Patent Application
20140295424
Chemical amplification of nucleic acids can be realized with the polymerase chain reaction (PCR), in which a DNA molecule (a template) can be duplicated via repeated thermal denaturation and enzymatic replication. Bead-based PCR is a variant of the PCR procedure that uses primers (short DNA fragments complementary to a specific region of the template) attached to microbeads. This procedure can result in bead-tethered template DNA duplicates. Therefore, it can serve as an analytical tool to simultaneously accumulate signals from DNA-based transducers and allow manipulation of DNA itself via solid-phase extraction (SPE) techniques.
Bead-based PCR has been used in applications including DNA sequencing, protein screening, and pathogenic DNA detection. For example, whole genome sequencing has been performed using bead-based PCR to facilitate the organization and detection of amplified sections of a fragmented E. coli genome. Compartmentalization of DNA in emulsions combined with bead-based PCR can allow for rapid screening of an entire genome for DNA binding proteins and cell-free protein synthesis.
Microfluidics technology can provide a rapid and efficient reaction platform due to efficient heat transfer properties. Microfluidics can also enable integrated chip-based systems that perform tasks such as sample pretreatment and post-amplification analysis, thereby improving reaction speed and test accuracy by shifting more operations to the microscale domain.
In bioanalytical assays, analytes of interest can be present in minute quantities and contaminated with impurities. Thus, sample preparation steps prior to analysis can be important for improving the resolution of detection results. In particular, isolation and enrichment of DNA molecules within dilute and complex samples can enable clinical detection of DNA markers linked to disease and synthetic selection of analyte-specific molecules such as aptamers.
Aptamers are oligonucleotides that display affinity for target molecules such as proteins, small molecules, nucleic acids, and whole cells, and can have applications to clinical diagnostics and therapeutics. The recognition abilities of aptamers have been employed with various transduction methods to generate novel diagnostic tools. In addition, aptamers have contributed to advances in therapeutics for diseases such as macular degeneration and various types of cancer. “Smart” aptamers can be generated which bind with specific equilibrium constants, kinetic parameters, and at specific temperatures.
Aptamer sequences can be developed by an evolutionary process known as Systematic Evolution of Ligands by Exponential Enrichment, or SELEX. However, it can be labor-intensive, and inefficient. Microchip-based devices for sample enrichment can reduce sample consumption and shorten assay times. Consequently, enrichment techniques can be implemented in microfluidic devices to separate and enrich low-Active concentration biological molecules from complex samples, for example, to improve various aspects of the SELEX process.
Genetic mutations take many forms, ranging from chromosome anomalies to single-base substitutions. Among them, single nucleotide polymorphisms (SNPs), which are single nucleotide variations in the genome between different individuals, are the most common form, occurring approximately once every 1000 bases. SNPs can be used as genetic markers to identify genes associated with complex disease. Therefore, accurate identification of SNPs can be of utility to disease diagnosis and prognosis.
Genotyping of SNPs can be based on enzymatic cleavage, allele specific hybridization, allele specific ligation or cleavage, and allele specific primer extension. Enzymatic cleavage can utilize thermostable flap endonucleases (FEN) and fluorescence resonance energy transfer (FRET) to recognize and detect SNP by the annealing of allele-specific overlapping oligonucleotides to the target DNA. This method is generally time-consuming and difficult to multiplex (i.e., to detect multiple SNPs in one reaction). There is therefore a need for new genotyping platforms to address these issues and offer improved accuracy, ability to multiplex, and increased throughput.
The disclosed subject matter provides techniques for isolation, selection, and amplification of nucleic acids, e.g., DNA molecules.
In certain embodiments, a method for amplifying a target DNA molecule using at least a first microchamber is provided. The microchamber can be formed as part of a MEMS-based microdevice, and can include at least one first primer immobilized on a solid phase. The first primer is suitable for amplifying the target DNA. A sample including the target DNA molecule can be introduced into the first microchamber, where the target DNA is hybridized onto the first primer. A complementary DNA of the target DNA can be produced in the first microchamber using the target DNA as a template, e.g., by using a PCR process and suitable PCR reagents and polymerase. The target DNA can then be separated from the complementary DNA. A second primer can be hybridized onto the complementary DNA, e.g., at a free end of the complementary DNA.
An amplification of the target DNA can be obtained using the complementary DNA as a template. Such amplified copy of the target DNA can be again separated from the complementary DNA, and the thermal cycling procedure repeated to produce a plurality of double-stranded DNA each including a copy of the target DNA and a copy of the complementary DNA.
In certain embodiments, the second primer can include a spectroscopically detectable tag, such as a fluorophore. Such a tag can be detected using, e.g., fluorescent spectroscopy. In some embodiments, the target DNA can be an aptamer.
In some embodiments, before introducing the sample containing the target DNA into the chamber for amplification, the sample can be purified. For example, a sample containing the target DNA and non-target DNA molecules can be into a second microchamber which includes an immobilized functional molecule that binds with the target DNA, such that the target DNA binds with the immobilized functional molecule in the second microchamber. The DNA molecules not bound with the functional molecule can be removed, e.g., by washing, and then the bound target DNA can be isolated from the functional molecule. The isolation can be performed by changing the temperature of the second chamber, e.g., raising the temperature. Alternatively, the isolation can be achieved using a chemical reagent, such as an alkali solution.
In certain embodiments, the target DNA can be transported from one microchamber to another microchamber electrophoretically, e.g., via a microchannel that connects the two microchambers and includes a gel suitable for electrophoresis of the target DNA. The transported target DNA can be amplified, using a PCR process, in the latter microchamber on-chip, and the amplified target DNA can be transported back into the former microchamber, e.g., electrophoretically via the same microchannel or another channel including a gel suitable for electrophoresis of the target DNA.
In some embodiments, the target DNA includes at least one polymorphic site, and the method further includes detecting such polymorphic site. After the amplification of the target DNA in a microchamber according to a bead-based PCR procedure (e.g., through multiple thermal cycles), the amplified copy of the target DNA can be separated from the complementary DNA. At least one an allele specific primer can be introduced into the microchamber, such that the primer anneals adjacent to a site of the complementary DNA corresponding to the polymorphic site. The allele specific primer can then be extended by one base to obtain an extended primer. The extended primer can then be isolated from the complementary DNA. The one base included in the isolated extended primer can then be detected, such as by MALDI-TOF mass spectroscopy, thereby determining the identity of the polymorphic site of the target DNA. The target DNA can include a plurality of polymorphic sites. In such a case, a plurality of allele specific primers can be used, each to anneal adjacent to one of the plurality of polymorphic sites. The multiple primers each can have different molecular weight. In such a manner, multiple polymorphic sites can be detected simultaneously.
In other embodiments, detecting a polymorphic site in a target DNA can be achieved as follows, using a microfluidic device having a first microchamber and a second microchamber in fluidic communication with the first microchamber. A sample including the target DNA is introduced into the first microchamber. At least one allele specific primer is introduced to anneal immediately adjacent to the polymorphic site of the target DNA. The allele specific primer is then extended by one base to obtain an extended primer. A plurality of copies of the extended primer are then generated in the first microchamber by one or more thermal cycles. The plurality of copies of the extended primer are transferred into the second microchamber which includes a solid phase having surface-attached functional molecules that bind with the extended primer such that the at least one of the plurality of copies of extended primer is captured by the solid phase.
The captured extended primer can then be isolated from the solid phase, e.g., by chemical cleavage; and the one base included in the isolated extended primer can be detected, e.g., using MALDI-TOF mass spectroscopy, to determine the identity of the polymorphic site of the target DNA. The target DNA can include a plurality of polymorphic sites. In such a case, a plurality of allele specific primers can be used, each to anneal adjacent to one of the plurality of polymorphic sites. The multiple primers each can have different molecular weight. In such a manner, multiple polymorphic sites can be detected simultaneously according to the above procedure.
