Patent Application
20170067091
Chemical amplification of nucleic acids can be realized with 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 PCR 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, and 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 DNA sequencing, protein screening, and pathogenic DNA detection. For example, certain 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 be used to provide integrated chip-based systems that perform tasks such as sample pretreatment and post-amplification analysis.
In bioanalytical assays, analytes of interest can be present in minute quantities and contaminated with impurities. Sample preparation prior to analysis can be important for improving the resolution of detection results. For example, isolation and enrichment of DNA molecules within dilute and complex samples can be used for 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. Certain aptamers have been employed with various transduction methods to generate diagnostic tools and/or used in therapeutics for diseases such as macular degeneration and various types of cancer. Certain so-called “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, which can be labor-intensive.
The disclosed subject matter provides techniques for isolation, selection, and amplification of aptamers, e.g., cell-targeting aptamers.
In certain embodiments, techniques for selecting and isolating cell-targeting aptamers are provided. In certain examples, cells can be cultured on a bottom portion of a selection chamber. A sample can be introduced into the selection chamber. The sample can include cell-targeting oligomers that bind to the cultured cells. Unbound oligomers can be removed from the selection chamber to isolate the cell-targeting oligomers. In accordance with exemplary embodiments of the disclosed subject matter, the cells can be cancer cells such as MCF-7 cells. The cell-targeting oligomer can be, for example, single-stranded DNA. The unbound oligomers can be removed by infusing a washing buffer into the selection chamber.
In accordance with some embodiments of the disclosed subject matter, the cell-targeting oligomer can be eluted and hydrodynamically transferred to an amplification chamber. Elution can be accomplished by raising the temperature in the selection chamber, e.g., by controlling a heater. Microvalves in a microchannel can be actuated to hydrodynamically transfer the cell-targeting oligomers to the amplification chamber.
In certain embodiments, primer-functionalized magnetic beads can be provided in the amplification chamber. For example, the primer-functionalized magnetic beads can be held in the amplification chamber by an external magnet. The primer-functionalized magnetic beads, e.g., streptavidin-coated magnetic beads, can be configured to capture the cell-targeting oligomers. The captured oligomers can then be amplified, e.g., by applying a polymerase chain reaction technique.
The cell-targeting oligomers can be hydrodynamically transferred from the amplification chamber to the selection chamber.
In accordance with another embodiment of the disclosed subject matter, microdevices for selecting and isolating cell-targeting oligomers are provided. The microdevice can include a selection microchamber formed in a cavity of a multilayered thin film structure. The microchamber can include cells immobilized on a bottom portion thereof. The microdevice can be made by culturing cells in the selection chamber. The cells can be cancer cells such as MCF-7 cells.
In accordance with embodiments of the disclosed subject matter, the microdevice can include a heater such as a resistive heater. The heater can be serpentine-shaped. The microdevice can also include a temperature sensor.
The microdevice can include an amplification chamber connected to the selection chamber via a microchannel. The microchannel can include one or more microvalves configured to hydrodynamically transfer cell-targeting oligomers from the selection chamber to the amplification chamber. For example, the microdevice can include a pneumatic control channel configured to actuate the one or more microvalves. The pneumatic control channel can include an oil-filled channel.
The amplification chamber can include a plurality of primer-functionalized magnetic beads such as streptavidin-coated beads. The beads can be held in the amplification chamber by an external magnet. A resistive heater can be located under the amplification chamber.
The microdevice can also include an additional microchannel including one or more microvalves configured to hydrodynamically transfer cell-targeting oligomers from the amplification chamber to the selection chamber.
In certain embodiments, techniques for isolating and amplifying oligomers using a selection chamber and an amplification chamber are provided. A first sample including oligomers can be introduced into the selection chamber, and the oligomers can be isolated. The oligomers can then be hydrodynamically transferred from the selection chamber to the amplification chamber. The oligomers can then be amplified in the amplification chamber, e.g., by applying a PCR technique. The oligomers can then be transferred from the amplification chamber to the selection chamber.
