Herein we present a lab-chip device for highly efficient and rapid detection of circulating tumor cells (CTCs) from whole blood samples. The device utilizes a microfabricated silicon microsieve with a densely packed pore array (10(5) pores per device) to rapidly separate tumor cells from whole blood, utilizing the size and deformability differences between the CTCs and normal blood cells. The whole process, including tumor cell capture, antibody staining, removal of unwanted contaminants and immunofluorescence imaging, was performed directly on the microsieve within an integrated microfluidic unit, interconnected to a peristaltic pump for fluid regulation and a fluorescence microscope for cell counting. The latter was equipped with a dedicated digital image processing program which was developed to automatically categorize the captured cells based on the immunofluorescence images. A high recovery rate of >80% was achieved with defined numbers of MCF-7 and HepG2 cancer cells spiked into human whole blood and filtered at a rapid flow rate of 1 mL min(-1). The device was further validated with blood drawn from various cancer patients (8 samples). The whole process, from sample input to result, was completed in 1.5 h. In addition, we have also successfully demonstrated on-microsieve fluorescence in situ hybridization for single cell molecular analysis. This simple method has great potential to supplant existing complex CTC detection schemes for cancer metastasis analysis.

A basic problem in gene synthesis is the acquisition of many short oligonucleotide sequences needed for the assembly of genes. Photolithographic methods for the massively parallel synthesis of high-density oligonucleotide arrays provides a potential source, once appropriate methods have been devised for their elution in forms suitable for enzyme-catalyzed assembly. Here, we describe a method based on the photolithographic synthesis of long (>60mers) single-stranded oligonucleotides, using a modified maskless array synthesizer. Once the covalent bond between the DNA and the glass surface is cleaved, the full-length oligonucleotides are selected and amplified using PCR. After cleavage of flanking primer sites, a population of unique, internal 40mer dsDNA sequences are released and are ready for use in biological applications. Subsequent gene assembly experiments using this DNA pool were performed and were successful in creating longer DNA fragments. This is the first report demonstrating the use of eluted chip oligonucleotides in biological applications such as PCR and assembly PCR.

Mutation and polymorphism detection is of increasing importance for a variety of medical applications, including identification of cancer biomarkers and genotyping for inherited genetic disorders. Among various mutation-screening technologies, enzyme mismatch cleavage (EMC) represents a great potential as an ideal scanning method for its simplicity and high efficiency, where the heteroduplex DNAs are recognized and cleaved into DNA fragments by mismatch-recognizing nucleases. Thereby, the enzymatic cleavage activities of the resolving nucleases play a critical role for the EMC sensitivity. In this study, we utilized the unique features of microfluidic capillary electrophoresis and de novo gene synthesis to explore the enzymatic properties of T7 endonuclease I and Surveyor nuclease for EMC. Homoduplex and HE DNAs with specific mismatches at desired positions were synthesized using PCR (polymerase chain reaction) gene synthesis. The effects of nonspecific cleavage, preference of mismatches, exonuclease activity, incubation time, and DNA loading capability were systematically examined. In addition, the utilization of a thermostable DNA ligase for real-time ligase mediation was investigated. Analysis of the experimental results has led to new insights into the enzymatic cleavage activities of T7 endonuclease I and Surveyor nuclease, and aided in optimizing EMC conditions, which enhance the sensitivity and efficiency in screening of unknown DNA variations.

In this report, an electrical detection scheme for the quantification of DNA using a nanogap sensor array is detailed. The prime objective is to develop a novel sensing procedure, based on the electronic transduction mechanism, which would mitigate the problems intrinsic to nanostructure-based biosensing devices. Design considerations of the sensor array take into account the feasibility of mass production in a cost-effective way by using standard silicon microfabrication technologies. The sensing mechanism relies on bridging the nanogap upon hybridization of the two termini of a target DNA with two different surface-bound capture probes, followed by a simple metallization step. About 2 orders of magnitude enhancement in conductance, as referred to a clean background (<1.0 pS) observed at a control sensor, was obtained in the presence of as little as 1.0 fM target DNA. This sensitivity is comparable to the best of electrochemical/electrical biosensors. A linear relationship between the conductance and the DNA concentration was obtained from 1.0 fM to 1.0 pM with an exceptional signal intensity of 2.1 x 10(4)% change per unit concentration. This change in conductivity is so large that it can unambiguously detect the concentration of DNA quantitatively and may obviate the need for target amplification used in current DNA tests. Moreover, the sensor array exhibited excellent single-base mismatch discrimination due to its unique vertically aligned nanostructure and the two-probe configuration.

Herein we present an integrated microfluidic device capable of performing two-step gene synthesis to assemble a pool of oligonucleotides into genes with the desired coding sequence. The device comprised of two polymerase chain reactions (PCRs), temperature-controlled hydrogel valves, electromagnetic micromixer, shuttle micromixer, volume meters, and magnetic beads based solid-phase PCR purification, fabricated using a fast prototyping method without lithography process. The fabricated device is combined with a miniaturized thermal cycler to perform gene synthesis. Oligonucleotides were first assembled into genes by polymerase chain assembly (PCA), and the full-length gene was amplified by a second PCR. The synthesized gene was further separated from the PCR reaction mixture by the solid-phase PCR purification. We have successfully used this device to synthesize a green fluorescent protein fragment (GFPuv) (760 bp), and obtained comparable synthesis yield and error rate with experiments conducted in a PCR tube within a commercial thermal cycler. The resulting error rate determined by DNA sequencing was 1 per 250 bp. To our knowledge, this is the first microfluidic device demonstrating integrated two-step gene synthesis.