WO2014078521A1 - Isolation and enrichment of nucleic acids on microchip - Google Patents

Abstract

Techniques for isolating target molecules using small molecules or cells are disclosed. The techniques can be used for isolating nucleic acids such as DNA. The target molecules are introduced into a selection microchamber and bond to a functional molecule located therein. The functional molecule can be, for example, cells or functional molecules immobilized on microbeads. The molecules that do not bind to the functional molecules can be removed. The target molecules can then be removed from the functional molecules, for example, by introduction of small molecules or by lysis caused when an electric field is applied to the microdevice.

Description

ISOLATION AND ENRICHMENT OF NUCLEIC ACIDS ON MICROCHIP

CROSS REFERENCES TO RELATED APPLICATIONS

This application claims priority to United States Provisional

Application No. 61/726,322, filed November 14, 2012; and Provisional Application No. 61/730,363, filed on November 27, 2012.. The disclosure of each of these applications is incorporated herein in its entirety.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

This invention was made with government support under CBET-

0854030, awarded by the National Science Foundation; RR025816-02 and

CA 147925-01, both awarded by the National Institutes of Health. The government has certain rights in this invention. BACKGROUND

Chemical amplification of nucleic acids can be realized with the polymerase chain reaction (PCR), in which a DN A 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 prior to analysis can be important for improving the resolution of detection results. 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, e.g., 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-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 a genotyping platform to offer improved accuracy, ability to multiplex, and increased throughput. SUMMARY

The disclosed subject matter provides techniques for isolation, selection, and amplification of target molecules.

In certain embodiments, methods for isolating a target using small molecules using a microchamber are provided. The microchamber can be formed as part of a MEMS-based microdevice, and can include an immobilized functional molecule that binds with the target, such that the target binds with the immobilized functional molecule in the microchamber. A sample including the target and non- target molecules can be introduced into the microchamber. Molecules not bound with the functional molecule can be removed. A solution including a small molecule that binds with the target can be introduced.

In accordance with exemplary embodiments of the disclosed subject matter, the target can be a nucleic acid such as DN A. The functional molecule can be, for example, a capture sequence. Molecules not bound to the functional molecule can be removed by rinsing the microchamber with a buffer solution. In accordance with embodiments of the disclosed subject matter, the small molecule can be glucose- boronic acid complex or deoxycholic acid. The target can be collected at an outlet of the microchamber. In accordance with other embodiments, the target can be transported to an amplification chamber.

In accordance with another aspect, methods for isolating a target using cells are provided. An example method includes providing a microdevice comprising a microchamber including a plurality of cells that bind with a target, introducing a first sample including the target and non-target molecules into the microchamber, removing molecules not bound to the cells, and applying an electric field to the microchamber. The cells can be, for example, MCF-7 cells. The target can be a nucleic acid such as UNA.

In accordance with a further aspect, a microdevice for isolating a target using cells is provided. The microdevice can include a selection microchamber including a plurality of cells and a first and second electrode for applying an electrode to the microdevice. The microdevice can further include a weir structure for retaining the plurality of cells in the selection microchamber.

In some embodiments, the microdevice further includes an

amplification microchamber in fluid connection with the selection microchamber. A microchannel filled with gel can connect the amplification microchamber to the selection microchamber. The microchannel can be filled with agarose gel. The microdevice can further include one or more processors coupled to the first and second electrode. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a flowchart describing an exemplary embodiment of a method for isolating a target using small molecules in accordance with the disclosed subject matter.

Figure 2 is a view of a microchamber that can be used for isolating a target using small molecules in accordance with one embodiment of the disclosed subject matter.

Figure 3 illustrates an exemplary embodiment of a method for isolating a target using small molecules in accordance with the disclosed subject matter.

Figure 4 depicts a microdevice that can be used for isolating a target using small molecules in accordance with one embodiment of the disclosed subject matter.

Figure 5 illustrates methods for maintaining the pH during electrophoresis in accordance with embodiments of the disclosed subject matter.

Figure 6 is a flowchart describing an exemplary embodiment of a method for isolating a target using cells in accordance with the disclosed subject matter.

Figure 7a depicts a top view of a microdevice that can be used for isolating a target using cells in accordance with one embodiment of the disclosed subject matter. Figure 7b is a side view of a microchamber that can be used for isolating a target using cells in accordance with one embodiment of the disclosed subject matter.

Figure 8 illustrates an exemplary embodiment of a method for isolating a target using cells in accordance with the disclosed subject matter. Figure 9 illustrates a microfluidic device that transports target molecules using flow control in accordance with an exemplary embodiment of the disclosed subject matter.

Figure 10 illustrates a second microfluidic device that transports target molecules using flow control in accordance with an exemplary embodiment of the disclosed subject matter

Figure 11 depicts a top view of a microdevice that can be used for isolating a target in accordance with one embodiment of the disclosed subject matter.

Figure 12 is a bar graph depicting band intensities of gel electropherograms of amplified eluents during ssDNA isolation using DC A, human IgE protein, and MCF-7 cells in accordance with embodiments of the disclosed subject matter.

Figure 13 shows fluorescence measurements for determination of binding affinity of isolated DNA pool (10 pmole) to human IgE protein in accordance with embodiments of the disclosed subject matter.

Figure 14 is a line graph showing changes in fluorescence of DNA- hybridized beads after 20 cycles of PCR in the amplification chamber in accordance with embodiments of the disclosed subject matter.

Figure 15 illustrates methods for isolating targets using proteins, small molecules, and cells, respectively, in accordance with embodiments of the disclosed subject matter.

Figure 16a, b, and c are bar graphs depicting band intensity for gel electropherograms obtained during selection of ssDNA using IgE, glucose-boronic acid complex, and MCF-7 cells, respectively, in accordance with embodiments of the disclosed subject matter.

