Niel Crews

Product differentiation during continuous-flow thermal gradient PCR

A continuous-flow PCR microfluidic device was developed in which the target DNA product can be detected and identified during its amplification. This in situ characterization potentially eliminates the requirement for further post-PCR analysis. Multiple small targets have been amplified from human genomic DNA, having sizes of 108, 122, and 134 bp. With a DNA dye in the PCR mixture, the amplification and unique melting behavior of each sample is observed from a single fluorescent image. The melting behavior of the amplifying DNA, which depends on its molecular composition, occurs spatially in the thermal gradient PCR device, and can be observed with an optical resolution of 0.1 degrees C pixel(-1). Since many PCR cycles are within the field of view of the CCD camera, melting analysis can be performed at any cycle that contains a significant quantity of amplicon, thereby eliminating the cycle-selection challenges typically associated with continuous-flow PCR microfluidics.

A hybrid CFD-mathematical model for simulation of a MEMS loop heat pipe for electronics cooling applications

A hybrid CFD-mathematical (HyCoM) model was developed to predict the performance of a micro loop heat pipe (MLHP) as a function of input heat rate. A micro loop heat pipe is a passive two-phase heat transport device, consisting of microevaporator, microcondenser, microcompensation chamber (CC) and liquid and vapor lines. A CFD model was incorporated into a loop solver code to identify heat leak to the CC. Two-phase pressure drop in the condenser was calculated by several two phase correlations and results were compared (Izenson and Crowley 1992 AIAA Paper A92-47847). Capillary tube correlations (Blevins 1984 Applied Fluid Dynamics Handbook (New York: Van Nostrand-Reinhold)) were used for pressure drop calculations in fluid lines. Effects of working fluid and change in geometry were studied. For a heat transport distance of 10 mm, the base model MLHP was 50 mm long, 16 mm wide and 1 mm thick. In the base model, widths of the grooves, liquid and vapor lines, evaporator and condenser were 55 µm, 200 µm, 750 µm, 2 mm and 4 mm, respectively.

Spatial DNA melting analysis for genotyping and variant scanning.

A continuous-flow, temperature gradient microfluidic device was used to demonstrate spatial DNA melting analysis with the resolution and reproducibility necessary for clinical SNP scanning and genotyping of human genomic DNA. With a steady-state temperature gradient of 20-30 degrees C across a sample, melting curves were constructed from a single fluorescence data acquisition. This technique was used to scan for heterozygotes and to fully genotype single base changes using unlabeled probes. Signal-to-noise ratios of 150-300 were achieved. The thermal effects of sample flow were examined, and temperature control was aided by inclusion of an isothermal channel inlet and thermal relaxation times in the experimental protocol. Human single base variants examined by spatial DNA melting analysis included rs354439, HTR2A 102T > C, and three alleles that affect appropriate warfarin dosage (CYP2C9*2, CYP2C9*3, and VKORC1 1173C > T). Heterozygote scanning was demonstrated with rs354439, while the other PCR targets were genotyped using unlabeled probes with T(m) differences of approximately 5 degrees C between genotypes. To validate the method, 12 blinded DNA samples were genotyped at the three warfarin-related sites by spatial DNA melting analysis with 100% accuracy.

Genotyping from saliva with a one-step microdevice

This paper presents a disposable microfluidic device for on-chip lysing, PCR, and analysis in one continuous-flow process. Male-female sex determination was performed with human saliva in less than 20 min from spit to finish, and requiring only seconds of manual sample handling. This genetic analysis was based on the amplification and detection of the DYZ1 repeat region unique to the Y-chromosome. The flow-through microfluidic chip consisted of a single serpentine channel designed to guide samples through 42 heating and cooling cycles. Cycling was performed by matching the local channel geometry to a steady-state temperature gradient established across the microfluidic chip. 38 channel segments were designed for rapid low volume PCR, and four were optimized for spatial DNA melting analysis. Fluorescence detection was used to monitor the amplification and to capture the melting signature of the amplicon was performed with a basic 8-bit CCD camera. The microfluidic device itself was fabricated from microscope slides and a double-sided tape. The simplicity of the system and its robust performance combine in an elegant solution for lab-on-a-chip genetic analysis.