The disclosed subject matter also provide microdevices, and fabrication methods thereof, for implementing the techniques described above.
a is a flowchart of an example process for isolating and amplifying a target DNA according to some embodiments of the disclosed subject matter.
b is a schematic diagram of a system for isolating and amplifying a target DNA according to some embodiments of the disclosed subject matter.
a-2e is a schematic diagram illustrating a process for isolating and enriching a target DNA from a library of DNAs using a microdevice having an isolation microchamber and an amplification microchamber according to some embodiments of the disclosed subject matter.
a-4d are schematic diagrams illustrating a process of detecting a polymorphic site on a DNA using a single chamber of a microdevice according to some embodiments of the disclosed subject matter.
a and 6b are schematic diagrams of the structure and dimensions of an example microdevice according to some embodiments of the disclosed subject matter.
a and 9b are images of gel electrophoresis analysis of tests using an 181 bp DNA segment of the B. pertussis genome, where (a) shows the results of a solution based test and (b) shows the results of a bead-based test according to an embodiment of the disclosed subject matter.
a-15c are micrographs of the microchamber illustrating the process of bead-based PCR according to one embodiment of the disclosed subject matter.
a and 17b are images of a microdevice without connections (a), and of the mixer being tested with a dye (b).
a and 20b are plots showing binding affinity comparison between enriched DNA and the starting random library according to some embodiments of the disclosed subject matter.
a-23j are schematic diagrams of an example fabrication process for the microchip as shown in
a is gel electropherogram of amplified eluents containing non-target DNA as obtained during the isolation process according to one embodiment of the disclosed subject matter;
a is gel electropherogram of amplified eluents obtained during in a control example;
a-29d depict electrophoretic transport of fluorescently labeled target DNA through the gel-filled microchannel under an electric field of 25 V/cm at different times according to some embodiments of the disclosed subject matter.
a is a gel electropherogram of eluents obtained from the enrichment chamber following PCR amplification;
a is a gel electropherogram of eluents obtained during the isolation according to one embodiment of the disclosed subject matter;
a is a gel electropherogram of eluents obtained during the isolation according to a control example;
a-36c show electrophoretic transport of fluorescently labeled target DNA under an electric field of 25 V/cm at different times according to one embodiment of the disclosed subject matter;
a is a gel electropherogram of eluents obtained from the enrichment chamber following PCR amplification according one embodiment of the disclosed subject matter;
a is a gel electropherogram of amplified eluents containing non-target DNA as obtained from the selection chamber in an example using the microdevice depicted in
a is a micrograph of beads in the PCR chamber according to one embodiment of the disclosed subject matter;
a-42c are fluorescence images of beads after (a) 0, (b) 10, (c) 20 PCR cycles according to some embodiments of the disclosed subject matter (scale bar: 100 μm.);
a and 44b are schematic diagrams of the structure of a microdevice according to some embodiments of the disclosed subject matter;
a is a bar graph showing characterization of bead-based PCR in a genotyping example using the microdevice as depicted in
a is a bar graph showing fluorescent intensity of beads before desalting, after desalting and after thermal elution in a genotyping example using the microdevice as depicted in
a is a MALDI-TOF mass spectrum of a mutated HBB gene in a genotyping test using the microdevice as depicted in
a is a cross-sectional schematic diagram of an SNP detection device according to one embodiment of the disclosed subject matter;
a is a plot showing a time-resolved tracking curve in a test of the temperature of SBE chamber of the device shown in
a is a MALDI-TOF mass spectrum of single base extension product in a test using the SNP detection device depicted in
The disclosed subject matter provides techniques for isolation, selection, and amplification of nucleic acids, e.g., DNA molecules on a microchip. More specifically, the disclosed subject matter provides for MEMS-based microdevice platform and associated methods for isolating and enriching desired DNA for genotyping and other applications.
In one aspect, the presently disclosed subject matter provides a method for amplifying a target DNA molecule using a microchamber including a first primer immobilized on a solid phase (e.g., microbeads) in the first microchamber. Referring to
The above procedure can be performed in a microchamber (or simply “chamber”) of a microdevice (also referred to as microchip) loaded with microbeads as the solid phase. The microdevice can be fabricated using standard microfabrication techniques, e.g., using PDMS soft lithography to create a chamber with desired shape and dimension. For example and not limitation, the microchamber can have a diameter of from about 0.1 mm to about 2 mm, and a depth of about 0.05 to 0.5 mm. Microheaters and temperature sensors, which can be used for temperature regulation in the PCR process, can be integrated into the microdevice, e.g., situated in a thin film layer underneath the microchamber. In connection with this embodiment and other embodiments as further described below, and for illustration and not limitation,
The target DNA can be from different sources, including synthetically generated DNA such as a randomized oligonucleotide library, or genomic DNA extracted from cells. Source locations can include off-chip processes as well as on-chip pre-processing of samples.
The microbeads are functionalized with a suitable primer for amplifying the target DNA (also referred to as “template DNA”). The microbeads can be polymer beads coated with streptavidin, which is known to have extraordinarily high affinity for biotin. The primer (e.g., a reverse primer) can be biotin-functionalized and immobilized onto the beads surface. When the sample including the target DNA is introduced into the chamber, the target DNA can hybridize to the bead-immobilized primers due to molecular recognition (e.g., Watson-Crick type base pairing). Other molecules in the sample, such as non-target DNA molecules, cells, small molecules, etc., are less likely to bind with the primers. Using the bead-immobilized primer and PCR reagents (including e.g., Tag polymerase, deoxynucleotide triphosphates, and buffer), a complementary DNA can be produced based on the target DNA, which together with the target DNA forms a double-stranded DNA (ds-DNA) tethered on the beads. Such ds-DNA can be denatured (or melted) at an elevated temperature, e.g., about 95° C., to separate the target DNA from the complementary DNA. A second primer, e.g., a forward primer, can be annealed onto the complementary DNA (e.g., at the free end of the complementary DNA) at a lowered temperature, e.g., at 50-62° C. Thereafter, using the contemporary DNA as a template, the second primer, and the PCR reagents, another copy of the target DNA can be produced, at a suitable chain extension temperature, e.g., about 72° C. Repeating the above temperature cycles (melting, annealing, and extension) can result in amplification of the target DNA, i.e., generation of exponentially increasing duplicate copies of the target DNA.
The untethered second primer can be labeled with a spectroscopically detectable tag (e.g., a fluorophore). In such a case, the result of the amplification after a number of PCR cycles can be fluorophore-labeled target DNA and unlabeled, bead-tethered complementary strands. Such labeled target DNA can be isolated for detection by fluorescent spectroscopy.
The above-described bead-based PCR procedure in a microchamber of a microdevice provides a convenient framework for detecting and manipulating DNA on-chip. Various processes concerning isolation, enrichment, evaluation of DNA utilizing such PCR procedure are contemplated. Some example embodiments are described below.
The sample including the target DNA (and other impurities) can be first processed such that the target DNA can be selectively captured by certain functional molecules that specifically bind with the target DNA. For example, molecular recognition of sequence-specific binding structures allows purification of such sequences from complex mixtures using a molecular analyte. In such a case, the functional molecule can be protein Immunoglobulin E (IgE), and the target DNA can be aptamer(s) specifically binding with IgE. This can be particularly useful for isolating and amplifying a target DNA in a sample which contains various other DNA sequences, e.g., an oligomer library.
An example procedure employing such pre-selection and follow-on amplification is illustrated in
The target DNA can be an aptamer. Aptamers can be developed for a broad spectrum of analytes with high affinity, possess well controlled target selectivity, and be synthesized to bind targets with predefined binding characteristics, such as temperature-sensitive binding. As such, external stimuli (such as temperature, pH, or ionic content) can be used to disrupt the aptamer-target binding complex. For example, chamber 220 can be set at a first temperature T1 for binding of the target aptamer, and after the removal of the unbound DNAs and other impurities, the temperature of the second chamber can be changed, e.g., raised to T2 which is higher than T1 such that the conformal structure of the aptamer is disrupted, thereby releasing the aptamer from the functional molecules. The temperature control can be achieved by integrated microheater and temperature sensor associated with the selection chamber. For certain aptamers, release temperature T2 can be lower than the capture temperature T1. In such cases, the lower temperature T2 can be achieved by thermoelectric cooling, e.g., by Peltier elements incorporated in the microdevice. Alternatively, the aptamer bound to the functional molecule can also be released using a reagent, such as an alkali solution.