Figure 17 shows fluorescence measurements taken during isolation of target molecules using IgE, MCF-7 cells, and glucose-boronic acid complex in accordance with exemplary embodiments of the disclosed subject matter. Figure 17a is a graph depicting fluorescence intensity measurements at the center of the gel-filled channel as IgE-binding fluorescently labeled ssDNA strands migrated from the selection chamber to the amplification. Figure 17b shows fluorescence measurements of solution collected at the amplification chamber following different durations of electro-kinetic transfer of DNA strands that bound to MCF-7 cells. Figure 17c shows fluorescence measurements of IgE-binding ssDNA strands transported and captured onto reverse primers-immobilized on beads. Figure 17d shows gel electropherogram intensities of electrokinetically transported ssDNA strands selected against glucose- boronic acid complexes captured on reverse primer-immobilized microbeads. Figure 17f shows fluorescence intensity of ssDNA strands amplified on beads following different numbers of PCR cycles applied in the amplification chamber. Figures 17g and 17h show intensities for gel electropherograms for eluents collected during the selection process for IgE and glucose-boronic acid, respectively, following the amplified strands on beads electrokinetically transported back in the selection chamber. Figure 17i shows fluorescence intensity measurements of MCF-7 cells following amplified strands on beads electrokinetically transported back into the selection chamber.

Figure 18a, b, and c are bar graphs depicting band intensity in the gel images for human IgE protein, glucose-boronic acid complex, and MCF-7 cells, respectively.

Figure 19 shows fluorescence-based binding affinity measurements of strands in accordance with embodiments of the disclosed subject matter.

DETAILED DESCRIPTION

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 system and method for isolating targets using small molecules. An exemplary embodiment of the method for isolating a target using small molecules is illustrated in Figure 1. A first sample including the target and non-target molecules can be introduced into a chamber including an immobilized functional molecules that binds with the target (at 102). The target can be, for example, a nucleic acid such as DNA. The target DNA can be, for example, an aptamer.

The sample including the target can first be processed such that the target can be selectively captured by certain small molecules that specifically bind with the target. The sample can be a solution that includes the target (e.g., target DNA) along with non-target molecules (e.g., non-target DNA as part of an oligomer library).

With reference to Figure 2, an exemplary embodiment of the chamber is shown (in Figure 2a). The chamber 202 includes a plurality of immobilized functional molecules 204. The unctional molecules can be capture sequences. The capture sequences can be, for example, biotinylated capture strands. Capture sequences are complementary to the sequence at one end of the DNA library strands. The capture sequences can be short (e.g., have a length of about 19 nt). In accordance with embodiments of the disclosed subject matter, the functional molecules can be immobilized on microbeads. The microbeads can be, for example, polymer microbeads coated with streptavidin. With further reference to Figure 2, the immobilized functional molecules can be maintained in the chamber 202 by a weir structure 206.

The target can bind to the immobilized functional molecules during incubation. Non-target molecules can also bind to the immobilized functional molecules. However, some non-target molecules do not bind to the functional molecules. Molecules not bound with the functional molecules can be removed (at 104). For example, the chamber can be rinsed with a buffer solution such as a HEPES buffer to remove the unbound molecules.

With further reference to Figure 1 , a solution including a small molecule that binds with the target can be introduced (at 106). The small molecule can be, for example, glucose, steroid, vasopressin, amino acids, cholesterol, or the like. For example, in embodiments of the disclosed subject matter the small molecule can be glucose-boronic acid complex or deoxycholic acid. Target molecules (e.g., target DNA strands) can release from the functional molecules and bind to the small molecule. Non-target molecules do no release from the functional molecules. The target molecules can be collected at an outlet of the chamber.

A further exemplary embodiment of the method for isolating targets using small molecules is illustrated in Figure 3. Microbeads are washed with buffer (at 302). The microbeads can be streptavidin microbeads. The microbeads can then be functionalized with a capture sequence 304, resulting in ca ture-runctionalized beads (at 306). The capture sequence can be biotylinated capture strands. The functionalized microbeads can then be incubated with target DNA 308, resulting in hybridized microbeads (at 310). The sample 312 can then be introduced, and the target DNA is released from the functional molecules (at 314). The non-target DNA 316 is not released. The target DNA binds with the small molecules (at 318).

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, Figure 4 depicts a microdevice 400 including a selection microchamber 402. The selection chamber 402 can include a bead inlet 404 for introducing (and removing) the functionalized microbeads. The microchamber 402 can be positioned above a heater 406 and a temperature sensor 408 that can be used to increase the temperature in order to remove non-target molecules from the chamber, without increasing the temperature such that target molecules are removed. As shown in Figure 2, the microchamber 402 can include a weir structure. The microdevice can have a volume of 5 μL and the weir structure can have a depth of 40μm. The bead inlet 404 can also be used for insertion of an electrode, such as a platinum wire electrode, for purposes of electrophoresis.

The selection microchamber 402 can be connected to an amplification microchamber 410. The amplification microchamber can be used 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. After selection a first sample including the target DNA molecule can be introduced into the amplification microchamber, where the target DNA is hybridized onto the first primer which is suitable for amplifying the target DNA. A complementary DNA of the target DNA is produced in the amplification microchamber using the target DNA as a template. The target DNA is then separated from the complementary DNA. A second primer is hybridized onto the complementary DNA. The target DNA is then amplified using the complementary DNA as a template. In example embodiments, the transporting of the target from the selection chamber to the amplification chamber can be accomplished by

electrophoresis. As illustrated in Figure 4, a microchannel 412 connecting the selection chamber 402 and the amplification chamber 410 includes a section filled with a gel 414. The gel can be any commonly used gel suitable for electrophoresis, such as agarose gel. After release (e.g., thermal release, where the heat can be supplied by the microheater 406 beneath the selection chamber 402) of the target from the functional molecule immobilized on beads, the target is transported by

electrophoresis through the gel 414, the electric field being supplied by a voltage applied between me positive electrode 416 and negative electrode 418. The transportation of the released target can only occur when a suitable electric field is applied through the gel. Thus, such arrangement can provide effective isolation between the enrichment chamber and the selection chamber, and therefore allow independent operation in the selection chamber (e.g., washing, elution) without the risk of contaminating the enriched products in the enrichment chamber.

