Low-power microwave-mediated heating for microchip-based PCR.

Microwave energy has been used to rapidly heat food and drinks for decades, in addition to assisting other chemical reactions. However, only recently has microwave energy been applied in microfluidic systems to heat solution in reaction chambers, in particular, the polymerase chain reaction (PCR). One of the difficulties in developing microwave-mediated heating on a microchip is the construction of the appropriate architecture for delivery of the energy to specific micro-areas on the microchip. This work employs commercially-available microwave components commonly used in the wireless communications industry to generate a microwave signal, and a microstrip transmission line to deliver the energy to a 1 µL reaction chamber fabricated in plastic microdevices. A model was developed to create transmission lines that would optimally transmit energy to the reaction chamber at a given frequency, minimizing energy usage while focusing microwave delivery to the target chamber. Two different temperature control methods were demonstrated, varying microwave power or frequency. This system was used to amplify a fragment of the lambda-phage genome, thereby demonstrating its potential for integration into a portable PCR system.

A modular microfluidic system for deoxyribonucleic acid identification by short tandem repeat analysis.

Microfluidic technology has been utilized in the development of a modular system for DNA identification through STR (short tandem repeat) analysis, reducing the total analysis time from the ∼6 h required with conventional approaches to less than 3h. Results demonstrate the utilization of microfluidic devices for the purification, amplification, separation and detection of 9 loci associated with a commercially-available miniSTR amplification kit commonly used in the forensic community. First, DNA from buccal swabs purified in a microdevice was proven amplifiable for the 9 miniSTR loci via infrared (IR)-mediated PCR (polymerase chain reaction) on a microdevice. Microchip electrophoresis (ME) was then demonstrated as an effective method for the separation and detection of the chip-purified and chip-amplified DNA with results equivalent to those obtained using conventional separation methods on an ABI 310 Genetic Analyzer. The 3-chip system presented here demonstrates development of a modular, microfluidic system for STR analysis, allowing for user-discretion as to how to proceed after each process during the analysis of forensic casework samples.

A simple method for the evaluation of microfluidic architecture using flow quantitation via a multiplexed fluidic resistance measurement.

Quality control of microdevices adds significant costs, in time and money, to any fabrication process. A simple, rapid quantitative method for the post-fabrication characterization of microchannel architecture using the measurement of flow with volumes relevant to microfluidics is presented. By measuring the mass of a dye solution passed through the device, it circumvents traditional gravimetric and interface-tracking methods that suffer from variable evaporation rates and the increased error associated with smaller volumes. The multiplexed fluidic resistance (MFR) measurement method measures flow via stable visible-wavelength dyes, a standard spectrophotometer and common laboratory glassware. Individual dyes are used as molecular markers of flow for individual channels, and in channel architectures where multiple channels terminate at a common reservoir, spectral deconvolution reveals the individual flow contributions. On-chip, this method was found to maintain accurate flow measurement at lower flow rates than the gravimetric approach. Multiple dyes are shown to allow for independent measurement of multiple flows on the same device simultaneously. We demonstrate that this technique is applicable for measuring the fluidic resistance, which is dependent on channel dimensions, in four fluidically connected channels simultaneously, ultimately determining that one chip was partially collapsed and, therefore, unusable for its intended purpose. This method is thus shown to be widely useful in troubleshooting microfluidic flow characteristics.

Towards an integrated microfluidic device for spaceflight clinical diagnostics Microchip-based solid-phase extraction of hydroxyl radical markers.

A microchip-based solid-phase extraction method for biological fluid small molecule analysis has been developed. Using a commercially available copolymer packed into a microchip channel, extraction and preconcentration of 2,3-dihydroxybenzoic acid (DHBA) and 2,5-DHBA from saliva was achieved. The metabolites, formed from salicylic acid by reactive oxygen species, can be used as markers of oxidative stress. The results show high recovery of both metabolites (>90+/-15% for spiked saliva) with an 80-fold concentration enhancement possible. The eluent is directly analyzed using capillary electrophoresis, with good resolution for the two metabolites. This study demonstrates the feasibility of future integrated microdevices for spaceflight small molecule biomarker analysis.

Pressure injection on a valved microdevice for electrophoretic analysis of submicroliter samples.

A recent report describes a reversible valve that can be used in series to achieve diaphragm pumping on chip (Grover, W. H.; Skelley, A. M.; Liu, C. N.; Lagally, E. T.; and Mathies, R. A. Sens. Actuators, B 2003, 89, 315-323). Here, the functionality of an integrated diaphragm pump on a hybrid PDMS-glass microchip to perform pressure injections for electrophoretic separations is demonstrated. A chip design that can perform both pressure and electrokinetic (EK) injection is described, and a mixture of fluorescein and ROX dyes in borate buffer is utilized as a model sample system. Multiple electrophoretic separations of sample injected with pressure and voltage are compared. Over multiple EK injections, an electrophoretic bias is observed and the injected analytes are not representative of the sample, with the peak area ratio changing 20% after 20 runs. Over multiple pressure injections, however, the sample composition is maintained, with a 3.6% CV over 20 runs. The data presented show the ability to alternate between injection types and pressure-inject a representative sample volume after a bias has already been observed with multiple EK injections. Multiple pressure injections have been performed on sample volumes as low as 500 nL while maintaining sample composition, supporting its use in integrated systems for small-volume sampling.