data acquisition and processing information specific to the type of DNA or RNA test to be conducted, for displaying the results on a monitor associated with the bio-drive, and/or saving the results on a hard drive, floppy disc, on the bio-disc itself, or on any other recordable media. A reflective disc format suitable for use in the present invention is disclosed in commonly assigned U.S. Provisional Application No. 60/249,391 entitled “Optical Disc Assembly for Performing Microscopy and Spectroscopy Using Optical Disc Drive,” hereby incorporated by reference in its entirety.
In an alternative embodiment, a transmissive disc format may be used in which the interrogation beam is transmitted through the target zone and detected by a top detector. Such a transmissive disc format is disclosed in commonly assigned U.S. Pat. No. 6,327,013 and in commonly assigned U.S. Provisional Applications Nos. 60/293,917; 60/303,437; and 60/323,405, entitled “Optical Discs and Assemblies for Detection of Microscopic Structures Using Focal Zone Control,” hereby incorporated by reference in their entireties.
Development of a DNA based assay for CD, CD-R, DVD, DVD-R, or any standard optical disc format and variations thereof according to the present invention, includes attachment of conjugated enzymes to the disc surface as a detection method. These enzymes are selected so as to yield, in the presence of a suitable substrate, reaction products that an interrogation beam of the drive can “see” or detect by a change in surface reflectivity or transmittance caused by the reaction products.
The enzymes are bound to the disc surface through binding agents including, for example, Streptavidin and biotin. A capture probe is attached to the disc in a capture zone, while a biotinylated target is allowed to hybridize with the capture probe. Once the target is hybridized with its respective capture probe, a Streptavidin conjugated enzyme introduced into the capture zone and allowed to bind to the biotin on the target. In this manner, the enzyme is attached to a disc surface. In a subsequent centrifugation (or wash) step, all unbound enzyme is removed. Substrate appropriate for the bound enzyme is added, and the enzymatic reaction is allowed to take place. The enzyme reaction products deposit on the disc surface at or near the bound enzyme, where they can be detected and quantitatively measured to provide both a qualitative and quantitative measurement of the analyte of interest.
In an alternative embodiment of the present invention, the enzymes are bound to the disc surface through DNA hybridization. A capture probe is attached to the disc, while the Streptavidin conjugated enzyme is attached to a biotinylated signal probe. Each of these probes is complementary to a different portion of the target sequence, but are not complementary to each other. In the presence of a target sequence, both capture and signal probes hybridize with the target. In this manner, the enzyme is attached to a disc surface within the capture zone. In a subsequent centrifugation (or wash) step, all unbound enzyme is removed. Substrate appropriate for the bound enzyme is added, and the enzymatic reaction is allowed to take place. The enzyme reaction products deposit on the disc surface at or near the bound enzyme, where they can be detected and quantitatively measured by a beam of electromagnetic radiation to provide both a qualitative and quantitative measurement of the analyte of interest.
The DNA capture probe can be bound to an active layer, bio-layer, or binding layer by passive adhesion or adsorption, electrostatic interaction (using a positively charged active layer), or covalent binding, achieved by using an activated active layer and a modified DNA wherein the modified DNA can covalently bind onto the active layer. For example, an aminated DNA can covalently bind onto a polystyrene co-maleic-anhydride active layer. This active layer may be formed from a variety of media including nitrocellulose, polystyrene, polycarbonate, gold, activated glass, modified glass, or modified media. The modified media includes anhydride groups, activated carboxylate groups, or carboxylic acid aldehyde groups.
After DNA hybridization, a neutravidin- or streptavidin-conjugated enzyme as, for example, alkaline phosphatase or horseradish peroxidase, is bound to the biotinlylated DNA as described above. Then a solution of enzyme substrate is added and reacts with the enzyme to form a reaction product in the form of insoluble pellets or precipitate, fluorescent, and/or colored product. The pellets stay localized on the reaction spots where the DNA probe was applied, even after centrifugation of the disc.