In accordance with other embodiments of the disclosed subject matter, techniques for isolating and amplifying an aptamer using a selection chamber and an amplification chamber can include introducing a first sample including an oligomers into the selection chamber, isolating the oligomers, transferring the oligomers from the selection chamber to the amplification chamber, amplifying the oligomers in the amplification chamber, and hydrodynamically transferring the oligomers from the amplification chamber to the selection chamber.
The selection chamber can include immobilized targets for selecting the oligomers. The immobilized targets can be, for example, cultured cells such as cancer cells. In accordance with other embodiments, the immobilized targets can be, for example, microbeads, e.g., IgE-functionalized microbeads. The microbeads can be retained in the selection chamber by a weir structure.
The oligomers can be transferred from the selection chamber to the amplification chamber via a first microchannel. For example, the oligomers can be hydrodynamically transferred from the selection chamber to the amplification chamber, e.g., using one or more microvalves. In accordance with other embodiments, the oligomers can be transferred from the selection chamber to the amplification chamber via electrophoresis.
In certain embodiments, microdevices for selecting and isolating cell-targeting oligomers are provided. A microdevice in accordance with an exemplary embodiment of the disclosed subject matter can include a selection microchamber, an amplification microchamber, a first microchannel between the selection chamber and the amplification chamber configured to transfer oligomers from the selection chamber to the amplification chamber, and a second microchannel between the selection chamber and the amplification chamber configured to transfer oligomers from the amplification chamber to the selection chamber. At least one of the first microchannel and the second microchannel can include one or more microvalves.
The selection chamber can include immobilized targets. For example, the immobilized targets can be cultured cells. In accordance with other embodiments, the immobilized targets can be microbeads retained on a weir structure. A heater and a temperature sensor can be positioned below the selection chamber.
One or both of the first microchannel and the second microchannel can include one or more microvalves configured to hydrodynamically transfer the oligomers between the selection chamber and the amplification chamber. The microdevice can include a pneumatic control channel configured to actuate the one or more microvalves. The pneumatic control channel can be, for example, an oil-filled channel.
In accordance with some embodiments of the disclosed subject matter, the amplification chamber can include primer-functionalized microbeads. An external magnet can be positioned to hold the microbeads in the amplification chamber.
In accordance with an exemplary embodiment of the disclosed subject matter, the second microchannel can include one or more microvalves configured to hydrodynamically transfer the oligomers from the amplification chamber to the selection chamber. The microdevice can include a pneumatic control channel configured to actuate the one or more microvalves. The pneumatic control channel can be, for example, an oil-filled channel. The first microchannel can include an agarose gel for transferring oligomers via electrophoresis.
The disclosed subject matter also provides microdevices, and fabrication methods thereof, for implementing the techniques described above.
The disclosed subject matter provides techniques for isolation, selection, and amplification of aptamers on a microchip. More specifically, the disclosed subject matter provides for MEMS-based microdevice platforms and associated methods for isolating and enriching aptamers for research, diagnostic, therapeutic, and other applications.
In one aspect, the presently disclosed subject matter provides a method for selecting and isolating cell-targeting aptamers. Referring to
The cells can be, for example, cancer cells. In accordance with other embodiments of the disclosed subject matter, the cells can be healthy cells. Cancer cells can be, for example, MCF-7 cells
With further reference to
Cell-targeting oligomers can include, but are not limited to, oligonucleotides between about 12 and 80 nucleotides in length. The cell-targeting oligomers can recognize a cell by specific affinity binding. Upon introduction of the sample, cell-targeting oligomers can strongly bind to the cells in the selection chamber, while non-cell-targeting oligomers can be unbound or weakly bound to the cells.
The cell-targeting oligomers can then be isolated by removing the non-cell-targeting oligomers from the selection chamber while the cell-targeting oligomers remain bound to the cells (at 106). For example, weakly bound ssDNA can be removed by washing using a washing buffer such as, for example, D-PBS. Multiple washes can be performed.