In example embodiments, the transporting of the target DNA from the selection chamber to the enrichment chamber can be accomplished by electrophoresis. As illustrated in
After the target DNA is transported into the enrichment chamber, further selection-transportation rounds can be performed, such that more target DNA will be accumulated in the enrichment chamber. Additionally or alternatively, the target DNA can be amplified in the enrichment chamber using the above-described procedure. The amplified products can be transported back into the selection chamber for further rounds of selection-transportation-amplification if desired. Such transportation from the enrichment chamber to the selection chamber can be again by electrophoresis, e.g., using the microchannel 340 and the gel 350 loaded therein (and a reverted electric field applied on the electrodes 360 and 365), or via a second gel-filled microchannel connecting the two chambers.
In other embodiments, the on-chip PCR procedure can be employed for detection of a polymorphic site in a target DNA (i.e., DNA having a single nucleotide polymorphism (SNP)), which is illustrated in
Alternatively, detection of a polymorphic site in a target DNA can be accomplished on a microdevice using different procedures not involving PCR. One example procedure for such detection is illustrated in
Further details of device structure, fabrication, and operation procedures of the above-described embodiments can be found in the following Examples, which are provided for illustration purpose only and not for limitation.
This example demonstrates bead-based PCR for isolating and amplifying a target DNA on a microchip including integrated heaters and temperature sensors.
As illustrated in
Devices were fabricated using standard microfabrication techniques. Chrome and gold layers for heater and temperature sensor (thicknesses 20 and 200 nm) were thermally deposited onto glass microscope slides and patterned using contact lithography and wet etching techniques. Patterning generated a 5.67 cm long by 200 μm wide heater having a resistance of 20Ω and covering an area of 0.242 cm2, and a 1.04 cm long by 40 μm wide resistive temperature sensor having a resistance of ˜30Ω. The thermal elements were then passivated with SiO2 (thickness 1 μm) formed using plasma-enhanced chemical vapor deposition, with openings for electrical connections formed using a shadow mask. The SiO2 not only passivates the electrical components, but also provides an efficient bonding surface for PDMS. To generate PDMS for the microfluidic chambers, PDMS prepolymer was mixed in the ratio of 10:1 with a curing agent and poured onto a clean silicon wafer, baked for 30 min at 75° C., and then peeled from the wafer.
Microfluidic chambers were defined by puncturing holes in the PDMS using a hole punch. The bottom PDMS piece was 1.3 mm thick with a 3.2 mm diameter hole, and the top piece was 1.3 mm thick with a 4.75 mm diameter hole (
In an alternative design, as illustrated in
In addition to designing and fabricating the microchip, the bead-based primers were designed to allow rapid and simple operation of the device. Biotin-streptavidin coupling was used in this example to attach the reverse primers to the beads, as this bond is both strong and formed spontaneously in the presence of both molecules. The reverse primers were synthesized with a dual-biotin label at the 5′ end, followed by a spacer molecule adjacent to the nucleotide sequence. The dual-biotin moiety can minimize the loss of signal due to thermal denaturation. Spacer molecules provide greater lateral separation between DNA on the beads, thereby reducing hybridization issues due to steric hindrance. Synthetically generated template DNA was used to obtain controlled, consistent results for characterization of DNA detection using bead-based PCR.
The bead-based PCR chip was applied to pathogenic DNA detection, demonstrated with a DNA sequence associated with B. pertussis. B. pertussis is a gram-negative bacteria that infects ˜48.5 million patients (with 300,000 fatalities) annually worldwide. While early detection is the key to the treatment of this disease, current methods (e.g., cell culturing) for detecting the B. pertussis bacterium take days or even weeks of turnaround time. This limitation can be addressed by the disclosed bead-based PCR microchip which allows rapid, sensitive, and specific detection of B. pertussis. Pathogenic DNA detection using bead-based PCR on a microchip can be accomplished as follows: Bead-tethered reverse primers loaded in the microchamber of the microchip can be exposed to a raw sample, such as cell lysate, which can include various impurities. Pathogenic DNA in the sample can then be captured onto the beads via its specific hybridization to the reverse primers. As the capture is based on the affinity between the DNA and the reverse primers, this also serves as a purification step. Thereafter, the pathogenic DNA can be mixed on-chip with PCR reactants and bead-based PCR of the pathogenic DNA can be performed using fluorescently labeled forward primers. This process can rapidly generate exponentially amplified, fluorescently labeled template copies on microbeads, which can be detected by fluorescent microscopy. The use of beads in the detection volume can generate an enhanced signal-to-noise ratio in comparison to amplification on flat solid surfaces due to a significant increase in the fluorophore-coated surface area. Finally, the labeled copies of the template can be released from their bead-bound complements by denaturation and eluted into pure buffer for further analysis, while the bead-bound complementary strands can be retrieved from the chip and stored as cDNA libraries.
For detection of DNA associated with B. pertussis, the following materials and reagents were used. All DNA was obtained in lyophilized form from Integrated DNA Technologies, Coralville, Iowa, USA. The primers are designed as a PCR assay for determination of B. pertussis infection. The DNA sequences used are as follows—forward primer: 5′-FAM-Spacer-GAT TCA ATA GGT TOT ATG CAT GGT T-3′ (SEQ ID NO:1), reverse primer: 5′-Double Biotin-Spacer-TTC AGG CAC ACA AAC TTG ATG GGC G-3′ (SEQ ID NO:2), and template: 5′-GAT TCA ATA GGT TGT ATG CAT GGT TCA TCC GAA CCG GAT TTG AGA AAC TGG AAA TCG CCA ACC CCC CAG TTC ACT CAA GGA GCC CGG CCG GAT GAA CAC CCA TAA GCA TGC CCG ATT GAC CTT CCT ACG TCG ACT CGA AAT GOT CCA GCA ATT GAT CGC CCA TCA AGT TTG TGT GCC TGA A-3′ (SEQ ID NO:3). The forward primer has been modified with the fluorescent label carboxyfluorescein at the 5′ terminus, while the reverse primer incorporates a dual-biotin modification at the 5′ end. Both molecules contain an inert spacer molecule between the 5′ modifications and the nucleotide sequence. PCR was performed using Tag enzyme, deoxynucleotide triphosphates (dNTPs), and PCR reaction mixture containing appropriate buffers (Promega GoTaq Plexi PCR Mix). Reverse primers were immobilized onto streptavidin coated polymer-based microbeads (Thermo Scientific Pierce Protein Research Products Ultralink Streptavidin Resin) averaging 80 μm in diameter. Concentration and purity measurements of DNA samples were conducted using UV/VIS (Thermo Scientific Nanodrop). Materials used in microfabrication included photoresist (Rohm & Haas Electronic Materials S1818, Microchem SU-8 2000), PDMS prepolymer (Dow Corning Sylgard 184), and Parylene C prepolymer (Kisko diX C).
PCR reaction mixture for B. pertussis DNA detection was prepared as follows. Each lyophilized DNA sample was suspended in deionized H2O and diluted to the desired concentration. The PCR mixture consisted of the following: 5×PCR Buffer (2 μL), 25 mM MgCl2 (0.6 μL), 10 mM dNTPs (0.4 μL), 50 μg/mL BSA (0.4 μL), 5% (by volume) Tween 20 (0.1 μL), microbeads (0.5 μL), water (4.1 μL), 25 μM forward primer (0.4 μL), 25 μM reverse primer (0.4 μL), and enzyme (0.1 μL).