The application of an electric field can cause an increase in the pH levels in the microchambers. Therefore, a buffer can be introduced into the microchambers to maintain appropriate pH levels. With reference to Figure 5, exemplary embodiments of methods for maintaining appropriate pH levels are shown. In Figure 5a, a buffer can be introduced at supplementary inlet 502 while the target

DNA travels from selection chamber 504 to amplification chamber 506. As shown in Figure 5b, a buffer can be introduced to a different supplementary inlet 508 when the target DNA travels back from the amplification chamber 506 to the selection chamber 504 in systems using multiple rounds of isolation and amplification. The buffer can be, for example, a HEPES buffer. The functional molecule can be, for example, a small molecule, a cell, or a protein.

A second embodiment of the method for maintaining the pH in accordance with the disclosed subject matter is shown in Figures 5c and 5d. With reference to Figure 5c, a buffer can be introduced at supplementary inlet 502 while the target DNA travels from selection chamber 504 to amplification chamber 506. Simultaneously, solution containing the target DNA can be injected into the selection chamber 504 at inlet 510. A buffer can be introduced through supplementary inlet 508 when the target DNA travels back from the amplification chamber 506 to the selection chamber 504 in systems using multiple rounds of isolation and amplification. The functional molecule can be, for example, a small molecule, a cell, or a protein..

A further embodiment of the method for maintaining the pH in accordance with the disclosed subject matter is shown in Figures Se and Sf. With reference to Figure Se, a buffer can be introduced at supplementary inlet 502 while the target DNA travels from selection chamber 504 to amplification chamber 506. A buffer can be introduced directly into the selection chamber 504 when the target DNA travels back from the amplification chamber 506 to the selection chamber 504 in systems using multiple rounds of isolation and amplification. The functional molecule can be, for example, a small molecule, a cell, or a protein.

In accordance with another aspect, the presently disclosed subject matter provides a system and method for isolating targets using cells. An exemplary embodiment of the method for isolating a target using cells is illustrated in Figure 6. A microdevice having a microchamber including a plurality of cells that bind with a target is provided (at 602). The cells can be, for example, cancer cells, stem cells, immune cells, blood cells, neuron cells, kidney cells, or the like. In accordance with an exemplary embodiment of the disclosed subject matter, the cells can be MCF-7 cells.

An exemplary embodiment of a microdevice in accordance with the disclosed subject matter is shown in Figure 7a. The microdevice 700 can include a selection chamber 702 and an amplification chamber 704. As shown in Figure 7b, the selection microchamber 702 includes a plurality of cells 706 retained in the

microchamber 702 by a weir structure 708. A microchannel 710 filled with a gel 712 (such as an agarose gel) connects the selection chamber 702 and the amplification chamber 704. The microdevice can also include microheaters 714, temperature sensors 716, and bead inlets 718. The microdevice 700 can also include electrodes, such as platinum (Pt) wire electrodes, at the bead inlets 718. The electrodes can be coupled to one or more processors including one or more electrical circuits to apply an electrical field to the microdevice 700.

With further reference to Figure 6, a first sample including the target and non-target molecules can be introduced into the microchamber (at 604). The target can be a nucleic acid such as, for example, DNA. The DNA can be an aptamer. The sample can also include non-target molecules such as, for example, non-target

DNA. With reference to Figure 8, the sample including target 802 and non- target molecules 804 can be incubated with the cells 806 (at 808). The target 802 binds to the cells 808, while the non-target molecules 804 do not bind to the cells 806. With further reference to Figure 6, molecules not bound to the cells can be removed (at 606). The molecules can be removed by washing with a buffer as shown, for example, in Figure 8 (at 810). An electric field can then be applied to the

microchamber (at 608). Lysis caused by generation of hydroxide ions during electrolysis causes the target to elute from the cells, as shown in Figure 8 (at 812).

In accordance with a further aspect, transportation of targets from the selection chamber to the amplification chamber (or vice versa) can be accomplished using flow control. Flow control can be provided can be controlled by a regulating channel.

With reference to Figure 9, an example microfluidic selection chip 900 is shown. The selection chip 900 includes a selection chamber 902 . Valves 904-916 can be closed by default, while a vacuum can be constantly applied to waste reservoir 918. Microbeads 920 can be introduced into the selection chamber 902 by opening valves 910 and 914, as vacuum-driven fluid flow is initiated from the microbead to the waste reservoirs. As the buffer enters the waste reservoir 918, the beads can be retained in the selection chamber 902 by the weir structure 922. A randomized library of ssDNA oligomers 924 can be introduced into the selection chamber 902 by opening valves 904, 908, and 914 to initiate a vacuum driven fluid flow. After an amount of fluid flow time which can be determined from the flow rate obtained from calibration, the flow can be stopped and the library can be incubated in the chamber at an appropriate temperature, allowing some of the ssDNA oligomers to bind to and be captured by the immobilized functional molecules. Unbound ssDNA strands remain in the solution and can be removed by rinsing, which can be initiated by opening valves 906, 908, and 914. To release captured ssDNA strands from the functional molecules, the chamber temperature can be changed to an appropriate value, by introducing a solution including small molecules, or by applying an electric field across the selection chip. Elution of the released ssDNA strands can be performed by leaving valves 906, 908, and 914 open.

An exemplary embodiment of a chip using flow control including a selection chamber 1002 and an amplification chamber 1004 is shown in Figure 10. The system can be vacuum driven from a common waste reservoir 1006, with flow control provided by pneumatic microvalves, all of which can be closed by default during operation. To operate the chip shown in Figure 10, in the initial SELEX round, the ssDNA library can be introduced into the selection chamber 1002, where the ssDNA can be captured by the bead-bound functional molecules 1010, purified, and thermally released. By opening valves 1016, 1018, 1026, 1028, 1032, and 1038, the released ssDNA can be transferred out of the selection chamber 1002 and mixed with the PCR mix. The ssDNA and PCR mix mixture then enters the amplification chamber 1004 and the ssDNA strands can be captured by immobilized DNA primers 1012 in the amplification chamber 1004. In this manner, timing of the fluid flow is not required while transferring targeted ssDNA between the selection and

amplification device elements. Hence, on-off flow control can be sufficient. The micro beads, while in general reusable for the next SELEX round, can be replaced by fresh beads by opening valves 1020 and 1022 to simplify temperature control requirements. PCR cycles can then be carried out, followed by removal of the used PCR mix and replacement of the amplification buffer with the selection buffer (from selection buffer 1044). The amplified ssDNA strands can be released by heating the amplification chamber, e.g., to about 95° C, and then transferred into the selection chamber by opening valves 1030, 1042, 1018, and 1024. The amplified ssDNA will be captured by the immobilized functional molecules in the selection chamber 1002. The primer beads in the amplification chamber can be replenished by opening valves 1034 and 1036. The next SELEX round can begin by selection of the amplified ssDNA again. Some of the amplified ssDNA can be driven into the collection well, and retrieved by pipetting for sequencing using standard procedures to determine whether to terminate the SELEX process. SELEX can be terminated when the strands specifically bind to their targets with high affinities.