The cell-targeting oligomers can then be eluted to break the bond between the cell-targeting oligomers and the cells (at 108). For example, the cell-targeting oligomers can be eluting by adjusting the temperature of the selection chamber. For example, the selection chamber can be set at a first temperature T1 for binding of the cell-targeting 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 cell-targeting aptamer from the cell.
The temperature control can be achieved by integrated microheaters and temperature sensors associated with the selection chamber. For example, a resistive heater and temperature sensor can be provided under the selection chamber. The microheater and the temperature sensor can be, for example, serpentine-shaped.
For certain oligomers, 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 oligomers bound to the cell can also be released using a reagent, such as an alkali solution.
With further reference to
After the cell-targeting oligomers are transferred from the selection chamber to the amplification chamber, the cells can be removed from the selection chamber. The selection chamber can be washed, e.g., using a buffer, and cell culturing can be re-started in the selection chamber.
Referring again to
A polymerase chain reaction (PCR) technique can be applied to amplify the cell-targeting oligomers. Using the bead-immobilized primer and PCR reagents (including e.g., Taq 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 complementary 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.
With further reference to
After the cell-targeting oligomers are transferred to the selection chamber, the used streptavidin beads can be removed from the amplification chamber. In accordance with certain embodiments of the disclosed subject matter, the external magnet can be removed and/or turned off. New streptavidin beads can be introduced into the amplification chamber and held in place (e.g., by replacing and/or turning on the external magnet).
In accordance with certain embodiments of the disclosed subject matter, the selection and amplification process can be repeated one or more additional times.
In another aspect, the disclosed subject matter provides a method for isolating and amplifying an aptamer. Referring to
With further reference to
Referring again to
With reference yet again to
In yet another aspect, the disclosed subject matter provides a method for isolating and amplifying an aptamer. Referring to
The oligomers can then be isolated (at 304). For example, the oligomers can be allowed to strongly bind with an immobilized target, e.g., cells or IgE-functionalized microbeads. The unbound and weakly bound ssDNA can then be removed by washing, e.g., using a washing buffer such as D-PBS. The oligomers can then be eluted in preparation for transfer to the amplification chamber. In accordance with an exemplary embodiment of the disclosed subject matter, the oligomers can be thermally eluted. For example, the temperature of the selection chamber can be raised using on-chip microheaters and temperature sensors. In accordance with another embodiment, the oligomers can be chemically eluted.
With further reference to
After the oligomers are transferred from the selection chamber to the amplification chamber, the immobilized target can be removed from the selection chamber. The selection chamber can be washed, e.g., using a buffer, and a new batch of immobilized targets can be loaded in the selection chamber.
The oligomers can be amplified in the amplification chamber (at 308). The amplification chamber can include primer-functionalized microbeads. For example, the primer-functionalized microbeads can be magnetic beads such as 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 surface of the beads. The magnetic beads can be held in the amplification chamber by an external magnet. For example, the magnet can be placed below a bottom portion of the amplification chamber. When the sample including the oligomer is introduced into the amplification chamber (e.g,. via the first microchannel), the oligomers 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 molecules, cells, small molecules, and the like, are less likely to bind with the primers.
A polymerase chain reaction (PCR) technique can be applied to amplify the oligomers. Using the bead-immobilized primer and PCR reagents (including e.g., Taq 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 complementary 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.
With further reference to
After the oligomers are transferred to the selection chamber, the used streptavidin beads can be removed from the amplification chamber. In accordance with certain embodiments of the disclosed subject matter, the external magnet can be removed and/or turned off. New streptavidin beads can be introduced into the amplification chamber and held in place (e.g., by replacing and/or turning on the external magnet).
In accordance with certain embodiments of the disclosed subject matter, the selection and amplification process can be repeated one or more additional times.