The specific enzymatic reaction products can be detected using different methods. These methods include microscopic analysis, measurement of fluorescence signal on the disc surface using a FluorImager (Molecular Dynamics), or detection of insoluble reaction product using an optical disc reader. For example, an event counting software useful for reaction product detection in a optical disc reader is disclosed in commonly assigned U.S. Provisional Application No. 60/291,233 entitled “Variable Sampling Control for Rendering Pixelization of Analysis Results in Optical Bio-disc Assembly and Apparatus Relating Thereto,” hereby incorporated by reference in its entirety. As discussed below in conjunction with FIG. 37 and in Example 5, the signal from an enzymatic reaction product, in one embodiment of the present invention, is concentration dependent using the event counting software to quantitate the data, thus making this a quantitative detection method. Moreover, a fluorescent enzymatic product may be detected by a fluorescent type disc reader while a colored product (chromagen) can be detected using a transmissive or reflective disc set-up described below. Alternatively, the signal detection and quantitation may be carried out using other methods of quantitation in conjunction the optical bio-disc reader with appropriate software. For example, the transmissive optical disc format may be used to quantify changes in light transmission or scattering as a result of generation of an enzymatic reaction product or reporter.
The DNA assay according to the present invention may be implemented in an open disc format as well as in a micro channel. In the open disc format, the reagents are spotted directly on the disc surface. Unbound reagents are removed by washing the disc. In the micro channel format, the capture probe binding is initially performed on an open disc substrate. After attaching the DNA capture probes, the channel is assembled by affixing adhesive and a cover disc or cap. Subsequent steps are performed in the closed channel which is filled with liquids such as buffer solutions, enzyme solutions, and DNA test samples which are analyzed for the presence of a target sequence.
Brief Overview of the Assay
In the assay according to the present invention, an enzyme reaction is used to detect the presence of an analyte (DNA or RNA) in a microchannel on an optical bio-disc. The analyte is immobilized on a capture layer on the surface, and the signal that is generated is localized and specific, as, for example, by the formation of an insoluble product of the enzyme reaction. The signal can be in the form of a pellet, a fluorescent product, and/or a colored product, and can be detected and quantified by an optical bio-disc reader utilized in conjunction with the inventions hereof. This assay is thus quantitative in nature. In addition, the formation of the pellet in one embodiment hereof is facilitated by a layer of nitrocellulose on the disc surface, which supports binding of the capture layer and formation and retention of the pellet.
Analytes
The present invention is directed to the detection and analysis of target nucleic acid sequences present in test samples. Target nucleic acids suitable for use with the present invention include both deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), including mRNA, rRNA, hnRNA and tRNA.
Target nucleic acid may be used directly from a biological sample, but preferably is amplified prior to testing via polymerase chain reaction (PCR) or isothermal amplification to generate amplicons. If using PCR for amplification, RNA may first be reverse transcribed into DNA using techniques well known in the art. Target nucleic acid may be single stranded or double stranded. If double stranded, the nucleic acid may be denatured prior to hybridization with capture DNA.
In one embodiment of the present invention, primers labeled with biotin are used in PCR reactions to yield biotin-labeled target DNA amplicons, which are then tested in the bio-disc assay as described below. Amplicons of various lengths are suitable for use in the present invention, with a preferred length from about 20 bases (or base pairs) to about 4000 bases (or base pairs), more preferably from about 200 to about 400 bases or base pairs.
The present invention may be used to detect specific nucleic acid sequences in a wide variety of biological samples, including but not limited to bodily fluids such as whole blood, serum, plasma, saliva, urine, lymph, spinal fluid, tears, mucous, semen and the like, agricultural products, food items, waste products, environmental samples, such as soil and water samples, or any other sample containing, or suspected of containing specific nucleic acid sequences of interest. For example, the present invention may be used to detect the presence of particular strains of microorganisms, such as viruses or bacteria, in body fluids or environmental samples, by detecting the presence of particular nucleic acid sequences in the sample. In another example, the present invention may be used to detect the presence of genetically modified agricultural products in food items. In yet another example, the present invention may be used to identify specific pulmonary infectious agents using a microarray format. Other uses of the present invention will be apparent to those of skill in the art.
