A dynamic liquid-phase microextraction technique combined with gas chromatography/mass spectrometry (GC/MS) is described for the extraction of 10 chlorobenzenes from water samples into 1 microL of organic solvent by using a conventional microsyringe. The effects of extraction solvent, plunger movement pattern, sampling volume, number of samplings, and salt concentration on the extraction performance were investigated. Good repeatabilities of extraction were obtained, with the RSD values below 5.3% except for hexachlorobenzene (9.3%). By using a sampling volume of 6 microL and 15 samplings, detection limits were found to be between 0.02 and 0.05 microgram/L under GC/MS-selective ion monitoring mode.

Low-pressure, high-temperature thermal bonding of polymeric microfluidic devices and their applications for electrophoretic separation

Effective microchip extraction of deoxyribonucleic acid (DNA) from crude biological matrixes has been demonstrated using silica beads or hybrid phases composed of beads and sol-gel. However, the use of monolithic sol-gels alone for extraction of human genomic DNA has been more difficult to define. Here we describe, for the first time, the successful use of monolithic tetramethyl orthosilicate-based sol-gels for effective micro-solid-phase extraction (muSPE) of DNA in a glass microchip format. A functional monolithic silica phase with micrometer-scale pores in the silica matrix resulted from addition of poly(ethylene glycol), a poragen, to the precursor mixture. This allowed a monolithic sol-gel bed to be established in a microchip channel that provided large surface area for DNA extraction with little flow-induced back pressure. DNA extraction efficiencies for simple systems (lambda-phage DNA) were approximately 85%, while efficiencies for the reproducible extraction of human genomic DNA from complex biological matrixes (human blood) were approximately 70%. Blockage of the sol-gel pores by components in the lysed blood was observed in repeat extraction on a single device as a decrease in the extraction efficiency. The developed muSPE protocol was further evaluated to show applicability to clinical samples and bacterial cultures, through extraction of PCR-amplifiable DNA.

In the past few years, much attention has been paid to the development of miniaturized polymerase chain reaction (PCR) devices. After a continuous flow (CF) PCR chip was introduced, several CFPCR systems employing various pumping mechanisms were reported. However, the use of pumps increases cost and imposes a high requirement on microchip bonding integrity due to the application of high pressure. Other significant limitations of CFPCR devices include the large footprint of the microchip and the fixed cycle number which is dictated by the channel layout. In this paper, we present a novel circular close-loop ferrofluid driven microchip for rapid PCR. A small ferrofluid plug, containing sub-domain magnetic particles in a liquid carrier, is driven by an external magnet along the circular microchannel, which in turn propels the PCR mixture through three temperature zones. Amplification of a 500 bp lambda DNA fragment has been demonstrated on the polymethyl methacrylate (PMMA) PCR microchip fabricated by CO(2) laser ablation and bonded by a low pressure, high temperature technique. Successful PCR was achieved in less than 4 min. Effects of cycle number and cycle time on PCR products were investigated. Using a magnet as the actuator eliminates the need for expensive pumps and provides advantages of low cost, small power consumption, low requirement on bonding strength and flexible number of PCR cycles. Furthermore, the microchip has a much simpler design and smaller footprint compared to the rectangular serpentine CFPCR devices. To demonstrate its application in forensics, a 16-loci short tandem repeat (STR) sample was successfully amplified using the PCR microchip.

No abstract

Effective DNA separations in microelectrophoretic systems are complicated by the need to passivate the surface dynamically or covalently. We describe the optimization and utilization of a novel buffer system for fast DNA separations by capillary and microchip electrophoresis without the need for any surface modification or conditioning prior to separation. At concentrations as high as 5%, hydroxypropyl cellulose (HPC) has a relatively low viscosity, allowing for microchip channel filling to be performed with ease. A MES/TRIS buffer system at pH 6.1 eliminates the need for surface preconditioning procedures due to the promotion of hydrogen bonding of HPC with the wall. An additional benefit with this buffer system is the low current observed at high fields when compared to other common DNA separation buffers. An artificial neural network (ANN) was used to model the data and to predict the optimum conditions. Utility of the ANN-optimized system for molecular diagnostic testing was demonstrated by performing microchip separations on DNA samples from patients suspected of having genetic mutations associated with Duchenne muscular dystrophy (DMD). Microchip analysis easily allowed for the patient samples positive for DMD mutations to be distinguished from patient samples negative for the disease.

No abstract

We report here a novel multichannel closed-loop magnetically actuated microchip for high-throughput polymerase chain reaction (PCR). This is achieved by designing a series of concentric circular channels on one microchip and exploiting a magnetic force to drive DNA samples flowing continuously through the closed loops. The magnetic force arises from an external permanent magnet through ferrofluid plugs inside the microchannels. The magnet enables simultaneous actuation of DNA samples in all the channels. As the samples go around the loops, they pass through three preset temperature zones. Parameters of PCR, such as incubation time, temperatures, and number of cycles, can be fully controlled and adjusted. High reproducibility was achieved for different channels in the same run and for the same channels in consecutive runs. Genetically modified organisms (GMOs) were amplified simultaneously using the developed device. This simple, reliable, and high-throughput PCR microchip would find wide applications in forensic, clinical, and biological fields.