Example 1

The microdevice used in this Example was prepared by spin-coating SU-8 photoresist layers on a silicon wafer. Each photoresist layer was baked on a hotplate, exposed to ultraviolet light through a photomask, and developed to realize a mold. A prepolymer of polydimethylsiloxane (PDMS) (Sylgard 184, Dow Corning) was then poured onto the SU-8 mold and baked at 75° C for 1 hour on a hotplate. A cured PDMS layer was peeled off and access holes for inlets and outlets were made. Meanwhile, chrome and gold layers were deposited on a clean glass substrate and patterned to form resistive heaters and temperature sensors using photolithography. The heater/sensor metal layer was passivated with silicon dioxide (thickness: 1 μm) using plasma-enhanced chemical vapor deposition, he PDMS layer was then bonded to the glass substrate following oxygen plasma treatment. Plastic tubes (Tygon, Cole Parmer) were connected to inlets and outlets for sample handling. Molten 3% agarose gel was injected to fill the microchannel through the gel inlet and solidi ied at room temperature for 15 minutes. A schematic depiction of the microdevice used in this Example is illustrated in Figure 11.

A library of fluorescently labeled 87-mer ssDNA having random sequences (5'-GCC TGT TGT GAG CCT CCT GTC GAA -N40- TTG AGC GTT TAT TCT TGT CTC CC-3'), forward (5'-GCC TGT TGT GAG CCT CCT GTC GAA-3') and reverse (5'-GGG AGA CAA GAA TAA ACG CTC AA-3') primers, and captured sequences were purchased from Integrated DNA Technologies. The mixtures of ssDNA for the experiments were prepared by mixing 1 μL of 100 μΜ random library in 99 of phosphate-buffered saline (PBS) buffer. Solutions of deoxycholic acid (DCA),(Sigma-Aldrich), human myeloma IgE (Athens Research& Technology), and MCF-7 breast cancer cells (American Type Culture Collection) were prepared by dissolving them in separate buffers. Streptavidin microbeads (Thermo Scientific) and NHS-activated (GE Healthcare) were functionalized with capture sequences and IgE, respectively, and stored in PBS buffer. Tris-boric acid (TB) buffer (89mM Tris 89 mM boric acid, and 100 mM NaCl) was used as an electrolyte for DNA electrophoresis on-ship. A PCR mixture was prepared.

Approximately 30% of the volume of the isolation chamber was filled with DNA- or target-functional ized microbeads or cells through the inlet The beads and cells were then washed with PBS buffer (20 μL/min) for S minutes using a syringe pump. For isolation of DCA-binding ssDNA, a random DNA mixture a random DNA mixture is introduced into the isolation chamber and washed with PBS buffer (10 μL/min). Then, a solution containing DCA was introduced into the isolation chamber (10 μL/min) and incubated with the beads for 10 minutes. The strands bound to DCA were collected at the outlet of the chamber in separate tubs (-33 μL). For isolation of ssDNA binding to IgE or MCF-7 cells, a random mixture of ssDNA was introduced into the isolation chamber (10 μL/min) filled with IgE- functionalized beads or the cells. Weakly bound strands to the target were washed with PBS buffer introduced to the chamber (40 μL/min) while the waste solutions were collected in separate samples at the outlet (-33 μL). Strongly bound DNA strands to targets are eluted by heating the chamber at 59° C for 5 minutes while the eluents are collected from the outlet in separate tubes (-33 μL).

For device characterization of DNA amplification, fluorescently labeled ssDN A and reversed-primer coated beads in TB buffer were filled in the isolation and amplification chambers, respectively. Platinum electrodes were inserted into the Pt inlets in each chamber and a SO V of potential difference was applied for 25 minutes. The DNA strands were then electrophoretically transported through the gel-filled channel and hybridized onto the reversed primers coated beads in the amplification chamber. PCR mixture was introduced into the amplification chamber using a micropipette. PCR amplification of DNA on the bead surfaces was induced by thermocycling using the integrated resistive heater and temperature sensor on-chip.

The collected eluents containing ssDNA were amplified using a conventional thermocycler and visualized with slab-gel electrophoresis. The relative amounts of residual DNA in the eluents were evaluated by comparing the band intensities for each sample in gel images using the ImageJ software (National

Institutes of Health freeware). A fluorescence microscope and spectrometer are used to measure the fluorescence intensities of microbeads.

Using a syringe, microbeads in PBS buffer were slowly introduced to fill an isolation chamber. The depth of the weir was 40 μm. Therefore, most of the microbeads injected (diameter: 45-165 μm) were trapped in the chamber while buffer could flow to exit through the chamber outlet. Figure 10(a) is a micrograph showing that beads can be densely loaded in the chamber. Similarly, MCF-7 cells (mean diameter: 15 μηι) in a buffer solution were injected into an isolation chamber using a micropipette and trapped by the weir structure (depth: 10 μm) having micropillars. The cells were captured by the structure while buffer solution passed through the spaces between the micropillars. Experimental results show that the amount of the beads and cells captured in the chamber can be controlled by varying the sample loading times.

With reference to Figure 12, gel electrograms were collected during the isolation experiments with different targets including DCA, human IgE proteins, and MCF-7 cells. The bands in lanes W and E represent amplified samples of eluents collected during ash and elution, respectively, while the numbers after the

abbreviations indicated the order in which eluents were collected. In the isolation experiment using DC A, band intensity generally decreases as washing continued, indicating that DNA strands did not bind to capture sequences were removed from the beads. The increase in the band intensity in lane E represents the eluted ssDNA strands from the beads that strongly bound to DCA. Similarly, weakly bound DNA strands to targets (i.e., human IgE and MCF-7 cells) being removed with buffer are represented by decreasing band intensities while strands strongly bound to the targets are indicated by the bands in lanes E. Band intensities shown in Figure 12 were measured using ImageJ.

