Robin Hui Liu

High sensitivity PCR assay in plastic micro reactors

Small volume operation and rapid thermal cycling have been subjects of numerous reports in micro reactor chip development. Sensitivity aspects of the micro PCR reactor have not been studied in detail, however, despite the fact that detection of rare targets or trace genomic material from clinical and/or environmental samples has been a great challenge for microfluidic devices. In this study, a serpentine shaped thin (0.75 mm) polycarbonate plastic PCR micro reactor was designed, constructed, and tested for not only its rapid operation and efficiency, but also its detection sensitivity and specificity, in amplification of Escherichia coli (E. coli) K12-specific gene fragment. At a template concentration as low as 10 E. coli cells (equivalent to 50 fg genomic DNA), a K12-specific gene product (221 bp) was adequately amplified with a total of 30 cycles in 30 min. Sensitivity of the PCR micro reactor was demonstrated with its ability to amplify K12-specific gene from 10 cells in the presence of 2% blood. Specificity of the polycarbonate PCR micro reactor was also proven through multiplex PCR and/or amplification of different pathogen-specific genes. This is, to our knowledge, the first systematic study of assay sensitivity and specificity performed in plastic, disposable micro PCR devices.

Development of Plastic Microfluidic Devices for Sample Preparation

Microfluidics technology shows excellent potential for applications in biotechnology, chemical sensing, and drug delivery. The use of microfluidics results in cycle time reduction, reagent cost and labor intensity savings due to the benefits of miniaturization, and functional integration. As a result, miniature DNA sample preparation and analytical devices may potentially become part of a point-of-care systems for rapid medical diagnoses. Similar diagnostic devices will benefit veterinary and food safety applications. In this paper, we discuss design, fabrication, and testing of plastic microfluidic devices for on-chip genetic sample preparation. These fabrication methods are being used to produce components of a complete genetic sample preparation micro-system. The detailed discussion on the development of micro-PCR (polymerase chain reaction) devices and bio-channel hybridization arrays is given. We also describe a path to further individual component integration.

Acoustic micromixer for enhancement of DNA biochip systems

Microfluidics is emerging as one of the fastest growing segments of micro-electro-mechanical system (MEMS) technologies due to its potential applications in biotechnology, chemical microreactors, and drug discovery. Micromixing is one of the most challenging problems in microfluidic systems, since it is a diffusion-limited process and can be very inefficient. A micromixing device based on an acoustic microstreaming principle is developed to enhance micromixing. The micromixer uses air bubbles as actuators that can be set into vibration by a sound field. The vibration of the air bubbles generates steady circulatory flows, resulting in global convection flows and thus rapid mixing. The time to fully mix dyed solutions in a 50-μL shallow chamber using acoustic microstreaming is significantly reduced from hours (a pure diffusion-based mixing) to 6 s. We demonstrate the use of this micromixer to enhance the performance of conventional DNA microarray biochips that often suffer from lengthy hybridization and poor signal uniformity due to a diffusion-limited hybridization process. Experiments showed that the acoustic micromixer results in five-fold hybridization signal enhancement with significantly improved signal uniformity, as compared to conventional diffusion-based biochips. Acoustic microstreaming has many advantages over most existing micromixing techniques, including a simple apparatus, ease of implementation, low power consumption (~ 2 mW), and low cost.

Integrated Microfluidic CustomArray Device for Bacterial Genotyping and Identification

The ongoing threat of the potential use of biothreat agents (such as Bacillus anthracis) as a biochemical weapon emphasizes the need for a rapid, miniature, fully automated, and highly specific detection assay. An integrated and self-contained microfluidic device has been developed to rapidly detect B. anthracis and many other bacteria. The device consists of a semiconductor-based DNA microarray chip with 12,000 features and a microfluidic cartridge that automates the fluid handling steps required to carry out a genotyping assay for pathogen identification. This fully integrated and disposable device consists of low-cost microfluidic pumps, mixers, valves, fluid channels, reagent storage chambers, and DNA microarray silicon chip. Microarray hybridization and subsequent fluid handling and reactions were performed in this fully automated and miniature device before fluorescent image scanning of the microarray chip. The genotyping results showed that the device was able to identify and distinguish B. anthracis from the other members of the closely related Bacillus cereus group, demonstrating the potential of integrated microfluidic and microarray technology for highly specific pathogen detection. The device provides a cost-effective solution to eliminate labor-intensive and time-consuming fluid handling steps and allows the detection and identification of biological warfare agents in a rapid and automated fashion.