The ingredients were mixed with target (template) DNA (1 μL of synthetic template DNA with a concentration range of 1 aM-100 pM) without the enzyme and the mixture was then degassed at −0.4 psi for 30 min in a darkened container (to prevent photobleaching of the fluorophore label). Testing in the PCR device was also performed under an enclosure to prevent excess light from reaching the DNA. Following degassing, enzyme was added to the mixture and the 10 μL PCR sample was pipetted into the chip, followed by 30 μL of mineral oil. During the testing with the integrated device (as depicted in
Following PCR, the sample was pipetted to a darkened 0.5 mL microcentrifuge tube. Beads were washed six times with 1×SSC buffer to remove excess labeled primers. As used herein, the “x” before a buffer refers to the concentration, overall, as compared to the literature value of a standard buffer (which are prescribed in literature). For example, a 10×SSC buffer is ten times as concentrated as would commonly be used, and stored that way so as to allow the preparation of 1× solutions by adding additional water or desired reagents in solution (for example, 1 mL 10× buffer can be added to 9 mL of a DNA sample to achieve a 1× buffer solution with a desired concentration of DNA). Bead washing was by mixing the sample with buffer, allowing the beads to settle via gravity, and removing the supernatant with a pipette. A five microliter aliquot of each test sample was pipetted into an individual 3.2 mm diameter PDMS well on a glass slide, and was observed using the fluorescent microscope. During integrated device testing, microbeads were washed by passing buffer through the chamber while being retained by the weirs prior to fluorescent measurement. The microscope was kept in an enclosure to prevent ambient light from interfering with the measurements or bleaching the fluorescent labels. The samples containing the beads with attached ds-DNAs were briefly excited with light using the fluorescent light source and the resulting emission was recorded using the CCD camera microscope attachment. Camera exposure times were optimized based on fluorescent signal intensity during device characterization to maximize the signal-to-noise ratio, defined here as the ratio of the measured fluorescence intensity to the intensity of background fluorescence. Digital images were analyzed using Image J software.
The resistive heater and sensor were first characterized for accurate on-chip temperature control. For this test, the microchip was placed in a temperature-controlled environmental chamber and its temperature varied. Chamber temperatures were measured using a platinum resistance temperature detector probe (Hart Scientific 5628) and on-chip resistances were measured with a digital multimeter (Agilent 34420A).
Resistance measurements of temperature sensors indicated a linear relationship between resistance and temperature. These data were used to calculate a TCR for the sensor of 2×10−3° C.−1. The heater was found to have a resistance of ˜20 Ω.
The accuracy of on-chip temperature measurements and the heating rate of the chip were then tested. A 1.5 mm diameter insulated K-type thermocouple probe (Omega Engineering) was inserted into the sample chamber along with a pure water sample. The chamber temperature was then controlled (heated cyclically) as would occur during a typical PCR test, but without the amplification reagents. According to the time course of temperature obtained during this control test (
The effects of test conditions, such as ambient light and temperature, on test results were also investigated. Biotin-streptavidin binding was chosen as a simple alternative to covalent methods for DNA immobilization, however, streptavidin molecules can denature as a result of the elevated temperature necessary to dehybridize DNA. Temperature cycling equivalent to typical PCR testing was performed on linkages between streptavidin and dual-biotin labeled DNA. Beads coated with streptavidin were mixed with 1 μM dual-biotin labeled primers and an equal concentration of fluorophores-labeled complementary strands. The solution was subjected to temperature cycling, returned to room temperature, and washed to remove any DNA in solution. In
During a series of PCR reactions, parameters were varied individually and the results examined to determine the parameters that would generate maximum signal intensity, thus lowering the detection limit of the device. First, solution-based PCR (i.e., excluding microbeads) was performed to confirm device calibration and sequences of both DNA primers (25 bases) and template (181 bases). Following amplification, gel electrophoresis was performed and results indicate that a strand of the expected length (181 bp) was produced (
An exemplary magnesium concentration was determined to be 1.5 mM, consistent with typical MgCl2 concentrations for PCR studies. A series of tests also determined that a consistent 20 s dwell time, or time spent at each temperature setpoint during a PCR cycle, would produce DNA most efficiently for the microdevice employed.
The effect of annealing temperature on the concentration and length of DNA generated during PCR was investigated. The annealing temperature can affect the hybridization of the primers to the template DNA; a higher annealing temperature can result in more specific hybridization (less erroneous hybridization of a primer to an unspecified DNA sequence), but less total hybridization of DNA (and therefore less product DNA after PCR). A series of bead-based PCR tests was conducted using the B. pertussis primer set in which the annealing temperature was varied. The results indicated that fluorescent intensity of the beads following PCR remained approximately consistent (
The annealing temperature test was then repeated with conventional solution-based PCR, and the results were analyzed using gel electrophoresis (
Next, the bead-based PCR detection device was optimized with respect to the concentration of beads in the reaction mixture. The presence of a solid surface in bead-based PCR introduces steric and geometric effects, affecting the reaction efficiency. Previous studies of solid-phase amplification focused on maximizing the final concentration of DNA, however, the primary concern of this study is the detection of DNA. The concentration of microbeads in the reaction mixture was therefore investigated and optimized to produce the greatest fluorescent signal. To test the effects of bead concentration on fluorescent signal intensity, bead-based PCR reactions were performed using conditions (58° C. annealing temperature, 1.5 mM MgCl2 concentration, 10 pM template concentration) and three different concentrations of beads (
The sharp peak in
The microdevice was then tested for detecting synthetic gDNA. Detection criteria included the limit of detection (the smallest concentration of DNA that could be detected) and the number of PCR cycles necessary for detection. The limit of detection of the device was investigated by performing a series of PCR reactions while changing the concentration of template DNA in the reactants. Concentrations ranging from zero templates to 1 pM (˜6×105 copies/μL) were tested. PCR reactions were run for 10 cycles (˜15 min) using the optimized test conditions discussed above (200 beads per microliter, 20 s dwell times, 58° C. annealing temperature, and 1.5 mM MgCl2). As seen in
In addition to measuring the detection limit of the device, the relationship between signal intensity and the number of PCR cycles performed prior to detection was also investigated. For this investigation, bead-based PCR reactions were performed as discussed in the previous section, but a 1 pM (˜6×105 copies/μL) concentration was consistently used and the number of PCR cycles was varied. As seen in
a-15c show micrographs of the microchamber illustrating the process of integrated microfluidic bead-based PCR. In
In this example, aptamers are selected and amplified using an integrated microchip including a selection chamber and an amplification chamber, as illustrated in
The microdevice (or microchip, chip) used in this Example includes two chambers, one of which (selection chamber) performs selection and separation of candidate aptamers, and the other (amplification chamber) amplifies and collects the candidate aptamers. A schematic diagram of the microdevice for this Example is shown in
All DNA used in the tests in this Example was purchased from Integrated DNA Technologies (IDT), with sequences as follows. Library: 5′-CTA CCT ACG ATC TGA CTA GCN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN GCT TAC TCT CAT GTA GTT CC-3r (SEQ ID NO:4). Forward primer: 5′-FAM-Spacer-C TAC CTA CGA TCT GAC TAG C-3′ (SEQ ID NO:5). Reverse primer: 5′-Dual Biotin-Spacer-G GAA CTA CAT GAG AGT AAG C-3′ (SEQ ID NO:6). Library and primer sequences are based on a conventional SELEX protocol targeting IgE. Reagents used for PCR include 5× GoTaq Flexi PCR mix (Promega), 25 mM MgCl2, 10 mM dNTPs (Promega), 50 μg/mL BSA (Sigma), GoTaq enzyme (Promega), and streptavidin-coated polymer microbeads (Streptavidin Plus Ultralink, Pierce). Beads used in the selection procedure were Bio-Rad Affi 10 Gel activated media with 80 μm average diameters. The functional molecules used to capture the target DNA (also referred to as target protein) used was human IgE (Athens Research).