In a further aspect, the disclosed subject matter provides a microdevice for selecting and isolating cell-targeting aptamers. An exemplary embodiment of a microdevice 400 in accordance with the disclosed subject matter is illustrated in
The microdevice 400 can further include a heater 408 and a temperature sensor 410. The microheater 408 can be a resistive heater and can be formed in a serpentine shape. The temperature sensor 410 can be a resistive temperature sensor can be formed in a serpentine shape. The heater 408 and temperature sensor 410 can be used to control the temperature in the selection chamber 402 using, for example, electronic control circuitry.
The microdevice 400 can further include an amplification chamber 412. The amplification chamber 412 can include an inlet 414 and an outlet 416, and the temperature of the amplification chamber 412 can be controlled by a heater 418 and temperature sensor 420, as described in connection with the selection chamber 402.
The selection chamber 402 and the amplification chamber 412 can be coupled via a first microchannel 422. The first microchannel 422 can include one or more microvalves configured to hydrodynamically transfer oligomers from the selection chamber 402 to the amplification chamber 412. The one or more microvalves can be actuated by a first pneumatic control channel 424. The first pneumatic control channel 442 can be filled with oil.
The one or more microvalves in first microchannel 422 can further be configured to hydrodynamically transfer oligomers from the amplification chamber 412 to the selection chamber 402. Alternatively, a second microchannel 426 between the selection chamber 402 and the amplification chamber 412 can be used. The second microchannel 426 can include one or more microvalves configured to hydrodynamically transfer oligomers from the amplification chamber 412 to the selection chamber 402. The one or more microvalves in second microchannel 426 can be actuated by a second pneumatic control channel 428.
The selection chamber 506 can include cultured cells 512. The cells 512 can be cultured on a bottom portion of the selection chamber 506 and can form a layer of cells that is immobilized on the bottom portion of the selection chamber. The cells can be, for example, cancer cells such as MCF-7 cells.
The amplification chamber can include primer-functionalized microbeads such as magnetic beads 514. The magnetic beads 514 can be, for example, streptavidin-coated polymer beads. The magnetic beads 514 can be held in place by an external magnet 516 positioned below the amplification chamber 508.
A microchannel 518 can connect the selection chamber 506 to the amplification chamber 508. One or more microvalves, which are not shown in
In another aspect, the disclosed subject matter provides a microdevice for isolating and amplifying an aptamer. An exemplary embodiment of a microdevice 600 in accordance with the disclosed subject matter is illustrated in
The selection chamber 602 and the amplification chamber 604 can be fabricated using standard microfabrication techniques, e.g., using PDMS soft lithography to create chambers with desired shape and dimension. For example and not limitation, the selection chamber 602 can have a semi-circular profile with a height of about 20 μm.
The microdevice 600 can include a selection chamber inlet 610 and a selection chamber outlet 612 for introduction and disposal of sample materials. For example, a randomized ssDNA library can be introduced via selection chamber inlet 610, while unbound and weakly bound ssDNA can be removed via the selection chamber outlet by washing. The microdevice can also include a selection chamber heater 614 and a selection chamber temperature sensor 616. The heater 614, which can be a resistive heater and be formed in a serpentine shape, and the temperature sensor 616, which can be a resistive sensor and be formed in a serpentine shape, can be located below the selection chamber 602 and can be used to control the temperature within the selection chamber 602. The microdevice can similarly include an amplification chamber inlet 618, an amplification chamber 620, an amplification chamber heater 622, and an amplification chamber temperature sensor 624.
The first microchannel 606 can be configured to transfer oligomers via electrophoresis as shown in
In accordance with another embodiment of the disclosed subject matter, the first microchannel 606 can be configured to hydrodynamically transfer oligomers from the selection chamber to the amplification chamber. The first microchannel can include one or more microvalves configured to hydrodynamically transfer aptamers from the selection chamber to the amplification chamber. The one or more microvalves can be actuated by a first pneumatic control channel. The first pneumatic control channel can be filled with oil.
The second microchannel 608 can be configured to hydrodynamically transfer aptamers from the amplification chamber to the selection chamber. For example, the second microchannel can include one or more microvalves configured to hydrodynamically transfer oligomers from the amplification chamber 604 to the selection chamber 602. The one or more microvalves can be actuated by a pneumatic control channel 628.