Ten-pmole of fluorescently labeled strands of random DNA library and the isolated ssDNA pool using human IgE were mixed separately in 100 μL of PBS buffers. IgE-coated beads (10 μL) were incubated with the DNA solutions and washed with pure PBS buffer by centrifugation. Remaining strands on the bead surfaces were thermally eluted and collected in buffer. The fluorescence intensities of the eluted strands in buffer were measured using a fluorescence spectrometer.

Measurement results illustrated in Figure 13 show that fluorescence intensity of the strands from eluted IgE-beads mat were incubated with the isolated DNA pool was notably stronger. This indicates that the isolated DNA strands strongly bound to IgE withstanding stringent wash. On the other hand, the random strands weakly bound and were released from IgE with wash represented by weak fluorescence intensity of the eluted DNA. Therefore, the binding measurements show that target-binding nucleic acids isolated using the device bind significantly strongly to targets than random strands.

One-pmole of fluorescently labeled ssDN A and reverse-primer coated beads in TB buffers were filled in the isolation and amplification chambers, respectively, he DNA strands were electrophoretically transported through the gel- filled channel and captured onto the reverse primers onto the beads in the

amplification chamber. Twenty-cycles of PCR were done in the chamber using the integrated resistive heater and temperature sensor. During the experiment, changes in fluorescence intensity of the beads were monitored using a fluorescence microscopy. The increase in fluorescence intensity of the beads after the electrophoresis shows that

DNA strands were electrophoretically transported and captured onto the beads in the amplification chamber. If the DNA strands were captured onto the bead surfaces by nonspecific adsorption rather than by hybridization to the reverse primers on the beads, DNA amplification would not occur that could result in an unchanged fluorescence intensity of the beads. However, after 20 cycles of PCR amplification fluorescence of the beads increased approximately 6 times, as shown in Figure 14. This shows that the DNA strands were initially hybridized onto the reverse-primers and effectively amplified on the bead surfaces via thermocycling.

Example 2

In this Example, multiple rounds of selection and amplification were carried out for aptamer isolation against various biological targets. Two chips were fabricated using photolithography in polydimethylsiloxane (PDMS) microfluidic layers bonded on glass substrates. Schematics of Chip I and Chip II are shown in Figures 15(a) and 15(b), respectively. Each chip consisted of two microchambers (volume: 5 μL) for selection and PCR amplification of target-binding nucleic acids, respectively. A resistive heater and a temperature sensor (Cr/Au: 5/10 0 nm) were integrated in the chambers for the temperate control during the selection and amplification processes. A microchannel filled with agarose gel (length: 7 mm, width: 1.4 mm, height: 300 μm) connects the two chambers to prevent cross-contamination while allowing transport of DNA strands with an electric field applied between the chambers. The channel sections on either side (length: 1.5 mm, width: 1 mm, height: 40 μm) of the gel-filled section kept the gel in place and separated it from the heater to prevent thermally induced damage. Platinum (Pt)-wire electrodes for electrokinetic transport of nucleic acids between the chambers were inserted into the bead inlets, he additional channels between the chambers and the gel-filled channel prevented the thermal damage of the gel from the heater chamber. Buffers and sample solutions were injected through inlets and eluents were collected at the outlets of the chips.

Each chamber had a weir structure for immobilizing microbeads (Chip I; weir height: 40 μm) or cells (Chip II; weir height: -8 μm) during operations.

The randomized ssDNA library and primer strands were purchased from Integrated DNA Technologies. Each strand of the DNA library used for experiments for IgE protein and MCF-7 cells was labeled with fluorescein

(Excitation/Emission: 495 nm 520 nm) and contained a random region of 40 bases flanked by 24- and 23 -base primer regions for the PCR amplification (5'- GCCTGTTGTGAGCCTCCTGTC GAA-40N-TTGAGCG TTTATTCTTGTCTCCC- 3'). The DNA library used for selection and amplification of glucose-boronic acid complex contains a random region of 30 bases flanked by 18- and 24-base primer regions (5'-GGAGGCTCTCGGGACGAC-30N-

GTCGTCCCGATGCTGCAATCGTA A). NHS-activated microbeads (diameter: 45 - 165 μτη, mean diameter: 90 |im) and human IgE protein were purchased from GE Healthcare Life Sciences and Athens Research, respectively. Chemicals to prepare buffers for protein-and small molecule-selection and amplification (44.5 mM Tris base, 44.5 mM boric acid, 50 mM NaCl, pH 8.5), and for cell-selection and amplification (14 mM HEPES, 14 mM NaOAc, 50 mM MgCl₂, pH 7.5) were purchased from Sigma-Aldrich, Inc. Elution buffers included 0.2 M NaOH in the selection and amplification buffer used for selection and amplification of each target. A microplate reader (Wallack Victor2, PerkinElmer) and a fluorescence microscope (LSM 510, Zeiss) were used for fluorescence measurements of solutions, microbeads, or cells. A power supply (E3631 A, Agilent Technologies) and a multimeter (34410A, Agilent Technologies) controlled by the LabVIEW software (National Instruments Corp.) running on a computer manipulated the temperature in the chip for PCR amplification on-chip. A conventional thermocycler (Eppendorf Mastercycler

Gradient, Eppendorf) was used to amplify DNA strands for the gel electropherogram and large scale PCR.

To prepare IgE functionalized beads, NHS activated beads (200 μL) were washed 3 times in a column with selection and amplification buffer. The beads were then incubated with 5.7 μM IgE (35 μL) at room temperature for 5 hr on a shaker and were washed 3 times with selection and amplification buffer. To block the NHS binding sites not occupied by IgE, the beads were incubated with 0.1M Tris-HCl buffer at room temperature for 1 hr followed by buffer wash. The IgE functionalized beads was stored in selection and amplification buffer in a refrigerator (4°C).