The microdevice was fabricated using contact lithography. Briefly, glass slides were coated with chrome and gold (15 nm and 150 nm thick, respectively), patterned using optical lithography, and etched to produce resistive heaters and temperature sensors. A 1 μm film of silicon dioxide was then applied via a plasma-enhanced chemical vapor deposition (PECVD), with a silicon hard mask defining openings for electrical connections. Molds for soft lithography were defined using optical lithography of layered SU-8 on silicon wafers. PDMS was then cast to produce microfluidic channels, chambers, weirs, and the mixer. The PDMS fluidic network was then bonded to the glass slide following oxygen plasma treatment, and the entire chip was coated with 1 μm of Parylene C via CVD prior to packaging (as shown in
Following fabrication, the resistive temperature sensors were calibrated in an environmental chamber and the mixer was tested using a dye to measure mixing efficiency (as shown in
Buffer containing candidate aptamers was mixed with PCR reagents and microbeads, and introduced to the amplification chamber. Remaining unamplified solution was separately removed from the chip and stored. With the amplification chamber filled, the inlets were sealed with wax and the solution thermally cycled. Fluorescence intensity of the beads confirms final surface concentration of DNA. The chamber was then held at 95° C. for 5 minutes to dehybridize bead-bound DNA, and buffer was then flowed at 1 μL/min to remove amplified ssDNA.
Binding analyses were performed in the selection chamber using a fresh chip containing fresh IgE-coated beads. A sample of enriched library DNA was further amplified off-chip, again using FAM-labeled forward primers. This was then purified with streptavidin-coated microbeads; ssDNA was eluted at 95° C., and resuspended in 1×PBSM. Sample concentration was then measured with UV/VIS absorption (ThermoScientific Nanodrop) prior to further analyses.
A portion of each buffer sample used for washing the beads in the selection chamber was removed and stored for testing following isolation and amplification. These samples were analyzed off-chip using conventional PCR, in parallel with on-chip amplification and collection. To measure the effect of on-chip aptamer isolation on the randomized pool, each of ten buffer aliquots which washed the IgE-coated beads at 37° C. was amplified off-chip and tested using gel electrophoresis. This generated a clearly defined gradient of concentration, with bands corresponding to initial washes fluorescing brighter than those corresponding to the following washes (
Further, the affinity of the enriched pool of aptamer candidates for IgE was tested. To generate a quantity of DNA large enough for testing, the enriched pool was amplified off-chip using conventional PCR. Following isolation and resuspension of the target ssDNA in 1×PBSM, the concentration of the sample was measured with UV/VIS and normalized to 1 μM. For comparison, randomized library was purchased containing a FAM modification at the 5′ terminus, and diluted to 1 μM in 1×PBSM. Samples of each (5 μL) were individually inserted into a microchamber containing microbeads freshly coated with IgE and maintained at 37° C., incubated for 5 minutes, washed with buffer, and micrographed during fluorescent excitation. The increased affinity of the enriched pool versus the randomized library was easily viewable using an optical microscope (
In addition to measuring the increased affinity of the enriched pool for IgE, the temperature-dependence of the affinity was measured. As with the affinity measurements, a 1 μM solution of fluorescently-labeled aptamer candidates in 1×PBSM was exposed to a chamber packed with microbeads coated with IgE until the fluorescent signal saturated. Pure buffer was then flowed at 1 μL/min, while the temperature of the chamber was changed in increments of 3° C. While buffer was continuously flowed, the chamber was maintained at each temperature for 5 minutes. The pool exhibited highly temperature-dependent binding to IgE, with maximum binding at 37° C. as desired (
In this example, isolation and enrichment of target DNA is demonstrated using a microfluidic chip having two chambers and a channel therebetween which includes a gel, as previously described in connection with
As shown in
The microchip used in this Example was fabricated from a polydimethylsiloxane (PDMS) microfluidic layer bonded onto a glass substrate patterned with a resistive heater and sensor using standard microfabrication techniques such as lithography (
To prepare IgE-functionalized microbeads, 200 μL of solution containing NHS activated microbeads (mean diameter: ˜100 μm, GE Healthcare) was washed 3 times with 1×PBS buffer modified to contain 1 mM of Mg2+ ions (8.1 mM Na2HPO4, 1.1 mM Na2HPO4, 138 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, pH 7.4) by centrifugation. Then the beads were incubated with 200 μL of 0.1 μM human myeloma IgE (Athens Research & Technology) for 5 hours at room temperature. After incubation, excess IgE molecules were discarded by washing the beads with fresh PBS buffer. To reduce nonspecific binding of ssDNA molecules to the beads, the surfaces that were not conjugated with IgE were passivated by incubating the beads in 0.1 M Tris-HCl buffer for 1 hour. The IgE-functionalized beads were stored in PBS buffer at 4° C. before use. An fluorescently labeled ssDNA library having random sequences (97-mer, TGT TGT GAG CCT CCT GTC GAA—50 random bases—TTG AGC GTT TAT TCT TGT CTC CC-3′) (SEQ ID NO:7), IgE-specific ssDNA aptamer D17.4 (78-mer, KD=10 nM, 5′-GCC TGT TGT GAG CCT CCT GTC GAA GCA CGT TTA TCC GTC CCT CCT AGT GGC GTG CTT GAG CGT TTA TTC TTG TCT CCC-3′) (SEQ ID NO:8), and forward (5′-GCC TGT TGT GAG CCT CCT GTC GAA-3′) (SEQ ID NO:9) and reverse (5′-GGG AGA CAA GAA TAA ACG CTC AA-3′) (SEQ ID NO:10) primers were purchased from Integrated DNA Technologies. A mixture of random ssDNA and aptamer D17.4 (1000:1, mole ratio) was used throughout the example to increase competition for IgE binding sites. The random ssDNA solution was prepared by mixing 1 μL of a 100 μM random ssDNA library and 1 μL of 0.1 μM aptamer D17.4 in 98 μL of 1×PBS buffer. The running buffer for electrophoretic transport of ssDNA in the microchannel and for a slab-gel electrophoresis was 0.5×TBE buffer (44.5 mM Tris base, 44.5 mM boric acid, 1.25 mM EDTA, pH 8.3). Three percent agarose gel (Difco Laboratories) for electrophoresis was prepared by dissolving 0.3 grams of agarose in 100 mL of 0.5×TBE buffer on a hotplate.
A schematic of the test setup is shown in
Isolation and enrichment of desired ssDNA molecules in the randomized ssDNA mixture was carried out as follows. The IgE-functionalized microbeads were loaded in the isolation chamber using a syringe through a bead inlet to fill approximately 30% of the chamber volume (˜3×104 beads). After loading, the beads were washed for 5 minutes with 1×PBS buffer at a flow rate of 40 μL/min using a syringe pump. The random ssDNA mixture (100 μL) was introduced to the chamber through the inlet at a flow rate of 20 μL/min and collected from the outlet in 3 separate plastic tubes (˜33 μL/tube). PBS buffer was injected to the chamber at 40 μL/min to wash weakly bound DNA strands from the IgE-beads, and the waste solution was collected in 10 separate tubes at the outlet (˜33 μL/tube). The two chambers were filled with 0.5×TBE buffer and then the isolation chamber was heated at 57° C. for 5 minutes via the resistive heater to elute strongly bound DNA strands from the beads.
As the thermal elution was occurring, Pt-wire electrodes were inserted into the chambers and an electric field of 25 V/cm was applied for 25 minutes. The DNA strands were then electrophoretically transported to the enrichment chamber through the gel-filled channel. To investigate the single round of isolation and enrichment of ssDNA, the two chambers were flushed with PBS buffer as eluents were collected in plastic tubes (˜33 μL/tube). For multiple rounds of DNA enrichment, the beads in the isolation chamber were discarded and the chamber was thoroughly washed with PBS buffer prior to the next round of the isolation and enrichment processes to remove undesired DNA molecules that might remain. Fresh IgE-functionalized beads were then introduced in the isolation chamber for the next round of DNA isolation and enrichment.