The microdevice 700 can include a substrate 710 such as a glass substrate. A passivation layer 712 can be situated between the substrate 710 an the interior of the selection chamber 702 and the amplification chamber 704. Temperature control elements 714 can be positioned below each of the selection chamber 702 and the amplification chamber 704.
The selection chamber 702 can include immobilized targets 716. For example, the immobilized targets 716 can be Immunoglobin E-functionalized microbeads, as shown in
The amplification chamber 704 can include primer-functionalized microbeads 718. The primer-functionalized microbeads 718 can be magnetic beads such as, for example, polymer beads coated with streptavidin. The magnetic beads can be held in the amplification chamber 704 by a magnet such as an external magnet 720 positioned below the amplification chamber 704.
The first microchannel 706 can be configured to transfer oligomers from the selection chamber 702 to the amplification chamber 704 via electrophoresis. For example, as shown in
With reference to
Selected aptamers can be used in a variety of applications, including research, diagnostic, and therapeutic applications. For example, aptamers selected by cancer cells can be used to identify those cancer cells in blood or tissue samples. In accordance with other embodiments, aptamers can be conjugated with a toxin and can be used to induce apoptosis in a target. Aptamers can also be used to block specific antigens to inhibit cellular functions, to separate out biomolecules of interest in chromatography applications, and as affinity probes in capillary electrophoresis (CE). In connection with the latter application, aptamer-target complexes can change the electrophoretic properties and can be detected through CE.
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.
A temperature control chip 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, which resulted in resistive temperature sensors and resistive heaters. Then, 1 μm of silicon dioxide was deposited using plasma-enhanced chemical vapor deposition (PECVD) to passivate sensors and heaters, the contact regions for electrical connections to which were opened by etching the oxide layer using hydrofluoric acid (
Finally, the surface of SBE microchanmber was coated with a thin layer of Parylene C via chemical vapor deposition (
All chemicals were purchased from Sigma-Aldrich (St. Louis, Mo.) unless otherwise indicated. ssDNA random library (5′-GCC TGT TGT GAG CCT CCT GTC GAA-40N-TTG AGC GTT TAT TCT TGT CTC CC-3′ (SEQ ID NO: 1)) and primers (Forward Primer: 5′-FAM-GCC TGT TGT GAG CCT CCT GTC GAA-3′ (SEQ ID NO: 2), and Reverse Primer: 5′-dual biotin-GG GAG ACA AGA ATA AAC GCT CAA-3′ (SEQ ID NO: 3)) were synthesized and purified by Integrated DNA Technologies (Coralville, Iowa). Deoxynucleotide triphosphates (dNTPs) and GoTaq Flexi DNA polymerase were obtained from Promega Corp. (Madison, Wis.). Minimum Essential Medium (MEM), fetal bovine serum (FBS), penicillin-streptomycin (P/S, penicillin 10,000 unit/mL, streptomycin 10,000 □g/mL), Dulbecco's phosphate-buffered saline (D-PBS), 0.25% Trypsin-EDTA and streptavidin coupled magnetic beads (Dynabeads® M-270 Streptavidin) were purchased from Invitrogen (Carlsbad, Calif.). MCF-7 cell line was obtained from the American Type Culture Collection (ATCC, Manassas, Va.). Closed-loop temperature control of both selection microchamber and amplification microchamber was achieved using two groups of temperature control units with a proportional-integral-derivative (PID) algorithm implemented in a LabVIEW (National Instruments Corp., TX) program on a personal computer 1002. Each temperature control unit contained a serpentine-shaped resistive temperature sensor and a resistive heater. The resistance of two temperature sensors was measured by a digit multimeter 1004 (34410A, Agilent Technologies Inc., CA) and a digit micro-ohm meter (34420A, Agilent Technologies Inc., CA), respectively. Each resistive heater was connected to an independent, dedicated DC power supply 1006 (E3631, Agilent Technologies Inc., CA) (
Fluid control was achieved using microfabricated pressure-driven valves 1008. Two oil-filled channels, each actuated by an air control valve (6464K16, McMaster-Carr, NJ), are connected to a nitrogen gas tank 1010 (Tech Air, NY) via a pressure regulator 1012 (CONCOA North America, VA). The microfluidic device inlets are connected to a set of syringes that contain samples, buffers and reagents driven by syringe pumps 1014 (KD210P, KD Scientific, MA).