A standard fluorescence binding assay was used to measure binding affinity of DNA strands to IgE. Fluorescently labeled strands with various

concentrations (0-100 nM) were prepared in selection and amplification buffer (total volume: 100 μL). IgE-functionalized beads in tubes (3□ 104/tube) were washed with selection and amplification buffer and incubated with the DNA strands at room temperature for 2 h. Following the incubation, the beads were washed with selection and amplification buffer three times to remove unbound strands. The tubes containing beads were heated at 95°C for 10 min using a thermocycler. Eluted strands from the beads were collected and their amounts were measured using a plate reader. The fluorescence intensity data were analyzed to estimate the dissociation constant by nonlinear curve fitting using the software Origin (Origin Lab Corp.).

For selection of protein-binding nucleic acids, microbeads

functionalized with a target protein were incubated with a randomized ssDNA library in the selection chamber. As shown in Figure 15(c), weak- and non-binding strands were removed with buffer wash, while strong binders were eluted from the target- beads by heating the chamber or incubating with buffer containing sodium hydroxide ( aOH). For the selection of small molecule-binding nucleic acids, random DNA strands are captured by short ssDNA immobilized on beads in the selection chamber. Upon the introduction of a solution containing target molecule into the chamber, target-binding strands were released from the beads, as illustrated in Figure 15(d). For selection of cell-binding nucleic acids, a random ssDNA library is incubated with target cells in the selection chamber. While strands that do not strongly bind to the cells are removed, strong-binders are eluted from the cells via cell lysis due to hydroxide ions (OH^") generated during the electrolysis in the chamber, as illustrated in Figure 15(e). The target-binding strands selected in the selection chamber were electrokinetically transferred into the amplification chamber with an electric field applied on the chips, as shown in Figure 15(f). As the strands entered the

amplification chamber, they were captured onto reverse primers immobilized on the beads retained in the chamber. The captured strands were then amplified on the bead surfaces via PCR and released from the beads using NaOH mixed in a buffer, as shown in Figure 15(g). The multiple copies of target-binding strands were then electrokinetically transported back into the selection chamber for additional rounds of selection and amplification. The selection, transport, and amplification can be repeated on a chip in a continuous fashion for enrichments of target-binding nucleic acids. A syringe infusion pump was used to introduce buffers and sample solutions into the chip. The temperature in a chamber was manipulated by a computer with a PID controller connected to a multimeter and a power supply ,as shown in Figure 15(h). An electric field for electrokinetic DNA transport was generated on the chip using platinum (Pt)-wire electrodes connected to a power supply.

Human IgE antibody, glucose-boronic acid complex, and MCF-7 cancer cells were used as protein, small molecule, and cell targets due to their clinical significance. For selection experiments using IgE and glucose-boronic acid complexes, approximately 50% of the selection chamber volume was filled with the functionalized beads. To investigate the selection of target-specific nucleic acids, control experiments were repeated using NHS beads not functionalized with IgE. Approximately 5,000 cells trapped in the selection chamber were used during selection of nucleic acids for MCF-7 cells. For verification of selection of target- binding strands in the chip, the experiment as repeated without the presence of the target cells as a control. Such a control was not necessary for the glucose-boronic acid complex because the strands released from the beads were a result of the binding interactions between the target and ssDNA. To demonstrate elimination of nonspecific binders, the counter selection process using a counter target (boronic acid) was included within the selection experiment for glucose-boronic acid target.

To evaluate the selection process, gel images were obtained using off- ship amplified eluents collected during the selection experiments for each target More than three repeated selection experiments were performed for each target. A bar graph depicting the relative band intensity for each target is shown in Figure 16.

Since the brightness of bands in a gel image represents the amount of ssDN A strands in the eluent loaded in the lane, the progress of the selection process can be investigated by comparing the band intensities. Bands in lanes W, E, and C represent amplified samples of eluent collected during washing, elution, and counter selection, respectively, while the numbers after the abbreviations of each process represent the order in which eluent samples were collected. For example, "1" in Wl means the first eluent sample collected during washing.

As shown in Figure 16(a), the band intensity associated with selection of IgE-binding nucleic acids decreased from lanes Wl to W10, indicating that ssDNA strands having low affinities to IgE were removed from the beads as the buffer wash continued. The increased band intensity in the elution lane suggests that ssDNA that strongly bind to IgE were released from the beads when the chamber was exposed to the elution buffer. For the control, the band intensity also gradually decreased from lanes Wl to W10. However, the absence of a visible band in lane E suggests that

DNA strands were almost completely removed from the beads during buffer was and more importantly the strands collected using IgE-functionalized beads were selected due to the binding interaction with IgE and not by non-specific adsorption to the chamber or bead surfaces.

With reference to Figure 16(b), the band intensity associated with selection of strands that bind to glucose-boronic acid complexes decreases from lanes Wl to W12, indicating that strands that were not captured onto the beads

functionalized with capture strands were washed with buffer. The strands that bind to boronic acids (counter target) were removed from the beads as the band shown in lane C suggests. A strong band in lane E indicates that strands that specifically bind to glucose-boronic acid complexes were eluted from the capture strands on beads.

With reference to Figure 16(c), the band intensities associated with selection of strands that bind to MCF-7 targets shows mat strands that did not bind to the cells were removed with buffer wash, while target-binding strands were isolated during the selection process. Successful selection of target-binding strands was also verified by the low band intensity shown in lane £ for the control experiment

In Chip I, fluorescently labeled ssDNA strands that bind to IgE-beads were separated from the beads using elution buffer in the selection chamber. An electric field of 25 V/cm was then generated between two chambers via Pt-wire electrodes to electrokinetically transport and capture the strands in the amplification chamber which was filled with primer-coated beads. To monitor DNA transfer, fluorescence images were taken at the center of the gel-filled channel with a 1 minute time interval as the fluorescently labeled strands migrated from the selection to amplification chambers through the gel-filled channel. The fluorescence intensities of each image were measured and plotted over the duration of DNA transport. The graph plotted shows that majority of the ssDNA strands reached to the monitoring site, which is the midpoint between two chambers, within approximately 10 minutes as the maximum fluorescence signal was observed at the time. Therefore, to maximize transport of ssDNA strands between two chambers, approximately 25 V/cm of electric field was generated on-chip for DNA transport for 30 minutes. No significant damage was observed in a gel following an exposure to the electric field. The amount of DNA strands electrokinetically transported into the amplification chamber did not increase beyond 30 minutes of an electric field application , as shown in Figure 17(a).

