Validation of a fully integrated microfluidic array device for influenza A subtype identification and sequencing.

Rapid detection and identification of influenza virus is becoming increasingly important in the face of concerns over an influenza pandemic. A fully integrated and self-contained microfluidic device has been developed to rapidly identify influenza A hemagglutinin and neuraminidase subtypes and sequence portions of both genes. The device consists of a DNA microarray with 12 000 features and a microfluidic cartridge that automates the fluidic handling steps required to carry out a genotyping assay for pathogen identification and sequencing. The fully integrated microfluidic device consists of microfluidic pumps, mixers, valves, fluid channels, reagent storage chambers, and DNA microarray silicon chip. Microarray hybridization and subsequent fluidic handling and reactions were performed in this fully automated and miniature device before fluorescent image scanning of the microarray chip. 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. 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 the detection and identification of influenza virus in a rapid and automated fashion.

Fully integrated miniature device for automated gene expression DNA microarray processing.

A DNA microarray with 12,000 features was integrated with a microfluidic cartridge to automate the fluidic handling steps required to carry out a gene expression study of the human leukemia cell line (K562). The fully integrated microfluidic device consists of microfluidic pumps/mixers, fluid channels, reagent chambers, and a DNA microarray silicon chip. 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 into the cartridge to provide pumping of liquid solutions. The device was completely self-contained: no external pressure sources, fluid storage, mechanical pumps, mixers, or valves were necessary for fluid manipulation, thus eliminating possible sample contamination and simplifying device operation. Fluidic experiments were performed to study the on-chip washing efficiency and uniformity. A single-color transcriptional analysis of K562 cells with a series of calibration controls (spiked-in controls) to characterize this new platform with regard to sensitivity, specificity, and dynamic range was performed. The device detected sample RNAs with a concentration as low as 0.375 pM. Experiment also showed that the performance of the integrated microfluidic device is comparable with the conventional hybridization chambers with manual operations, indicating that the on-chip fluidic handling (washing and reaction) is highly efficient and can be automated with no loss of performance. The device provides a cost-effective solution to eliminate labor-intensive and time-consuming fluidic handling steps in genomic analysis.

Integrated microfluidic biochips for DNA microarray analysis.

A fully integrated and self-contained microfluidic biochip device has been developed to automate the fluidic handling steps required to perform a gene expression study of the human leukemia cell line (K-562). The device consists of a DNA microarray semiconductor chip with 12,000 features and a microfluidic cartridge that 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. A single-color transcriptional analysis of K-562 cells with a series of calibration controls (spiked-in controls) was performed to characterize this new platform with regard to sensitivity, specificity and dynamic range. The device detected sample RNAs with a concentration as low as 0.375 pM. Detection was quantitative over more than 3 orders of magnitude. Experiments also demonstrated 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.

Self-contained, fully integrated biochip for sample preparation, polymerase chain reaction amplification, and DNA microarray detection.

A fully integrated biochip device that consists of microfluidic mixers, valves, pumps, channels, chambers, heaters, and DNA microarray sensors was developed to perform DNA analysis of complex biological sample solutions. Sample preparation (including magnetic bead-based cell capture, cell preconcentration and purification, and cell lysis), polymerase chain reaction, DNA hybridization, and electrochemical detection were performed in this fully automated and miniature device. Cavitation microstreaming was implemented to enhance target cell capture from whole blood samples using immunomagnetic beads and accelerate DNA hybridization reaction. Thermally actuated paraffin-based microvalves were developed to regulate flows. Electrochemical pumps and thermopneumatic pumps were integrated on the chip to provide pumping of liquid solutions. The device is completely self-contained: no external pressure sources, fluid storage, mechanical pumps, or valves are necessary for fluid manipulation, thus eliminating possible sample contamination and simplifying device operation. Pathogenic bacteria detection from approximately milliliters of whole blood samples and single-nucleotide polymorphism analysis directly from diluted blood were demonstrated. The device provides a cost-effective solution to direct sample-to-answer genetic analysis and thus has a potential impact in the fields of point-of-care genetic analysis, environmental testing, and biological warfare agent detection.

Hybridization enhancement using cavitation microstreaming.

Conventional DNA microarray hybridization relies on diffusion of target to surface-bound probes, and thus is a rate-limited process. In this paper, a micromixing technique based on cavitation microstreaming principle that was developed to accelerate hybridization process is explained. Fluidic experiments showed that air bubbles resting on a solid surface and set into vibration by a sound field generated steady circulatory flows, resulting in global convection flows and, thus, rapid mixing. The time to fully mix dyed solutions in a 50-microL chamber using cavitation microstreaming was significantly reduced from hours (a pure diffusion-based mixing) to 6 s. Cavitation microstreaming was implemented to enhance DNA hybridization in both fluorescence-detection-based and electrochemical-detection-based DNA microarray chips. The former showed that cavitation microstreaming results in up to 5-fold hybridization signal enhancement with significantly improved signal uniformity, as compared to the results obtained in conventional diffusion-based biochips for a given time (2 h). Hybridization kinetics study in the electrochemical detection-based chips showed that acoustic microstreaming results in up to 5-fold kinetics acceleration. Acoustic microstreaming has many advantages over most existing techniques used for hybridization enhancement, including a simple apparatus, ease of implementation, low power consumption (approximately 2 mW), and low cost.