Capture DNA
Capture DNA oligonucleotides, or probes, are immobilized onto a bio-disc and are hybridized to target DNA or RNA to thereby “capture” the target nucleic acid in the target zone for detection. Capture DNA may be single stranded or partially double stranded near the attachment point to the active layer on the bio-disc. One preferred embodiment of the capture DNA includes a partially double stranded DNA. The double strand us located at the reactive end of the probe, such as the amino end, because the double strand has been found to more effectively project the capture probe erectly or upwardly from the active layer as compared to ssDNA in some instances. An extension or spacer including, for example, ssDNA and PEG, may be employed so as to increase the hybridization efficiency of the capture probe with the target. The sequence of the capture DNA is selected so as to hybridize directly with target DNA or RNA, thereby forming a complex comprising capture DNA, target DNA or RNA and conjugated enzyme bound thereto as depicted below in
In an alternative embodiment described below, signal DNA is also be present in this complex. In this embodiment, the signal DNA sequence is complementary to a portion of the target DNA or RNA, but is not complementary to the capture DNA. As one portion of the target DNA hybridizes with capture DNA, while a different portion hybridizes with signal DNA, a complex forms in which the target DNA acts as a “bridge” between the capture DNA and signal DNA as described below in conjunction with
Signal DNA
In one embodiment of the present invention, target DNA hybridizes directly with capture DNA bound to the active layer of a bio-disc. In an alternative embodiment, target DNA is used as a “bridge” between signal DNA and the capture DNA. In the alternative embodiment, the sequence of the signal DNA is selected so as to contain a region that is complementary to the target DNA, but which contains no sequence complementarity with the capture DNA, such that the signal DNA will not form a complex with the capture DNA in the absence of target DNA. The target DNA contains a first region of complementary sequence to the capture DNA, permitting hybridization of the first region of the target DNA to the capture DNA, and a second region of complementary sequence to the signal DNA, permitting hybridization of the signal DNA to the second region of the target DNA, thereby linking or bridging the signal DNA to the capture DNA.
The target DNA may be of any length suitable to effectively immobilize itself and the signal DNA to the capture DNA. Typically, signal DNA and capture DNA are from about 10 bases to about 100 bases in length, preferably from about 20 bases to about 60 bases in length. Typically, the target DNA amplicons have an overlap of from about 20 bases to about 40 bases with the signal DNA and an overlap of from about 20 bases to about 40 bases with the capture DNA. Preferably, the target DNA amplicon has a GC (guanine and cytosine) content greater than 50%, within the areas of overlap between the capture probe, target, and signal probe. Although one skilled in the art will appreciate that GC content and length of the target DNA amplicon may be modulated to effectuate stable hybridization to the signal and capture DNA.
In one embodiment of the present invention, signal DNA or the target DNA is labeled with an affinity agent, such as biotin, to permit binding to conjugated enzymes via biotin/streptavidin interactions with streptavidin-conjugated enzymes.
Capture Layer Preparation
Capture DNA probes are bound to the surface of the disc through non-covalent adsorption to a layer of nitrocellulose, which is spin-coated on the disc. The layer of nitrocellulose can be applied to different types and surfaces of discs. After attaching the capture probes, microchannels may be assembled and prepared for sample application as shown and described below in conjunction with
Blocking Non-specific DNA, RNA and Protein Adsorption
After the disc is assembled, the channels are blocked with a DNA/protein blocking solution to prevent non-specific binding of target nucleic acid, signal probes and/or enzymes on the target zone. The blocking solution may be a buffer containing, for example, bovine serum albumin (BSA), salmon sperm DNA, and/or Denhardt's solution.
Sample Application
When a sample is injected into the microchannel, any target RNA or DNA present in the sample binds to the capture probe through hybridization. In one embodiment, target DNA is generated in an amplification reaction using biotinylated primers, resulting in biotinylation of the target DNA. Following hybridization, unbound amplicon DNA is removed with a wash step.
In another embodiment of the assay, the target RNA or DNA is not directly biotinylated. Rather, a biotinylated signal DNA probe is used. In this embodiment, target DNA is amplified by PCR or isothermal amplification using non-biotinylated primers (target RNA is similarly generated by isothermal amplification). The target DNA is then hybridized to a biotinylated signal DNA probe.
Signal Generation
In one embodiment, a solution of streptavidin- or neutravidin-conjugated enzyme, such as horseradish peroxidase, is injected into the microchannel, where the enzyme binds to the biotinylated amplicon or signal probe via the streptavidin- or neutravidin-biotin interactions. Excess enzyme is removed through a wash step and the microchannel is filled with a solution of an enzyme substrate, such as TMB (3,3,5,5 tetramethylbenzidine in stable peroxide buffer), that is converted to an insoluble product, becomes luminescent or fluorescent, changes color through the enzyme reaction, or otherwise generates a detectable signal. In one embodiment, the enzyme reaction product is an insoluble precipitate that adheres to the active layer, forming a detectable precipitate as described below in conjunction with FIGS. 21 and 22A-22D.