Development of aptamers in the microfluidic device starts from culturing MCF-7 cells in the selection chamber for a sufficiently long time (>4 hours) to ensure cell attachment and surface biomarker regeneration (
The temperature sensor was first calibrated using an environmental test chamber (9023, Delta Design Inc., CA) maintained at a series of temperatures which are measured with high accuracy temperature reference probes (5628, Fluke Calibration, UT). The measured resistance (R) of the thin-film gold temperature sensor was observed to vary linearly with temperature (T), represented by the relationship R=R0[1+α(T−T0)], where R0 is the sensor resistance at a reference temperature T0, and α is the temperature coefficient of resistance (TCR) of the sensor.
Fitting this relationship to the measurement data allowed determination of the parameter values, which were used to determine the chamber temperature from the measured sensor resistance during the selection, elution and amplification process. The temperature sensor under the amplification chamber had a measured resistance of 136.42 Ω at a reference temperature of 25.0° C. with a TCR of 3.10×10-3 1° C. The temperature control of the chamber was then characterized during thermal cycling. Time-resolved tracking of on-chip thermal cycling showed that the amplification chamber temperatures attained specified setpoints via control of the on-chip heater and off-chip fan quickly and precisely (
The on-chip cell culture in the selection chamber was then investigated. 2 μL of MCF-7 cell suspension at 1×107 cells/mL in complete culture media was introduced into the selection chamber, which was then kept at 37° C. in a humidified incubator containing 5% CO2 for 5 hours. The selection chamber was next rinsed using D-PBS at 10 μL/min for 1 min to remove unattached and dead cells, and a phase contract image was taken with an inverted microscope (IX81, Olympus Corp., PA) equipped with a digital camera (C8484, Hamamatsu Corp., NJ). Cells were attached well on the bottom surface (
To characterize the isolation of cell-binding ssDNA from a random library, the temperature of selection chamber was kept at 37° C. for the whole procedure by using the temperature control unit located beneath. 100 pmol of ssDNA library in 20 μL binding buffer was infused into the chamber at 1 μL/min for 20 min. Then, cells were washed with 9 aliquots of washing buffer at 10 μL/min, each for 3 min, to remove undesired ssDNA. Waste from each buffer wash were collected, amplified using PCR, and analyzed using polyacrylamide gel electrophoresis (PAGE), as shown in
To demonstrate the thermal elution of strongly bound ssDNA after washing, the microchamber temperature was raised to 60° C. using the same temperature control unit. The cells were then rinsed with 3 aliquots of washing buffer (10 μL) at 1 μL/min, 5 μL/min and 10 μL/min, respectively. The high band intensity of lane E1 (1 μL/min), E2 (5 μL/min) and E3 (10 μL/min) indicates successful enrichment of cell-binding ssDNA (
To characterize the bead-based amplification of cell-binding ssDNA, 10 μL thermal eluent was driven to the amplification chamber with primer-coated magnetic beads at 1 μL/min and room temperature for 10 min. The magnetic beads were gently stirred by the permanent magnet to achieve a better capture efficiency. Then, 2 μL of PCR reactants were introduced and thermally cycled. After washing, the fluorescent images of beads with and without cycling were taken (
To collect the amplified cell-binding ssDNA, the magnetic beads were rinsed at 95° C. and 1 μL/min for 10 min. The rinsed beads (
