Polymerase chain reaction-capillary electrophoresis genetic analysis microdevice with in-line affinity capture sample injection.

An integrated polymerase chain reaction (PCR)-capillary electrophoresis (CE) microdevice with an efficient in-line affinity-based injector has been developed for genetic analysis. Double stranded DNA PCR amplicons generated in an integrated 250 nL PCR reactor are captured, purified, and preconcentrated by an oligonucleotide probe immobilized in an in situ polymerized gel matrix followed by thermal release and injection into the CE-separation channel. This in-column injector employs a photopolymerized oligonucleotide-modified acrylamide capture gel to eliminate band broadening and increase the injection efficiency to 100%. The on-chip generated PCR amplicons processed on this microdevice exhibit a 3-5 fold increase in signal intensities and improved resolution compared to our previous T-shaped injector. Multiplex analysis of 191-bp amplicons from Escherichia coli O157 and 256-bp amplicons from E. coli K12 is achieved with a 6-fold increase in resolution. These advances are exploited to successfully detect E. coli O157 in a 500-fold higher background of E. coli K12. This microdevice with in-line affinity capture gel injection provides an improved platform for low-volume, high sensitivity, fully integrated genetic analysis.

Integrated microfluidic bioprocessor for single-cell gene expression analysis.

An integrated microdevice is developed for the analysis of gene expression in single cells. The system captures a single cell, transcribes and amplifies the mRNA, and quantitatively analyzes the products of interest. The key components of the microdevice include integrated nanoliter metering pumps, a 200-nL RT-PCR reactor with a single-cell capture pad, and an affinity capture matrix for the purification and concentration of products that is coupled to a microfabricated capillary electrophoresis separation channel for product analysis. Efficient microchip integration of these processes enables the sensitive and quantitative examination of gene expression variation at the single-cell level. This microdevice is used to measure siRNA knockdown of the GAPDH gene in individual Jurkat cells. Single-cell measurements suggests the presence of 2 distinct populations of cells with moderate (approximately 50%) or complete (approximately 0%) silencing. This stochastic variation in gene expression and silencing within single cells is masked by conventional bulk measurements.

Integrated affinity capture, purification, and capillary electrophoresis microdevice for quantitative double-stranded DNA analysis.

A novel injection method is developed that utilizes a thermally switchable oligonucleotide affinity capture gel to mediate the concentration, purification, and injection of dsDNA for quantitative microchip capillary electrophoresis analysis. The affinity capture matrix consists of a 20 base acrydite modified oligonucleotide copolymerized into a 6% linear polyacrylamide gel that captures ssDNA or dsDNA analyte including PCR amplicons and synthetic oligonucleotides. Double stranded PCR amplicons with complementarity to the capture probe up to 81 bases from their 5' terminus are reproducibly captured via helix invasion. By integrating the oligo capture matrix directly with the CE separation channel, the electrophoretically mobilized target fragments are quantitatively captured and injected after thermal release for unbiased, efficient, and quantitative analysis. The capture process exhibits optimal efficiency at 44 degrees C and 100 V/cm with a 20 microM affinity capture probe (TM = 57.7 degrees C). A dsDNA titration assay with 20 bp fragments validated that dsDNA is captured at the same efficiency as ssDNA. Dilution studies with a duplex 20mer show that targets can be successfully captured and analyzed with a limit of detection of 1 pM from 250 nL of solution (approximately 150,000 fluorescent molecules). Simultaneous capture and injection of amplicons from E. coli K12 and M13mp18 using a mixture of two different capture probes demonstrates the feasibility of multiplex target capture. Unlike the traditional cross-injector, this method enables efficient capture and injection of dsDNA amplicons which will facilitate the quantitative analysis of products from integrated nanoliter-scale PCR reactors.

Multichannel reverse transcription-polymerase chain reaction microdevice for rapid gene expression and biomarker analysis.

A microdevice is developed for RNA analysis that integrates one-step reverse transcription and 30 cycles of PCR (RT-PCR) amplification with capillary electrophoresis (CE) separation and fluorescence detection of the amplicons. The four-layer glass-PDMS-glass-glass hybrid microdevice integrates microvalves, on-chip heaters and temperature sensors, nanoliter reaction chambers (380 nL), and 5-cm-long CE separation channels. The direct integration of these processes results in attomolar detection sensitivity (<11 template RNA molecules or approximately 0.1 cellular equiv) and rapid 45-min analysis, while minimizing sample waste and eliminating contamination. Size-based electrophoretic product analysis provides definitive amplicon-size verification and multiplex analysis. Multiplexed differential gene expression analysis is demonstrated on mdh and gyrB E. coli transcripts. RNA splice variant analysis of the RBBP8 gene is used to identify tumorigenic tissue. RT-PCR microdevice analysis of normal breast tissue RNA generates the expected 202-bp normal splice isoform; tumor breast tissue RNA samples generate a 151-bp amplicon signifying the presence of the tumorigenic splice variant. The ability to perform RNA transcript and splice variant biomarker analysis establishes our RT-PCR microdevice as a versatile gene expression platform.

Multichannel PCR-CE microdevice for genetic analysis.

We have developed a fully integrated multichannel polymerase chain reaction-capillary electrophoresis (PCR-CE) microdevice with nanoliter reactor volumes for highly parallel genetic analyses. Resistance temperature detectors and heaters made out of Ti/Pt are integrated on the microchip using a scalable radial design to provide precise temperature control of the four parallel PCR-CE reactor systems. Heating rates of >15 degrees C s(-1) and cooling rates of >10 degrees C s(-1) allow cycle times of 50 s and 30 complete PCR cycles in <27 min. PDMS membrane valves control and localize PCR reagents in the 380-nL reactors. By directly integrating PCR reactors with the CE separation system, efficient coupling of amplification with separation is achieved. The microdevice demonstrates good amplification uniformity and sensitivity down to 10 initial template copies in the 380-nL reactor (approximately 43 aM) with signal-to-noise ratio greater than 10. Parallel PCR-CE multiplex amplification and genetic analyses of four different samples with (1) both M13mp18 control template and E. coli K12 cells, (2) only M13mp18 template, (3) only E. coli K12 cells, and (4) negative control are completed in less than 30 min in a single run.

Microfluidic device for electric field-driven single-cell capture and activation.

A microchip that performs directed capture and chemical activation of surface-modified single cells has been developed. The cell capture system is comprised of interdigitated gold electrodes microfabricated on a glass substrate within PDMS channels. The cell surface is labeled with thiol functional groups using endogenous RGD receptors, and adhesion to exposed gold pads on the electrodes is directed by applying a driving electric potential. Multiple cell types can thus be sequentially and selectively captured on desired electrodes. Single-cell capture efficiency is optimized by varying the duration of field application. Maximum single-cell capture is attained for the 10-min trial, with 63 +/- 9% (n = 30) of the electrode pad rows having a single cell. In activation studies, single M1WT3 CHO cells loaded with the calcium-sensitive dye fluo-4 AM were captured; exposure to the muscarinic agonist carbachol increased the fluorescence to 220 +/- 74% (n = 79) of the original intensity. These results demonstrate the ability to direct the adhesion of selected living single cells on electrodes in a microfluidic device and to analyze their response to chemical stimuli.