In another embodiment, the conjugated enzyme is first dried onto a pad or membrane, which is deposited into a side chamber in fluid communication with the microchannel as illustrated in
Enzymes and Substrates
Enzymes useful in the practice of the present invention include any enzyme that may be adapted to interact with a specific nucleic acid probe, as, for example, through the interaction of an enzyme conjugated with streptavidin- or neutravidin- and with a biotin labeled DNA. The enzyme produces a detectable signal in the presence of a suitable substrate. For example, conjugated horseradish peroxidase (HRP; Pierce, Rockford, Ill.) may be used with the substrate 3,3,5,5-tetramethylbenzidine (TMB; Calbiochem cat. no. 613548, CAS-54827-17-7) in the presence of hydrogen peroxide to produce an insoluble precipitate. Horseradish peroxidase can also be used in conjunction with CN/DAB (4-chloronaphthol/3,3′-diaminobenzidine, tetrahydrochloride), 4-CN (4-chloro-1-napthol), AEC (3-amino-9-ethyl carbazol) and DAB (3,3-diaminobenzidine tetrahydrochloride) to form insoluble precipitates. Similarly, the enzyme alkaline phosphatase (AP) can be used with the substrate bromochloroindolylphosphate in the practice of the present invention. Other suitable enzyme/substrate combinations will be apparent to those of skill in the art.
Detection
The signal from the enzyme reaction can be read with an optical bio-disc reader. Either a bottom detector on a disc with a reflective cover, or a top detector with a transmissive disc can be employed with the optical bio-disc reader for the assay and disc inventions disclosed herein and described in detail below in connection with
Disc Implementation
The assays and methods of the present invention may be advantageously implemented on an analysis disc, modified optical disc, or bio-disc. The bio-disc may include a flow channel having target or capture zone, a return channel in fluid communication therewith, and in some embodiments a mixing chamber and/or a side chamber in fluid communication with the flow channel.
The bio-disc may be implemented on an optical disc including an standard information encoding format such as CD, CD-R, DVD, DVD-R or a modified version thereof. The bio-disc may include encoded information for performing, controlling, and post-processing the test or assay. For example, such encoded information may be directed to controlling the rotation rate of the disc. Depending on the test, assay, or investigational protocol, the rotation rate may be variable with intervening or consecutive sessions of acceleration, constant speed, and deceleration. These sessions may be closely controlled both as to speed and time of rotation to provide, for example, mixing, agitation, or separation of fluids and suspensions with agents, reagents, DNA, RNA or antibodies.
Drive Implementation
A bio-disc drive assembly or reader may be employed to rotate the disc, read and process any encoded information stored on the disc, and analyze the samples in the flow channel of the bio-disc. The bio-disc drive is thus provided with a motor for rotating the bio-disc, a controller for controlling the rate of rotation of the disc, a processor for processing return signals form the disc, and an analyzer for analyzing the processed signals. The drive or disc may include software specifically developed for performing the assays disclosed herein.
The rotation rate of the motor is controlled to achieve the desired rotation of the disc. The bio-disc drive assembly may also be utilized to write information to the bio-disc either before, during, or after the test material in the flow channel and target zone is interrogated by the read beam of the drive and analyzed by the analyzer. The bio-disc may include encoded information for controlling the rotation rate of the disc, providing data acquisition, and processing, reporting and recording information specific to the type of genetic test to be conducted, and for displaying the results on a display monitor associated with the bio-drive in accordance with the assay methods relating hereto.
Further objects of the present invention together with additional features contributing thereto and advantages accruing therefrom will be apparent from the following description of preferred embodiments of the present invention which are shown in the accompanying drawing figures with like reference numerals indicating like components throughout.
Reflective Disc
The cap portion 116 includes an inlet port 122, a vent port 124, and, optionally, an enzyme buffer port 141. The cap portion 116 may be formed from polycarbonate and is preferably coated with a thin reflective surface 146 on the bottom thereof as viewed from the perspective of FIG. 2A. The reflective surface 146 is best illustrated in FIG. 2C.
In a preferred embodiment, trigger markings 126 are included on the reflective surface. Trigger markings 126 may include a clear window in all three layers of the bio-disc, an opaque area, or a reflective or semi-reflective area encoded with information that sends data to a processor 166, as shown in
The second element shown in
Some of the fluidic circuits 128 illustrated in
The third element illustrated in
Referring now to
Transmissive Disc
The second element shown in
The third element illustrated in
Referring next to
With reference now to
Referring next to
As illustrated in this embodiment, the plastic adhesive member 118 is applied over the active layer 144. The exposed section of the plastic adhesive member 118 illustrates the cut out or stamped U-shaped form that creates the fluidic circuits 128. The final principle structural layer in this embodiment of the present bio-disc 110 is the clear, non-reflective cap portion 116 that includes the inlet 122, vent port 124, and, optionally, the buffer port (not shown) as described above in FIG. 3C.