Chip II was then used to investigate electrokinetic transfer of strands that bind to MCF-7 cells in which fluorescently labeled ssDNA strands bound to cells in a chamber were eluted and electrokinetically transferred into another chamber. Following different durations of the electric field application, fluorescence intensities of the buffer were measured in the chamber to which DNA strands were transferred. Approximately 25 minutes would be sufficient to electrokinetically transfer ssDNA that bind to MCF-7 cells between two chambers in Chip II , as shown in Figure 17(b). Following the transfer DNA strands eluted from IgE-runctionalized beads, the reverse primer-immobilized beads filled in the amplification chamber were washed with buffer and their fluorescence intensities were measured using a fluorescence microscope. The average fluorescence intensity of the beads following an electrokinetic transfer (positive) was significantly higher than the intensity of bare beads (control) indicating that the ssDNA strands were captured onto the reverse primers functionalized on the beads by hybridization following their migration into the amplification chamber, as shown in Figure 17(c). In separate experiments using Chip I, the capture of electrokinetically transported ssDNA strands selected against glucose-boronic acid complexes in the selection chamber onto reverse primer-coated microbeads in the amplification chamber was shown. To verify the capture of the strands, the beads were washed with buffer and captured strands were separated from the beads using elution buffer. The strong band shown in lane S suggests that strands were captured by reverse primers on beads during the electrokinetic transfer, as shown in Figure 17(d). In addition, electrokinetic transfer and capture of MCF-7 cell binding strands to the primer-coated microbeads in the amplification chamber of Chip II was also verified by the increase in the fluorescence intensity of the beads, as shown in Figure 17(e).

Following the capture of electrokinetically transported strands, the beads in the chamber were washed with buffer. Then the chamber was filled with buffer containing PCR reagents and thermocycling was induced using the resistive heater and temperature sensor integrated in the chamber. Fluorescently labeled forward primers were used for PCR so that fluorescence signals of the beads, which correspond to the generation of double-stranded DNA (dsDNA), could be measured using a fluorescence microscope . As illustrated in Figure 17(f), the fluorescence intensity curve showed that during the initial 10 PCR cycles the captured DNA strands were amplified exponentially on the bead surfaces doubling the strands each cycle. As the number of PCR cycles increased to from 10 to 20, reaction components such as reverse primers on the beads were being consumed and amplification slowed down. Thus, the fluorescence intensity increased more linearly. Beyond 25 PCR cycles, the fluorescence intensity did not further increase as most reverse primers on the bead surfaces were consumed and no more PCR products were being generated. The amplification chamber in a single chip can be used multiple times without generation of significant amount of strands that might remain in the chamber from previous amplification process.

The strands amplified on beads in the amplification chamber were separated from the dsDNA into ssDNA following incubation with elution buffer and were electrokinetically transferred to the selection chamber. The strands were then incubated with fresh IgE-functionalized beads or capture-immobilized beads in Chip I, or MCF-7 cells in Chip II loaded in the selection chamber and strands that did not bind to the target were removed with buffer wash. To evaluate the outcomes of the experiments, a gel image was obtained using the eluents collected during the buffer wash from Chip I while the fluorescence intensity of the cells was directly measured from Chip II.

The bands seen in lanes Wl-W10.as shown in Figure 17(g), and Wl- W12, as shown in Figure 17(h), indicate that the amplified strands on beads were successfully separated and transported back into the selection chamber in Chip I. Note that the bands seen in the gel images show that DNA strands were transferred electrokinetically in the selection chamber. In addition, the band intensity generally decreases as the weakly bound strands to the IgE-functionalized beads were

progressively removed with the buffer wash. A significant increase in the

fluorescence signal observed in the cells following an experiment indicates that strand separation and transport of amplified DNA strands could be also achieved in Chip II, as shown in Figure 17(i). These experimental results indicate that a single chip can be used for continuous rounds of selection and amplification for a given target.

The results obtained in experiments and theoretical analysis for the chip characterization demonstrate that processes involving multiple rounds including the selection, transport, amplification, and strand separation of target-binding strands could be achieved continuously in the chips. To confirm these observations, multiple rounds of selection and amplification were performed using a chip for each target without interruption between each process for verification. Eluents, however, were collected during removal of unbound strands on functionalized beads or target cells during the selection process while the eluents of the enriched final DNA pools for each target were also sampled at the end of a multi-round process. Using the eluents, gel images were obtained from which band intensities were compared to investigate overall enrichment processes of target-binding strands for each target. Chip I was used for the enrichment of target-binding strands for IgE proteins in which 3 rounds were performed continuously. One additional selection process using human Immunoglobulin G (IgG), to which the enriched DNA strands were not desired to bind (counter selection), was conducted before the final enriched

DNA pool was collected at the outlet of the selection chamber. Chip I was also used for the enrichment of target-binding strands for glucose-boronic acid complex binding in which 3 rounds were performed. Counter selection processes using boronic acids were added in the 2nd and 3rd rounds to maximize the selection of strands that bind to the target complexes but not to boronic acids (counter target). For the enrichment of MCF-7 cell-binding strands, Chip II was used and 3 rounds were performed continuously. Typical process time was approximately 15 hours.

Bar graphs depicting band intensity are shown in Figure 18. Gel images were obtained using eluents collected during the enrichment of IgE-binding strands. As indicated by the distinct band shown in the lane Wl for each round in Figure 18(a), all the necessary processes such as selection, transfer, and amplification were successfully carried out during the continuous experiment. In general, the band intensity decreased from Wl to W10 as non- or weak-binding strands were removed from bead surfaces with buffer wash in each round. During the 3rd round, relatively smaller amount of DNA was removed from the beads during wash as the weak band intensity shown in lane Wl . This could be because the binding affinity of the enriched pool increased and thus the individual strands in the pool bound more strongly to the targets. Target-binding strands collected following the multiple rounds and an additional counter selection were obtained at the end of the experiment as a strong band shown in lane E.