To analyze the results from the example, representative eluent samples from each step were amplified off-chip by polymerase chain reaction (PCR) using a thermal cycler (Mastercycler Personal, Eppendorf). The PCR procedure included denaturation of DNA at 95° C. for 3 minutes followed by 20 cycles of amplification. Each cycle consisted of denaturation at 95° C. for 15 seconds, annealing at 59° C. for 30 seconds, and extension at 72° C. for 45 seconds. Following the amplification, 7 μL of PCR product was mixed with 7 μL of 2×DNA loading dye containing bromophenol blue and xylene cyanol (Thermo Scientific) and loaded into each lane of a 3% agarose gel. Electrophoresis was then carried out at 100 V for 30 minutes in 0.5×TBE buffer using a slab gel apparatus (Mupid-exU, Advance). The gel was then stained with ethidium bromide in deionized water for 5 minutes. The bands in the gel representing the concentration of DNA in each eluent sample were visualized using a UV illuminator (AlphaImager 3400, Alpha Innotech). A fluorescence microscope was used to monitor the electrophoretic transport of ssDNA through the gel-filled channel. The intensities of gel-bands and fluorescence from images obtained were analyzed using the Image J software (National Institutes of Health freeware).
In the isolation-enrichment procedure, the IgE-binding ssDNA was first isolated from the randomized ssDNA mixture in the isolation chamber. IgE isolation was effected by exposing the chamber to samples of randomized DNA and then washing with pure buffer to remove unbound DNA. These buffer samples containing residual DNA were collected following washing, and were amplified with PCR and visualized with slab gel electrophoresis to determine the effectiveness of the isolation procedure.
The upper and lower bands seen in lanes P and I1-E1 represent amplified samples of the 97 bp random ssDNA and 78 bp D17.4 aptamer, respectively. The upper bands are brighter than the lower bands as a result of the 1000:1 molar ratio of random ssDNA to D17.4 aptamer in the DNA mixture used for the isolation. No bands are seen in lane N, indicating that the reagents used were not contaminated by undesired DNA molecules. In addition, no bands are seen in lane EC, indicating that the gel-filled microchannel effectively prevented contamination of the enrichment chamber with unwanted ssDNA from the isolation chamber during the capture of the target DNA strands.
A bar graph depicting the band intensity of the 97-mer random ssDNA (the upper band) from lane I1 to lane E1 is plotted to show the progress of the isolation of IgE-binding ssDNA (
To verify that the isolated ssDNA strands were specifically bound to IgE, the example was repeated using fresh NHS beads with no protein coupled to the surfaces. The gel image (
1×PBS and 0.5×TBE buffers were tested as exemplary electrolytes for the electrophoretic transport of DNA through the gel-filled microchannel. PBS buffer is a strong electrolyte (electrical conductivity: 15 mS/cm), and as it is commonly used for other steps in the process its use in electrophoresis can simplify the enrichment process. Alternatively, TBE buffer (approximate electrical conductivity: 350 μS/cm) can be used (as it is an electrolyte commonly used in gel electrophoresis applications).
As shown in
Time required to electrophoretically transport ssDNA from the isolation to enrichment chambers was determined. The fluorescence micrographs obtained during electrophoretic transport of fluorescently labeled ssDNA strands at different times monitored at the center of the gel-filled channel are shown in
An entire round of isolation and enrichment of IgE-binding ssDNA in a single microchip was performed. Random ssDNA library was exposed to IgE-coated beads, weakly bound strands were washed away, and aptamer candidates were thermally eluted and electrophoretically transported to the enrichment chamber. To analyze the results, eluent was collected from each step (i.e., incubation: I, washing: W, elution: E) as well as the buffer used to wash the two chambers (i.e., isolation chamber: IC, elution chamber: EC) after the processes were completed. These eluents were then chemically amplified using PCR and visualized using slab gel electrophoresis. The electropherogram visualizing the amplified eluents are shown in
To investigate the ability of the microchip to enrich IgE-binding DNA, multiple rounds of ssDNA enrichment were performed on the chip. The isolated IgE-binding ssDNA strands were repetitively enriched via electrophoretic transport. A gel electropherogram of amplified eluents collected after 1 round (lane 1), 2 rounds (lane 2), and 3 rounds (lane 3) of enrichment in the microchip is shown in
This Example demonstrates the use of a microdevice having a similar structure as that in Example 3, but using chemical elution rather than thermal elution to release the bound candidate aptamers.
The microdevice used in this Example includes a selection/isolation chamber 3210 and an enrichment chamber 3220 each having a depth of 200 μm and a volume of 5 μL, the chambers being connected by a microchannel 3240 (1 mm×7.8 mm×40 μm) (as shown in
In the example, human myeloma IgE (Athens Research & Technology) was dissolved in 1× phosphate-buffered saline (PBS buffer) to a final concentration of 1 μM. NHS-activated microbeads were purchased from GE Healthcare and functionalized with IgE. An 87-mer ssDNA library having random sequences (5′-GCC TGT TGT GAG CCT CCT GTC GAA-N40-TTG AGC GTT TAT TCT TGT CTC CC-3′) (SEQ ID NO:11), and fluorescently labeled IgE-specific ssDNA aptamer D17.4 primers were purchased from Integrated DNA Technologies. The mixtures of ssDNA were prepared by mixing 1 μL of the random library (100 μM) and 1 μL of the aptamer D17.4 (0.1 μM) in 98 μL of PBS buffer.
Isolation and enrichment of desired ssDNA molecules in a randomized ssDNA mixture was carried out as follows. The IgE-beads were loaded in the isolation chamber using a syringe through a bead inlet to fill approximately 30% of the chamber volume. After loading, the beads were washed for 5 minutes with PBS buffer at a flow rate of 20 μL/min using a syringe pump. Throughout the examples, a DNA mixture having 0.1% of IgE-specific aptamer D17.4 was used. The ssDNA mixture was introduced to the isolation chamber and collected from the outlet of the isolation chamber in 3 separate samples (sample volume: ˜33 μL). PBS buffer was introduced to the chamber at 20 μL/min to release weakly bound DNA strands from the IgE-beads, and the waste solution is collected in 10 separate samples at the outlet (sample volume: ˜30 μL). To elute strongly bound DNA strands from the beads, 5 μL of 0.1 M NaOH in 0.5×TBE buffer was introduced into the chamber and incubated for 5 minutes. Platinum electrodes were inserted into the Pt inlets in each chamber and an electric field of 25 V/cm was applied. The strands were then electrophoretically transported to the enrichment chamber through the gel-filled channel. Following electrophoretic DNA transport, the isolation chamber was washed with PBS buffer to remove undesired DNA molecules. To concentrate the IgE-binding ssDNA in the enrichment chamber, the isolation and electrophoretic transport processes was repeated. Samples of eluent containing ssDNA molecules were collected throughout the example and amplified using polymerase chain reaction (PCR) off-chip. Slab-gel electrophoresis was used to evaluate the concentration of DNA eluted throughout the process by comparing the band intensities for each sample. A fluorescence microscope was used to monitor the electrophoretic transport of ssDNA through the gel-filled channel.
IgE-binding ssDNA was first isolated from the randomized ssDNA mixture in the isolation chamber.