Referring now to
With reference now to
In the transmissive bio-disc 111, on the other hand, the transmitted beam 156 is detected by a top detector 158 and is analyzed for the presence of signal agents. In this embodiment, a photo detector may be used as a top detector 158.
Referring now to
Reference in now made to
With reference now to
Referring to
The substrate 120 in this embodiment includes a series of grooves 170. The grooves 170 are in the form of a spiral extending from near the center of the disc toward the outer edge and are implemented so that the interrogation beam 152 may track along the spiral.
Open Disc Formats
Referring to
In this embodiment, a capture DNA 172 may be depended downwardly when the disc is loaded in the drive (FIG. 1). In this open-face format, the other assay reactants are brought into proximity with the capture DNA 172 by a variety of different methods which include, for example, depositing a test sample on the disc with a pipette. In this embodiment, the target zones 140 are simply formed by the application of a small volume of capture DNA 172 solution to the active layer 144 to form clusters of capture DNA 172 in desired locations on the active layer 144 as illustrated.
Referring next to
In this transmissive (or semi-reflective) open-face format, the assay reactants are brought into proximity with the capture DNA 172 by a variety of different methods which include, for example, depositing a test sample on the disc with a pipette. In this alternative embodiment, the target zones 140 are simply formed by the application of a small volume of capture DNA 172 solution to the active layer 144 to form clusters of capture DNA 172 in desired locations on the active layer 144 as illustrated. Detection of the beam carrying information about the analyte for this embodiment is achieved by use of a top detector 158.
Attaching Capture DNA, Target DNA, and Conjugated Enzyme
Referring to
Referring next to
Referring now to
Using a Signal DNA to Attach Conjugated Enzyme to the Target Zone
With reference to
The Enzyme-Substrate Reaction and the Formation of an Insoluble Precipitate
Referring to
Referring now to
Referring next to
Next,
Target Detection Methods
Referring next to
With reference now to
Referring now to
Although the disc composition between the different disc formats may vary, the biochemical interactions remain the same. In
After hybridization, the flow channel 130 may be washed to clear the target zone 140 of any unattached DNA in the sample. After removing the unattached DNA in the sample, enzymes 178 with binding agent 180 are introduced in the channel, as shown in FIG. 33C. In one embodiment, enzymes are introduced via the input port 122, as shown in FIG. 33C. In an alternative embodiment, enzyme buffer is introduced through an enzyme buffer port into a side chamber (not shown), which is in fluid communication with the flow channel and which contains a pad or membrane onto which enzyme has been dried as described above in conjunction with FIG. 4. The buffer solubilizes the enzyme, which then flows into the flow channel 130.
As the flow channel 130 is filled with enzyme solution, the enzymes 178 begin to flow or move down the flow channel 130 as illustrated in
After enzymes 178 bind to the affinity agent 176, the flow channel 130 may be washed to clear the target zone 140 of any unattached enzyme 178. As shown in
The interrogation beam 152 may then be scanned through the target zone 140 to determine the presence of signal agents as illustrated in
Referring next to
In
After hybridization, the disc may be washed to clear the target zone 140 of any unattached DNA or RNA sample. Upon removing the unattached DNA or RNA in the sample, signal DNA probes 184 containing an affinity agent 176 are introduced into the flow channel 130, as shown in FIG. 34B. As the flow channel 130 is further filled with signal DNA 184, the signal DNA begins to flow or move down the flow channel 130 as illustrated in
After hybridization, the flow channel 130 may be washed to clear the target zone 140 of any unattached signal DNA 184 probes. As shown in
As the flow channel 130 is filled with enzyme solution, the enzymes 178 begin to flow or move down the flow channel 130 as illustrated in FIG. 34D. When the enzyme 178 comes into close proximity with the hybridized signal DNA/target DNA/capture DNA complex in the target zone 140, the enzymes 178 bind to the signal DNA 184 via the interaction between the affinity agent 176 and binding agent 180, best shown in FIG. 20 and as further illustrated in FIG. 34E.