To demonstrate the feasibility of multiple-round, closed-loop cell specific aptamer generation, a three-round ssDNA selection, enrichment and amplification process was carried out. The weakly bound ssDNA of each wash in all three rounds, and the thermally eluted strongly bound ssDNA in the third round were collected from the selection chamber (
The ssDNA isolated from the microfluidic SELEX process was tested for their affinity towards MCF-7 cells using a fluorescence binding assay. The background-subtracted average fluorescence intensity of cells incubated with the enriched aptamer candidate pool was 27-fold higher than that of cells incubated with randomized ssDNA (
The device includes two (selection and amplification) microchambers (1.7 μL each) connected via two microchannels (
To investigate oligomer binding to IgE, amplified eluents (16 cycles via PCR) were collected during ssDNA library introduction and washing, and visualized with gel electrophoresis (
Bead based PCR was confirmed by introducing the ssDNA library into the amplification chamber containing reverse primer functionalized beads and using the integrated resistive temperature sensor and heater to perform PCR (
The device includes two (selection and amplification) microchambers each of 1.7 uL volume. The microchambers are equipped with electrode ports for the insertion of platinum wires which generate an electric field for electrokinetics. The selection microchamber features a weir structure for capturing microbeads. The selection and amplification microchambers are connected via two microchannels (as shown in
All chemicals were purchased from Sigma-Aldrich (St. Louis, Mo.) unless otherwise indicated. Deoxyribonucleotide triphosphates (dNTPs) and GoTaq Flexi DNA polymerase were obtained from Promega Corp. (Madison, Wis.). Randomized oligomer library (5′-GCC TGT TGT GAG CCT CCT GTC GAA-45N-TTG AGC GTT TAT TCT TGT CTC CC-3′ (SEQ ID NO: 4)) and primers (Forward Primer: 5′-FAM-GCC TGT TGT GAG CCT CCT GTC GAA-3′ (SEQ ID NO: 2), and Reverse Primer: 5′-dual biotin-GG GAG ACA AGA ATA AAC GCT CAA-3′ (SEQ ID NO: 3)) were synthesized and purified by Integrated DNA Technologies (Coralville, Iowa). Human Myeloma Immunoglubulin E (IgE) was purchased from Athens Research and Technology (Athens, Ga.), and NETS-activated microbeads were purchased from GE Healthcare (Little Chalfont, Buckinghamshire, United Kingdom). Dulbecco's phosphate-buffered saline (D-PBS), and streptavidin coupled magnetic beads (Dynabeads® M-270 Streptavidin) were purchased from Invitrogen (Carlsbad, Calif.).
The microfluidic device was fabricated using conventional multi-layer soft-lithography techniques. First, a layer of AZ-4620 positive photoresist (Clariant Corp. Somerville, N.J.) was spin-coated on a silicon wafer (Silicon Quest International, Inc., San Jose, Calif.), exposed to ultraviolet light through photomasks, developed, and baked to form the round-shaped flow channel that can be sealed completely. Then, using the same silicon wafer, SU-8 (MicroChem, Newton, Mass.) layers were spin-coated and developed to define the flow layer. In parallel, a layer of SU-8 photoresist was patterned on another silicon wafer to establish the control. Meanwhile, chrome (10 nm) and gold (100 nm) thin films were thermally evaporated on a glass slide, patterned through photolithograph, and wet etched to form heaters and temperature sensors. The heater and sensor were passivated by spin-coating PDMS prepolymer solution and curing at 72° C. for 30 minutes.