The band intensity profile of the 87-mer ssDNA during incubation, consecutive washes, and elution is shown in
The enrichment chamber was washed with buffer just prior to electrophoretic migration of isolated DNA. To determine if the gel-filled channel prevents contamination of the enrichment chamber with unwanted DNA from the isolation chamber, the buffer was PCR amplified and analyzed using gel electrophoresis (
To verify that the eluted 87-mer ssDNA is IgE-specific, the isolation was repeated using fresh NHS beads without functionalizing the surfaces. Following all buffer washes, samples of buffer were PCR amplified using the same conditions as with the previous test (
The electrophoretic transport of isolated ssDNA through the gel-filled microchannel was then investigated. Two Pt electrodes connected to a DC power supply were inserted in the Pt inlets of the isolation and enrichment chambers to form a cathode and an anode, respectively. The electrophoretic transport of the ssDNA through the gel-filled channel was monitored using an inverted epifluorescence microscope. The fluorescence micrographs obtained at different times at the center in the channel are shown in
Following the electrophoretic transport of IgE-binding ssDNA for 20 minutes, the two chambers were thoroughly washed with PBS buffer and the waste solutions were collected. A gel electropherogram of the collected eluents is shown in
To investigate the ability of the chip to enrich DNA, multiple rounds of ssDNA isolation and transport were performed on a single chip. Following a round of isolation and transport, fresh IgE-beads were introduced to the isolation chamber. The captured ssDNA was then concentrated in the enrichment chamber by repetitive isolation and transport. A gel electropherogram for eluents collected after 1 round (lane 3), 2 rounds (lane 4), and 3 rounds (lane 5) of enrichment in different chips is shown in
In this Example, gel-based electrophoretic nucleic acid transport, bead-based nucleic acid isolation, and polymerase chain reaction (PCR) are combined to simplify microchip design, fabrication, and operation by eliminating the need for complex flow handling components. The process includes the selection and electrophoretic transport as described above in connection with
Amplified eluents from each step were visualized using gel electrophoresis and intercalating dyes, as shown in
In further tests, the amplified strands were separated (e.g., by eluting) from the beads in the PCR chamber and electrophoretically transported back into the isolation chamber, such that additional selection-amplification rounds can be performed. During multiple rounds of the aptamer selection-transport-amplification process, no damage was observed to the agarose gel in the channel indicating that the gel physically separated the two chambers while eliminating cross-contamination of buffers by selectively transporting nucleic acids via electrophoresis. The binding affinity of the randomized nucleic acid pool to IgE significantly improved to 13 nM after 4 rounds of aptamer selection. An enriched mixture of 87-mer and 78-mer nucleic acids was used for the binding affinity measurements. Multiple rounds of isolation and enrichment of aptamers against steroid and MCF-7 cells on the microchip were also tested. Results showed this on-chip approach can be simple yet versatile enough to select aptamers against a variety of functional molecules.
This Example illustrates a MEMS-based SNP genotyping method that performs PCR, SBE, and desalting reactions on microbeads in a single microchamber, as previously described in connection with
The microfluidic device used in this Example includes a microchamber 4410 formed by PDMS situated on a microheater 4430 and temperature sensor 4440 (
The temperature control part of the microchip was fabricated using standard microfabrication techniques. Briefly, a glass slide 4460 (Fisher HealthCare, Houston, Tex.) was cleaned by piranha. Chrome (10 nm) and gold (100 nm) thin films 4462 were deposited by thermal evaporation and patterned by wet etching. Then, a passivation layer of 1 μm of silicon dioxide 4464 was deposited using plasma-enhanced chemical vapor deposition (PEC VD). Finally, contact pads for wire bonding, connecting the instruments to the on-chip sensor and heater, were opened by etching the oxide layer using hydrofluoric acid (
Separately, the microfluidic chamber was fabricated from PDMS (Sylgard 184, Dow Corning Inc. Midland, Mich.) using soft lithography techniques. SU-8 photoresist 4472 (MicroChem. Corp., Newton, Mass.) was spin-coated and patterned on a silicon wafer 4470 to form mold-defining microfluidic features. Next, a PDMS prepolymer solution (base and curing agent mixed in a 10:1 ratio) was cast onto the mold and cured on a hotplate at 72° C. for 1 hour (
Subsequently, the inlet and outlet were punched on the resulting sheet bearing the microfluidic features, which was then bonded to the temperature control chip after treatment of the bonding interfaces with oxygen plasma for 15 seconds (
All chemicals used in this Example were purchased from Sigma-Aldrich (St. Louis, Mo.) unless otherwise indicated. Streptavidin beads (Pierce Streptavidin Plus UltraLink Resin) were obtained from Thermo Fisher Scientific Inc. (Rockford, Ill.). Dideoxynucleotide triphosphates (ddNTPs) were purchased from Jena Bioscience GmbH (Jena, Germany). Deoxynucleotide triphosphates (dNTPs) and GoTaq Flexi DNA Polymerase were obtained from Promega Corp. (Madison, Wis.). Thermo Sequenase was purchased from GE Healthcare (Piscataway, N.J.). Template DNA, including a mutated type (5′-CCT CAC CAC CAA CTT CAT CCA COT TCA CCT TGC CCC ACA GGG CAG TAA CAG GAG TCA GAT GCA CCA TOG TOT CTG TTT GAG GTT GCT AGT GAA CAC AGT TGT GTC AGA AGC AAA TGT AAG CAA TAG ATG GCT CTG CCC TGA CT-3′ (SEQ ID NO:12), the SNP site is underlined and SBE primer annealing site is italic) and an unmutated type (5′-CCT CAC CAC CAA CTT CAT CCA CGT TCA CCT TGC CCC ACA GGG CAG TA
T CAG GAG TCA GAT GCA CCA TGG TGT CTG TTT GAG GTT GCT AGT GAA CAC AGT TGT GTC AGA AGC AAA TGT AAG CAA TAG ATG GCT CTG CCC TGA CT-3′ (SEQ ID NO:13), the SNP site is underlined and SBE primer annealing site is italic) of the HBB gene, double biotin modified reverse primer (5′-double biotin-AGT CAG GGC AGA GCC ATC TA-3′) (SEQ ID NO:14), fluorescein (FAM) modified forward primer (5′-FAM-CCT CAC CAC CAA CTT CAT CC-3′, M.W.=6651) (SEQ ID NO:15), and SBE primer (5′-ACG GCA GAC TTC TCC-3′, M.W.=4513) (SEQ ID NO:16) were synthesized and purified by Integrated DNA Technologies (Coralville, Iowa).
Closed-loop temperature control of the microchamber was achieved using the integrated temperature sensor, heater, and a fan 4650 under the microchip 4600 with a proportional-integral-derivative (PID) algorithm implemented in a LabVIEW (National Instruments Corp., TX) program on a personal computer 4610. The resistance of the sensor was measured by a digital multimeter 4640 (34420A, Agilent Technologies Inc., CA), and the heater and fan were connected to two DC power supplies 4620 (E3631, Agilent Technologies Inc., CA) respectively. The inlet was connected to a syringe that contained reaction buffer or washing buffer driven by a syringe pump 4630 (KD210P, KD Scientific Inc., MA). The outlet was connected to a microcentrifuge tube 4670 for collection of genotyping product to MALDI-TOF MS or waste. All fluorescent images of beads were taken using an inverted epifluorescence microscope (Diaphot 300, Nikon Instruments Inc., NY) with a CCD camera (Model 190CU, Micrometrics, NH), after removing the device from the fan (
The streptavidin beads in the microchamber were rinsed with binding and washing (B&W) buffer (5 mM Tris-HCl, 0.5 mM EDTA, 1 M NaCl, and 0.01% Tween 20, pH=7.5). The reverse primer (50 pmol) in B&W buffer was introduced and incubated with the beads for 30 min, followed by washing with B&W buffer at 10 μL/ruin for 10 min.
Bead-based PCR was performed for 30 thermal cycles as follows: 95° C. for 15 s, 56° C. for 30 s, and 72° C. for 30 s. A 5 μL sample of PCR reactants was introduced twice, prior to cycling and between 15th and 16th cycle, and each sample consisted of 0.08 pmol of template, 8.33 pmol of forward primer, 1× GoTaq Flexi Buffer, 0.83 units of GoTaq Flexi DNA Polymerase, 1.67 nmol of dNTP and 6.25 nmol of MgCl2 (1.25 mM). The microbeads were then rinsed with 0.15 mM NaOII in B&W buffer at 5 μL/min for 10 min to elute template ssDNA, followed by a rinse of pure B&W buffer at 10 μL/min for 10 min, leaving complementary ssDNA on the beads.
To perform SBE, the SBE primer targeting the SNP on the complementary sequence of exon 1 of the HBB gene was extended by a single base in the microchamber using ddNTPs. A 5 μL sample of SBE reactants was introduced to the microchamber twice, prior to SBE and between 5th and 6th thermal cycle, and underwent 10 thermal cycles as follows: 90° C. for 15 s, 40° C. for 30 s, and 70° C. for 30 s. Each SBE reactant consisted of 6.67 pmol of primer, 16.67 pmol of ddNTP, 1× Thermo Sequenase reaction buffer and 2.67 units of Thermo Sequenase. The microchamber was then rinsed using B&W buffer at 5 μL/min for 10 min, followed by desalting with DI water at 5 μL/min for 20 min. Finally, the microchamber was incubated at 95° C. for 1 min, followed by a rinse with DI water at 20 μL/min and 95° C. for 3 min, to elute the hybridized primer.