After enzyme binding, the flow channel 130 may be washed to clear the target zone 140 of any unattached enzyme 178. After removing unbound enzymes 178, the enzyme substrate 186 is introduced in the channel, as illustrated in FIG. 34F. As the flow channel 130 is filled with enzyme substrate 186, the enzyme substrate 186 begin to flow or move down the flow channel 130. When the enzyme substrate 186 comes in contact with the enzyme 178 bound to the target zone 140 via the signal DNA/target DNA/capture DNA complex, the enzyme substrate reaction 194 occurs, which results in the production of a signal agent, as shown in FIG. 34G. The signal agent may be a color production, luminophore production, fluorescence, or the formation of insoluble precipitate 190 as illustrated in FIG. 34H and
The interrogation beam 152 is then scanned through the target zone 140 to determine the presence of signal agents, as illustrated in
With reference now to
In
As the flow channel 130 is further filled with test sample solution, the target RNA or DNA 174, the signal DNA 184 probe (if present), and the enzymes 178 begin to flow or move down the flow channel 130 as illustrated in FIG. 35B.
When target RNA or DNA 174 of a specific sequence is present in the test sample, the target RNA or DNA 174 hybridizes with the capture DNA 172 as shown in FIG. 35C. In this manner, the enzymes 178, attached to the target DNA 174 as described in
After hybridization, the flow channel 130 may be washed to clear the target zone 140 of any unattached target RNA or DNA, after which enzyme substrate 186 is introduced into the channel, as shown in FIG. 35D. As the flow channel 130 is filled with enzyme substrate 186, the enzyme substrate 186 begin to flow or move down the flow channel 130. When the substrate comes in contact with the enzyme 178, the enzyme substrate reaction 194 occurs, producing a signal agent, as shown in FIG. 35E. The signal agent may be color production or luminophore production. The signal agent may also be the formation of a precipitate 190, as illustrated in FIG. 35F and above in
The interrogation beam 152 is scanned through the target zone 140 to determine the presence of signal agents, as illustrated in
Data Generated from the Assay
Referring to
The test results of any of the test methods described above may be readily displayed on the display monitor 114 shown in FIG. 1. The optical bio-disc 110 or 111 according to the present invention may include encoded software that interacts with the drive, the controller 164, the processor 166, and the analyzer 168 as shown in
Applications of the Present Invention
As will be appreciated, the enzyme-based assay for detecting and analyzing nucleic acids in test samples has a number of potential applications. For example, this assay can be used to test for the presence of specific disease causing agents, such as viruses or bacteria, in biological samples taken from patients. Capture DNA probes specific for various disease causing organisms can be deposited in specified target zones on a bio-disc in an ordered array (or microarray). Multiplexed PCR reactions, using mixed primer sets directed to these various disease causing organisms, can be conducted on patient samples, and the resulting amplicons used as target DNA probes in the bio-disc assay system. Enzyme reactions can be monitored as described above to determine which target zones in the ordered array produce a detectable signal. These target zones can then be correlated to the specific capture DNA probe deposited at that location, allowing clinicians to quickly identify which target DNA molecules are amplified from the patient sample and, accordingly, which disease causing organism is likely present in the patient.
Similarly, the present invention can be used to test water and soil samples for specific microorganisms and is also helpful in monitoring agricultural products as, for example, in testing for the presence of genetically modified organisms (GMO). For example, crop samples can be amplified using primer sets specific for a marker gene in a genetically modified plant and for intrinsic plant genes, such as lectin or zein. The amplified target DNA can then be used on bio-discs containing capture probes for both the marker gene and for intrinsic plant genes. Calibration controls can also be run simultaneously, in which known concentrations of the GMO are amplified for use as target DNA. By comparing the reaction products of the unknown sample with the calibration curve, the presence of genetically modified organisms in a sample can be detected and quantified.
An overview of how the present invention can be used in detecting and quantifying GMOs is shown in
An optical bio-disc containing at least five flow channels is prepared with five target zones in each flow channel (as will be appreciated, these numbers may be varied depending on the particular assay): two for the marker gene capture probe, two for the lectin capture probe (as positive controls), and one with a non-specific probe, as a negative control. Each of these flow channels is depicted in FIG. 38. Five separate PCR reactions are performed to produce target DNA using primer sets directed to both the marker gene and the lectin gene. Four of the reactions are performed using template DNA from calibration standards containing known quantities of GMO (e.g., 0%, 0.1%, 0.5% or 1% GMO); the fifth reaction is performed using the unknown sample. Each set of DNA sample is hybridized to one of the flow channels and the enzyme reaction is performed as described herein. The detectable signal, as, for example, measured by the amount of an insoluble precipitate or pellet formation, for the unknown sample is compared to those of the calibration curve, allowing the presence (and amount) of GMO in the unknown sample to be determined. If the signal in the unknown sample is too high to permit quantification, it may be necessary to repeat the procedure with dilutions of the unknown sample.