Subsequently, PDMS prepolymer solution (base and curing agent mixed in a 10:1 ratio) was spin-coated onto the silicon wafer bearing the flow layer, and cured on a hotplate at 72° C. for 15 minutes. Another PDMS prepolymer solution was cast onto the control layer silicon wafer and cured on a hotplate at 72° C. for 30 minutes. The resulting control layer PDMS slab was peeled off from the mold, punched to form a pneumatic inlet, and bonded to the PDMS membrane on the silicon mold bearing the flow layer features. The bonded slab was then peeled from the flow layer wafer. After punching inlets and outlets, the slab was bonded to a glass slide bearing the heater and temperature sensor. Finally, molten agarose gel was injected in the gel inlet of the device to and cured at room temperature to form the electrokinetic transfer channel. A fabricated device can be seen in
NHS-activated microbeads are functionalized with protein by incubation with IgE. The functionalized microbeads are then introduced into the selection chamber of the device until approximately 40% of the selection chamber volume was occupied by beads. Selection of oligomers is then performed by infusing randomized library (1 uM) into the device (10 uL/min) for 10 minutes, followed by multiple washes with PBS buffer (20 uL/min) to remove weakly binding oligomers for 15 minutes. Next, primer functionalized magnetic beads are introduced into the amplification chamber of the device and held by an external magnet. Tris-boric acid electrolyte buffer is then injected into the device and platinum wires are inserted into the electrode inlets of each chamber with a 50 V potential difference applied between them for 35 minutes. Meanwhile, strongly bound oligomers remaining in the selection chamber are thermally eluted (50° C.) using the integrated heater and temperature sensor. The 25 V/cm electric field induced by the platinum wires electrokinetically transfers the thermally eluted oligomers to the positive electrode in the amplification chamber where the oligomers then hybridize to the reverse primers immobilized on the magnetic bead surfaces. The platinum wires are removed from the device eliminating the electric field, PCR reagents are introduced into the amplification chamber and bead-based PCR progresses utilizing the heater and temperature sensor located beneath the amplification chamber. A PCR process of 95° C. for 10 seconds, 59° C. for 30 seconds, and 72° C. for 10 seconds is used. After 20 cycles of PCR thermocycling, the IgE-functionalized microbeads are removed from the selection chamber and replaced with new IgE-functionalized microbeads. The valve is then opened and oligomers are released from the bead surfaces by heating to 95° C. The released oligomers are transported back to the selection chamber through the opened valve via pressure-driven flow (20 uL/min) for further affinity selection with the replenished microbeads. This closed-loop process is repeated for a total of four affinity selections and four 20-cycle PCR amplifications.
To demonstrate multi-round, closed-loop affinity selection and amplification, washing waste from four rounds of selection and the strongly bound thermally eluted ssDNA from the fourth round were collected, amplified (16 cycles PCR) and imaged with gel electrophoresis (
The enriched aptamer pool collected from the thermal elution of the fourth selection round was further investigated for its affinity and specificity using a fluorescence binding assay. Six different concentrations (100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM and 3.125 nM) of fluorescently tagged oligomers (enriched aptamer pool or randomized library) were incubated with IgE-functionalized beads in triplicate 100 μL volumes. After incubating the oligomers with the beads for 30 minutes, the beads were washed and the bound oligomers were thermally eluted (95° C.). The eluted oligomers were collected and their relative amounts were determined with a Wallac EnVision Multilabel Reader fluorescent spectrometer.
When the enriched pool was incubated with IgE-immobilized beads, washed, and bound oligomers were thermally eluted and measured, the fluorescent intensity rapidly increased until reaching an asymptote (
When the fluorescently tagged randomized library used to initiate aptamer selection was incubated with IgE-immobilized beads representing the amount of targetbound oligomers, increased without reaching an asymptote (
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 of International Patent Application No. PCT/US2015/022044, filed Mar. 23, 2015, which claims priority from U.S. Provisional Application No. 61/968,919, filed Mar. 21, 2014; Provisional Application No. 62/068,554, filed Oct. 24, 2014; and Provisional Application No. 62/109,596, filed Jan. 29, 2015, each of which is incorporated by reference herein in its entirety. The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Nov. 17, 2016, is named 070050_5798_SL.txt and is 1,696 bytes in size.
This invention was made with government support under CBET-0854030, awarded by the National Science Foundation; 8R21GM104204, 5U19 AI067773, RR025816-02, and CA 14 7925-01 awarded by the National Institute of Health. The government has certain rights in this invention.
| Number | Date | Country | |
|---|---|---|---|
| 61968919 | Mar 2014 | US | |
| 62068554 | Oct 2014 | US | |
| 62109596 | Jan 2015 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2015/022044 | Mar 2015 | US |
| Child | 15269494 | US |