The temperature-resistance relationship of the thin-film gold temperature sensor was calibrated following fabrication. The calibration data showed that the measured resistance (R) of the sensor exhibited a highly linear relationship with temperature (T), which can be fitted to R=R0[1+α(T−T0)], where R0 is the sensor resistance at reference temperature T0, and α is the temperature coefficient of resistance (TCR) of the sensor. The TCR was determined to be 3.06×10−3° C.−1 for a typical chip, which had a reference resistance of 83.44Ω at a reference temperature of 21.9° C. Time-resolved tracking of on-chip thermal cycling showed that the chamber temperatures attained specified setpoints via control of the on-chip heater and off-chip fan quickly and precisely (
To characterize bead-based PCR, reactants were thermally cycled on-chip and fluorescent bead intensity was then measured and compared to control tests. To obtain consistent results under controlled conditions, template DNA was used as the target sequence for the characterization. After B&W buffer washing, the fluorescent intensity of beads was significantly higher than those without thermal cycling, enzyme or templates, which were only 5%, 7% and 16% of the original test (
Prior to SBE, template ssDNA generated during PCR were removed from the bead-bound complementary DNA. To test the efficiency of the chemical elution method, the template ssDNA was first amplified using fluorescently labeled forward primers and double biotinylated reverse primers in a conventional thermal cycler, and the amplification product was immobilized onto the streptavidin beads, which were packed in the microchamber afterwards. The fluorescent intensity of the beads was then measured before and after rinsing with buffer. The fluorescent intensity of rinsed beads was 87% lower than that of pre-elution beads (
To generate a DNA solution prior to detection with MALDI-TOF MS, hybridized primers were desalted and then thermally eluted into DI water. The effect of desalting and the efficiency of the thermal elution method were tested to ensure that DNA loss during this step would not compromise detection by MS. The fluorescently labeled forward primer in B&W buffer was first hybridized to the ssDNA on the beads and desalted with DI water. The fluorescent intensity of the beads was then measured before and after rinsing at 95° C., and the elution product was manually pipetted to a MALDI plate and tested using MALDI-TOF MS. During desalting, the microchamber was rinsed with DI water, and the fluorescent intensity remained at 95.5% of pre-desalting intensity (
Having tested the individual procedures necessary for SNP detection, the procedures were integrated and the SBE products were analyzed using MALDI-TOF MS. Theoretically, the mass of extended primer can be calculated according to the equation inp=mr+mn−mb, where mr is the mass of extended primer, mr is the mass of unextended primer, mn is the mass of corresponding ddNTP and mb is the mass of bond formation (175 m/z). SNPs on both mutated HBB gene and unmutated HBB gene were detected. As the target nucleotides of the mutated and unmutated template DNA are adenosine and thymidine, a single dideoxyadenosine triphosphate (ddATP, M.W.=472) and dideoxythymidine triphosphate (ddTTP, M.W.=463) were incorporated into each primer, respectively. Thus the mass of the product for mutated and unmutated HBB gene were respectively expected to be 4810 Daltons (4513+472−175), as shown by the distinct peak at 4810 m/z in
In this example, an alternative approach for detecting polymorphic site of a target DNA, as previously described in connection with
The microdevice used in this Example, as illustrated in
The device was fabricated using standard microfabrication techniques. Briefly, gold (100 nm) and chrome (5 nm) thin films were thermally evaporated onto the glass substrate, and patterned by photolithography and wet etching. This resulted in resistive temperature sensors and resistive heaters, which were then passivated by deposition of silicon dioxide (1 μm) using plasma enhanced chemical vapor deposition (PECVD). Next, the PDMS sheet was bonded to the temperature control chip irreversibly after treatment with oxygen plasma. Finally, the inner surface of the device was coated with a thin layer of Parylene C via chemical vapor deposition.
The temperature-resistance relationship of a resistive temperature sensor is calibrated following fabrication to provide accurate temperature control. The resistance of thin film gold resistor is linearly dependent on temperature, as given by R=R0 (1+α(T−T0)), where R is the sensor resistance at temperature T, R0 is the sensor resistance at reference temperature T0, and α is the sensor's temperature coefficient of resistance (TCR). Measurements of SBE sensor resistances at varying temperatures are shown in
The temperature tracking history was tracked in a test and shown in
A primer (5′-GATAGGACTCATCACCA-3′, 5163 m/z) (SEQ ID NO:17) targeting exon 8 of the cancer suppressor gene p53 was extended by a single base (ddUTP-N3-biotin) in the SBE chamber. 10 μL of SBE solution was introduced to the SBE chamber and underwent 10 thermal cycles as follows: 90° C. for 10 s, 40° C. for 60 s, 70° C. for 30 s. SBE solution including 20 pmol of synthetic DNA template, 40 pmol of primer, 60 pmol of ddUTP-N3-biotin (M.W.=1189), 1× Thermo Sequenase reaction buffer and 2 units of Thermo Sequenase. Almost 100% of the primer molecules were extended, as shown by a single product peak (6177 m/z) in the mass spectrum (shown in
SBE product (10 μL from the commercial thermal cycler, Eppendorf Mastercycler® Personal, using the same SBE solution and parameters) terminated with ddUTP-N3-biotin was successfully captured by streptavidin-coated microbeads packed in the SPC channel and released upon incubation in tris(2-carboxyethyl)phosphine (TCEP, 100 mM, pH=9) at 65° C. for 10 min, as evidenced by a subsequently detected single mass spectral peak at 5713 m/z (
To characterize desalting efficiency, as low as 0.5 pmol primer molecules in TCEP were introduced into the C18 bead-packed desalting channel, followed by washing with deionized water and elution with 50% acetonitrile. Effective desalting was observed from a distinct mass spectral peak at 5163 m/z (
For SNP detection, 10 μL of SBE solution was introduced as before, followed by SBE, SPC, chemical cleavage and desalting in series in the microdevice. The SNP site was detected successfully, as shown in the mass spectrum (
The description herein merely illustrates the principles of the disclosed subject matter. Various modifications and alterations to the described embodiments will be apparent to those skilled in the art in view of the teachings herein. Accordingly, the disclosure herein is intended to be illustrative, but not limiting, of the scope of the disclosed subject matter.
This application is a continuation application from PCT/US12/056,888, filed Sep. 24, 2012, which claims priority from U.S. Provisional Application No. 61/538,774, filed Sep. 23, 2011; Provisional Application No. 61/542,124, filed Sep. 30, 2011; Provisional Application No. 61/588,078, filed Jan. 18, 2012; Provisional Application No. 61/588,079, filed Jan. 18, 2012; Provisional Application No. 61/588,082, filed Jan. 18, 2012; Provisional Application No. 61/590,458, filed Jan. 25, 2012; Provisional Application No. 61/674,187, filed Jul. 20, 2012; Provisional Application No. 61/674,191, filed Jul. 20, 2012; Provisional Application No. 61/674,192, filed Jul. 20, 2012; and Provisional Application No. 61/683,977, filed Aug. 16, 2012, the disclosure of each of which is incorporated herein in its entirety.
This invention was made with government support under CBET-0854030, awarded by the National Science Foundation; RR025816-02 and CA147925-01, both awarded by the National Institutes of Health. The government has certain rights in this invention.
| Number | Date | Country | |
|---|---|---|---|
| 61538774 | Sep 2011 | US | |
| 61542124 | Sep 2011 | US | |
| 61588078 | Jan 2012 | US | |
| 61588079 | Jan 2012 | US | |
| 61588082 | Jan 2012 | US | |
| 61590458 | Jan 2012 | US | |
| 61674187 | Jul 2012 | US | |
| 61674191 | Jul 2012 | US | |
| 61674192 | Jul 2012 | US | |
| 61683977 | Aug 2012 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US12/56888 | Sep 2012 | US |
| Child | 14221596 | US |