The gel image obtained for glucose-boronic acid complexes also indicates that a continuous on-chip selection and amplification process properly performed as distinct bands are shown in the lanes Wl in Figure 18(b). A relatively strong band in lane Wl 0 in the 1 st round could be due to the excess amount of randomized ssDNA strands introduced initially that were not captured by the capture strands on microbeads. Discernible bands shown in the lane C for 2nd and 3rd rounds indicate that strands that could bind to boronic acid molecules as well as strands did not bind to capture strands were removed from the selection chamber. Nevertheless, and intensities of lanes W10 are weaker than the ones of lanes Wl for later rounds (2nd and 3rd rounds). A band shown in the lane E suggests that the chip is capable of enriching strands that bind to glucose-boronic acid complexes.

Bands shown in lanes Wl in Figure 18(c) obtained for MCF-7 cell also show that the on-chip selection and amplification was carried out properly. Very weak bands are seen in lanes W10 indicating strands that did not bind strongly to the cells were effectively removed with buffer wash. Although its intensity is weak, the band shown in the lane E suggests that strands that bind to target cells were enriched using the chip.

The binding affinities of enriched DNA pools obtained following the continuous rounds on-chip against the targets was investigated. In addition, the enriched pools were cloned and the sequences of some of randomly picked strands were identified for binding affinity measurements. A standard fluorescence binding assay was used to test binding affinities of strands enriched against IgE proteins. Affinity measurements for strands that bind to glucose-boronic acid complex were performed using a method slightly modified from a gel electrophoresis-based measurement. Flow cytometry was used to measure the affinity against MCF-7 cells. The dissociation constants (KD) of target binding strands were estimated using a single site binding model relating fluorescence intensity (r) to DNA concentration ([DNA_f]) as

(l-r ) /r [DNAf]

Binding affinities of the DNA pool enriched against IgE protein to the target were measured and compared with that of random pools. As shown in Figure 19(a), the enriched pool shows significantly stronger signal intensities than the random pool indicating that the affinity of the ssDNA pool to the target considerably improved following the enrichment process using the device. The binding affinity of a strand (SIGE.S), an identified sequence in the enriched pool, to IgE protein target, was then measured. As shown in Figure 19(b), the strand also shows strong binding affinity to IgE as the fluorescence intensity rapidly increased at lower DNA concentrations and reached constant values at higher DNA concentrations. On the other hand, the strand did not bind to IgG protein (counter target) as the fluorescence intensity increases very slowly with the increased DNA concentration. The dissociation constant (KD) of the strand was measured to be approximately 10 nM, which is comparable with die existing IgE aptamers (KD = -10-33 nM). The computer generated secondary structure of the sequence shows that the strand forms a hairpin loop structure which could be responsible for its strong binding affinities to IgE protein. To assess the binding affinity of DNA strands enriched against glucose- boronic acid complexes, microbeads were functionalized with individual strands via capture strands. Then, different concentrations of target solutions were incubated with beads to allow binding reactions between the strands on beads and the target molecules. Strands released from beads following the incubation with different target concentrations were amplified via PCR for gel electropherogram. Band intensities measured from the gel images shown in Figure 19(c) show that the enriched DNA pool binds significantly stronger to the target molecule ( D = ~ 5 μΜ) than the random pool. Furthermore, dissociation constant of a sequence in the enriched pool (SGB.2) was ~2 uM (as shown in Figure 19(d)), which is in the range of values for aptamers binding to small molecules.

Flow cytometry measurements showed that the average fluorescence intensity of cells incubated with the fluorescently labeled enriched DNA pool was significantly stronger than the cells incubated with the random DNA library as indicated by the fluorescence intensity curve which is shifted to the right (i.e., higher intensity) when compared to the signal obtained from bare cells. Note that all cells possess some intrinsic level of autofluorescence, which is commonly caused by nicotinamide adenine dinucleotide (NADH), riboflavins, and flavin coenzymes in the cells, as shown in Figure 9(e). In addition, the affinity of an aptamer candidate for MCF-7 cells was KD = 20 nM (as shown in Figure 19(f)), which is within ranges for aptamers reported against other cancer cells.

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.

Claims

1. A method for isolating a target using small molecules, the method comprising:

(a) introducing a first sample including target and non-target molecules into a first microchamber including an immobilized functional molecule that binds with the target;

(b) removing molecules not bound with the functional molecule; and

(c) introducing a solution comprising a small molecule that binds with the target.

2. The method of claim 1 , wherein the target comprises a nucleic acid.

3. The method of claim 2, wherein the target comprises DN A.

4. The method of claim 1 , wherein the functional molecule comprises a capture sequence.

5. The method of claim 1 , wherein removing molecules not bound with the functional molecule comprises rinsing the microchamber with a buffer solution.

6. The method of claim 1 , wherein the small molecule comprises one of glucose- boronic acid complex and deoxycholic acid.

7. The method of claim 1 , further comprising collecting the target at an outlet of the microchamber.

8. The method of claim 1 , further comprising transporting the target to an amplification microchamber.

9. A method for isolating a target using cells, the method comprising:

(a) providing a microdevice comprising a microchamber including a plurality of cells that bind with a target;

(b) introducing a first sample including the target and non-target molecules into the microchamber;

(c) removing molecules not bound to the cells; and

(d) applying an electric field to the microchamber.

10. The method of claim 9, wherein the cells comprise MCF-7 cells.

1 1. The method of claim 9, wherein the target comprises a nucleic acid.

12. The method of claim 11 , wherein the target comprises DNA.

13. A microdevice for isolating a target using cells, the microdevice comprising: a selection microchamber comprising a plurality of cells; and

a first and second electrode for applying an electric field to the microdevice.

14. The microdevice of claim 13, further comprising a weir structure for retaining the plurality of cells in the selection microchamber.

15. The microdevice of claim 13, further comprising an amplification

microchamber in fluid connection with the selection microchamber.

16. The microdevice of claim 15, further comprising a microchannel connecting the amplification microchamber and the selection microchamber, the microchannel being filled with a gel.

17. The microdevice of claim 16, wherein the gel comprises an agarose gel.

18. The microdevice of claim 13, wherein the microdevice comprises one or more processors in coupled to the first and second electrode.