Development of integrated microfluidic system for genetic analysis

Biotechnology, in conjunction with semiconductor and microelectronics, would have a tremendous impact on new solutions in gene and drug discovery, point-of-care systems, pharmacogenomics, and environmental and food safety applications. A combination of microfabrication techniques and molecular biology procedures have the potential to produce powerful, inexpensive, and miniature analytical devices (e.g., microfluidic lab chips), aiding further development of genetic analysis. Microfluidics for biotechnology applications require development of inexpensive, high-volume fabrication techniques and reduction of biochemical assays to the chip format. We discuss design, fabrication, and testing of plastic microfluidic devices for on-chip genetic sample preparation and DNA microarray detection. Plastic microfabrication methods are being used to produce components of a complete microsystem for genetic analysis. A detailed discussion on the development of micromixers, microvalves, cell capture, micro-polymerase chain reaction (PCR) devices, and biochannel hybridization arrays is given. We also describe a path to further individual component integration.

Highly parallel integrated microfluidic biochannel arrays

Microfluidic biochannel arrays that integrates massively parallel microfluidic channels with Motorola glass-based microarray biochips have been successfully developed. We demonstrated that such a device allows DNA hybridization assays to be performed in a miniaturized and highly parallel fashion. The biochannel arrays are a new DNA diagnostics platform that has several desirable features such as single-use and cost-effective, highly parallel (up to 52 protocols in parallel), and processed rapidly. Additionally, only nanoliter-volumes (/spl sim/100 nL) of DNA sample/reagents are consumed per array.

Plastic In-Line Chaotic Micromixer for Biological Applications

A plastic 3D “L-shaped” serpentine micromixer is developed to enhance mixing of biological samples. Both numerical simulation and experiments show such a device has high mixing efficiency and low shear strain field.

FULLY INTEGRATED MICROFLUIDIC BIOCHIPS FOR DNA ANALYSIS

Microfluidics-based biochip devices are developed to perform DNA analysis from complex biological sample solutions. Microfluidic mixers, valves, pumps, channels, chambers, heaters, and DNA microarray sensor are integrated to perform magnetic bead-based rare cell capture, cell preconcentration and purification, cell lysis, polymerase chain reaction, DNA hybridization and electrochemical detection in a single, fully automated biochip device. No external pressure sources, mechanical pumps, or valves are necessary for fluid manipulation, thus eliminating sample contamination and simplifying device operation. Pathogenic bacteria detection and single-nucleotide polymorphism analysis directly from blood are demonstrated. The device with capability of on-chip sample preparation and DNA detection provides a cost-effective solution to direct sample-to-answer genetic analysis, and thus has potential impact in the fields of point-of-care genetic analysis and disease diagnosis.

Self-Contained, Fully Integrated Biochips for Sample Preparation, PCR Amplification and DNA Microarray Analysis

Rapid developments in back-end detection platforms (such as DNA microarrays, capillary electrophoresis, real-time polymerase chain reaction and mass spectroscopy) for genetic analysis have shifted the bottleneck to front-end sample preparation where the ‘real’ samples are used. In this chapter, we present a fully integrated biochip device that can perform on-chip sample preparation (including magnetic bead-based cell capture, cell preconcentration and purification and cell lysis) of complex biological sample solutions (such as whole blood), polymerase chain reaction, DNA hybridization and electrochemical detection. This fully automated and miniature device consists of microfluidic mixers, valves, pumps, channels, chambers, heaters and DNA microarray sensors. 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 ≈ mL 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.

The Use of Microelectronic-Based Techniques in Molecular Diagnostic Assays

Publisher Summary The convergence of molecular biology and high microelectronic technology has led to the development of integrated multifunctional genetic analytical systems, which have high sensitivity, specificity, speed, and cost-effectiveness. This chapter reviews the use of microelectronic-based techniques in molecular diagnostic assays with a focus on microfabrication, cell sorting, molecule preconcentration, sample preparation techniques, DNA/RNA amplification on chips, electronic-assisted hybridization assays, and examples of commercially available platforms. The ability of delivering electric and magnetic signals to the localized portions of the biochip permitted exploitation of fundamental dielectric and magnetic properties of biological molecules and cells. The interaction between an external electric field and molecules is used for their manipulation and concentration. Dielectrophoretic techniques allow for sorting, separation, and isolation of cells from different organisms and tissues. The ability to monitor electron transfer between the molecule and the external measurement system form the basic principles for electronic detection of DNA. An arsenal of microfabrication capabilities developed in the microelectronic industry are leveraged towards on-chip assay applications, where integration of conductive, heating, and magnetic elements has resulted in the development of micro-PCR devices, microarray DNA chips containing thermal gradients, and dielectrophoretic and magnetic devices for cell and molecule preconcentration. These developments allow for a design of localized sensors analyzing single cells or molecules immobilized in distinct locations on the chip. Further progression in the capability of these techniques will be achieved through the emerging field of nanotechnology.