Optical Bio-discs with Egui-radial Fluidic Circuits
The optical bio-disc systems and methods for detecting specific sequences of oligonucleotides, as described above, may be readily implemented in an optical bio-disc with an equi-radial fluidic circuit. Details regarding optical bio-dics with equi-radial fluidic circuits is disclosed in, for example, commonly assigned co-pending U.S. Provisional Patent Application Ser. No. 60/353,014 entitled “Optical Discs Including Equi-Radial and/or Spiral Analysis Zones and Related Disc Drive Systems and Methods” filed Jan. 29, 2002, which is incorporated herein by reference in its entirety.
Experimental Examples
Having generally described the invention, the same will be more readily understood through reference to the following examples, which are provided by way of illustration, and are not intended to be limiting of the present invention.
Fresh polystyrene solution was prepared by adding 3 g polystyrene pellets (Sigma cat. no. 182427; molecular weight=280,000) to 310 ml toluene and stirring for 1 hour using a teflon stir bar and a stir plate. After the polystyrene was completely dissolved in the toluene, 68 ml reagent grade isopropanol was slowly added while stirring.
A stock solution of nitrocellulose was prepared by diluting a nitrocellulose collodium solution (4-8% in ethanol/diethylether, Fluka cat. no. 09986, lot no. 389973/1 30299) 1:5 in reagent grade ethanol. Prior to spin coating, the stock solution was diluted 1:10 with reagent grade ethanol and filtered using a 0.2 μm syringe filter.
A polycarbonate disc having a 200 Angstrom gold semi-reflective layer (BTI Optical Bio-disk Set FDL21:E001308) was placed on a “spin coater,” or modified centrifuge, with the reflective surface up. While rotating the disc on the spin coater, the reflective surface was cleaned with reagent grade alcohol.
The spin coater was set to start spinning at 2500 rpm, followed by acceleration to 4000 rpm within 10 seconds. During this 10-sec acceleration, a steady stream of polystyrene solution was applied to the disc using a pasteur pipette, with the polystyrene solution applied from the outer edge to the inner side in one smooth stroke.
The spin coater speed was then adjusted to 1500 rpm, and the diluted and filtered nitrocellulose solution was applied onto the inner portion of the disc in a steady stream using a pasteur pipette.
The disc from Example 1 was placed on a CD assembler/spindle with the nitrocellulose layer up. Between about 0.5 to 2.0 μl of 1 μM oligo probes (capture DNA) in 1 M NH4OAc were applied to the disk at defined target zones. The droplets of capture DNA were dried onto the nitrocellulose at 37° C.
A cover disc containing U-shaped fluidic circuits (50 μM adhesive; Fraylock, DBL243a) was applied using a disk assembler spindle, and the disc was run through a wringer to seal the two disks.
DNA blocking solution (1% bovine serum albumin [BSA], 5× Denhardt's solution, 0.1 mg/ml salmon sperm DNA, 200 mM KCl, 10 mM MgCl2, 50 mM Tris, pH 7.4) was degassed in a vacuum desiccator and injected into the fluidic circuits of a bio-disc prepared as in Example 2, taking care that no air bubbles remained in the circuits. The bio-disc was then incubated at room temperature for 30 to 60 minutes.
The DNA blocking solution was removed, and the fluidic circuits washed with hybridization buffer (200 mM NaCl, 10 mM MgCl2, 50 mM Tris, pH 7.4) injected into the fluidic circuits using a syringe. PCR amplicons (target DNA amplified using biotinylated primer sets, purified using the Qiagen QIAquick PCR Purification Kit, cat. no. 28104, lot no. 10927932, and eluted using hybridization buffer) were denatured at 95° C. for 5 minutes and immediately placed on ice for 5 minutes.
The denatured amplicons were added to the appropriate fluidic circuits (10 μl per fluidic circuit) and allowed to hybridize for 1.5 to 2 hours at room temperature. Following hybridization, the fluidic circuits were washed with hybridization buffer using a syringe.
Neutravidin-Horseradish Peroxidase Conjugated enzyme (N-HRP; Pierce product no. 31001, lot no. BK46404) was diluted 1:5000 in hybridization buffer, and 12 μl was applied to each fluidic circuit. The disc was then incubated at room temperature for 15 minutes.
The fluidic circuits were then washed with hybridization buffer using a syringe, and 12 μl of TMB Substrate in Stable hydrogen peroxide buffer (Calbiochem cat. no. 613548, lot B34202) was added to each fluidic circuit. The enzyme reaction was allowed to proceed for 5 mintues, after which the reaction was stopped by flushing the fluidic circuits with distilled water using a syringe.
