Integrated Microfluidic CustomArray™ Biochips for Gene Expression and Genotyping Analysis

Abstract

DNA microarray technology has become one of the most promising analytical tools in molecular biology. It has been widely used for studying mRNA levels and examining gene expression in biological samples. It is becoming a powerful tool in the arena of diagnostics and personalized medicine. In this chapter, we present a fully integrated and self-contained microfluidic biochip device that has been developed to automate the fluidic handling steps required to carry out microarray-based gene expression or genotyping analysis. The device consists of a semiconductor-based CustomArray™ chip with 12,000 features and a microfluidic cartridge. The CustomArray™ was manufactured using a semiconductor-based in situ synthesis technology. The oligonucleotides were synthesized on an array of electrodes on a semiconductor chip using phosphoramidite chemistry under electrochemical control. The microfluidic cartridge consists of microfluidic pumps, mixers, valves, fluid channels and reagent storage chambers. Microarray hybridization and subsequent fluidic handling and reactions (including a number of washing and labeling steps) were performed in this fully automated and miniature device before fluorescent image scanning of the microarray chip. Electrochemical micropumps were integrated in the cartridge to provide pumping of liquid solutions. A micromixing technique based on gas bubbling generated by electrochemical micropumps was developed. Low-cost check valves were implemented in the cartridge to prevent cross talk of the stored reagents. Gene expression study of the human leukemia cell line (K562) and genotyping detection and sequencing of influenza A subtypes have been demonstrated using this integrated biochip platform. For gene expression assays, the microfluidic CustomArray™ device detected sample RNAs with a concentration as low as 0.375 pM. Detection was quantitative over more than three orders of magnitude. Experiment also showed that chip-to-chip variability was low indicating that the integrated microfluidic devices eliminate manual fluidic handling steps that can be a significant source of variability in genomic analysis. The genotyping results showed that the device identified influenza A hemagglutinin and neuraminidase subtypes and sequenced portions of both genes, demonstrating the potential of integrated microfluidic and microarray technology for multiple virus detection. The device provides a cost-effective solution to eliminate labor-intensive and time-consuming fluidic handling steps and allows microarray-based DNA analysis in a rapid and automated fashion.