Each fluidic circuit was sealed with tape, and the bio-disc was then placed in a disc-reader, similar to that shown in
A bio-disc with 6 target zones was prepared as in Example 2, with 1.6 μl of 10 μM DNA oligonucleotides specific to one of the Brucella strains applied to three of the target zones, as indicated in Table 2, below. One target zone contained a mix of all three Brucella species, one target zone contained biotinylated DNA (positive control), and one target zone contained no capture DNA (background).
Brucella sp. genomic DNA was subjected to PCR amplification using forward/reverse primer sets directed to B. melitensis to generate target B. melitensis amplicons. Each reaction contained 1 ng/μl Brucella DNA, 0.2 μM biotinylated forward and reverse primers, 0.2 mM dNTPs, 0.05 U/μl Taq polymerase, 3.0 mM MgCl2 and 1× PCR buffer (Qiagen; 15 mM MgCl2). The thermocycle conditions were:
The enzyme DNA assay was performed as described in Example 3, both without the addition of the target B. melitensis amplicons (results indicated in Table 2) and with the addition of purified target B. melitensis amplicons (results indicated in Table 3). Values from Tables 2 and 3 are event counts collected using an optical disc reader using the reflective disc format. The counting area was a rectangle, 940 μm×2300 μm; event count amplitude was between 225-500.
B. abortus
B. melitensis
B. suis
B. abortus
B. melitensis
B. suis
As shown above, the B. melitensis target zone produces a much higher signal than the other Brucella species when the B. melitensis amplicon is used in the present invention.
Bio-discs were prepared as in Example 2, with NosT 52-mer oligonucleotide capture probes applied to the target zones. NosT is a marker gene for genetically modified plant material.
GMO reference materials, consisting of soya bean powder containing different mass fractions of powder from genetically modified soya beans, were obtained from Fluka BioChemika, certified reference material IRMM-410R (0, 0.1, 0.5, 1, 2, 5% Roundup Ready Soya). DNA was extracted from each of the six reference materials, using the WIZARD® Magnetic DNA Purification System for Food (Promega, Madison, Wis.).
The extracted DNA was subjected to PCR amplification using biotinylated forward/reverse primer sets directed to NosT to generate biotinylated 280-mer target NosT amplicons. Each reaction contained 50 ng extracted DNA, 0.2 μM biotinylated forward and reverse primers, 0.2 mM dNTPs, 0.05 U/μl Taq polymerase, 3.0 mM MgCl2 and 1× PCR buffer.
The enzyme assay was performed as in Example 3, using the NosT amplicons as target probes. The results are shown in Table 4.
As shown in Table 4, the detectable signal (event counts) increased as the percentage of GMO material increased. Between 0% and 1% GMO, there was a 90% correlation between the amount of GMO material and the resulting signal.
Concluding Summary
All patents, patent applications, and other publications mentioned in this specification are incorporated herein in their entireties by reference.
While this invention has been described in detail with reference to a certain preferred embodiments, it should be appreciated that the present invention is not limited to those precise embodiments. Rather, in view of the present disclosure that describes the current best mode for practicing the invention, many modifications and variations would present themselves to those of skill in the art without departing from the scope and spirit of this invention. For example, the optical bio-disc molecular diagnostic-enzyme based detection methods and systems, of the present invention, may be readily modified and implemented to test for proteins, cells, cell surface markers, or any antigen in an immunoassay format. In this immunoassay implementation of the present invention, the capture probe may be an antibody having specific affinity to an analyte. The analyte may be an antigen including proteins, glycoproteins, cells, cell surface markers, or any antigen. The enzyme would then be conjugated to a second (signal) antibody, having an affinity to a different epitope on the same antigen and no affinity for the capture probe, and the enzyme substrate reaction and signal detection carried out as described above. Alternatively, labeled or tagged signal antibodies may be used as the reporter instead of the precipitate or pellet formation of the enzyme-substrate system described above. Labels may include, for example, fluorescent molecules and microparticles. The scope of the invention is, therefore, indicated by the following claims rather than by the foregoing description. All changes, modifications, and variations coming within the meaning and range of equivalency of the claims are to be considered within their scope.
This application claims the benefit of U.S. Provisional Applications, Ser. Nos. 60/292,110, filed May 18, 2001, and 60/313,917, filed Aug. 21, 2001, the contents of which are hereby incorporated by reference in their entireties.
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