Flow-induced thermal effects on spatial DNA melting

Continuous-flow temperature gradient microfluidics can be used to perform spatial DNA melting analysis. To accurately characterize the melting behavior of PCR amplicon across a spatial temperature gradient, the temperature distribution along the microfluidic channel must be both stable and known. Although temperature change created by micro-flows is often neglected, flow-induced effects can cause significant local variations in the temperature profile within the fluid and the closely surrounding substrate. In this study, microfluidic flow within a substrate with a quasi-linear temperature gradient has been examined experimentally and numerically. Serpentine geometries consisting of 10 mm long channel sections joined with 90 degrees and/or 180 degrees bends were studied. Infrared thermometry was used to characterize the surface temperature variations and a 3-D conjugate heat transfer model was used to predict interior temperatures for multiple device configurations. The thermal interaction between adjacent counter-flow channel sections, which is related to their spacing and substrate material properties, contributes significantly to the temperature profile within the microchannel and substrate. The volumetric flow rate and axial temperature gradient are directly proportional to the thermal variations within the device, while these flow-induced effects are largely independent of the cross-sectional area of the microchannel. The quantitative results and qualitative trends that are presented in this study are applicable to temperature gradient heating systems as well as other microfluidic thermal systems.

Thermal Gradient PCR in a Continuous-Flow Microchip

A new continuous-flow PCR microchip has been developed that operates by cycling a prepared sample within a spatial temperature gradient. This design allows for minimal thermal residence times - a key feature of the protocols used by the fastest commercial PCR equipment. Since thermal gradients are a natural effect of heat dissipation, the appropriate temperature distribution for PCR can be generated by a minimum of one heater held at a steady state temperature. With a thermal gradient of more than 3°C/mm across the width of the chip, each complete PCR cycle requires approximately 2cm of channel length. These glass chips were manufactured using standard glass microfabrication methods as well as the Xurographic rapid prototyping technique. Targets of 110bp and 181bp were amplified from &Fgr;X174 plasmid DNA on these thermal gradient chips as well as on commercial PCR equipment, then subsequently analyzed by gel electrophoresis. Visual inspection of fluorescent images of the stained gels shows that the amplicon size and yield for the systems are comparable.

Thermoelectric lab-on-a-chip ELISA

We report a new, thermoelectric method for performing enzyme-linked immunosorbent assay (ELISA) in a microfluidic device. The concentration of the analyte is determined by measuring the heat of an enzymatic reaction between glucose and glucose oxidase using thin-film antimony/bismuth thermopile. The feasibility of lab-on-a-chip thermoelectric ELISA is demonstrated by measuring the concentration of 8-hydroxy-2-deoxyguanosine (8OHdG) in urine samples from amyloid precursor protein transgenic mice. The detection method is based on formation of a complex between 8OHdG, anti-8OHdG capture antibody and glucose oxidase linked IgG antibody. The complex is immobilized at the lower channel wall of the microfluidic device, over the measuring junctions of the thermopile. The amount of heat detected by the thermoelectric sensor is inversely proportional to the concentration of 8OHdG. Standard calibration curve was created using synthetic 8OHdG. The regression line equation of the standard calibration curve was used to estimate the concentration of 8OHdG in mouse urine.

Microscale Thermal Biosensor: Critical Design Considerations and Optimization

Calorimetric biosensors have been used for detecting various bioprocesses such as enzyme-substrate activity, protein binding activity, DNA reactions and cell metabolism. The majority of the microcalorimeter applications were proof-of-concept in nature, but having a strong potential for development for actual clinical, scientific, or commercial need. Success of these emerging experimental methodologies will be determined by such factors as the sensitivity and speed of these analyses when compared with existing technologies. These performance metrics are fundamentally related to the thermal transport through the microsystems. In this study, we present the design and fabrication of a microcalorimeter. We also characterize the impact of flow velocity affecting the thermal time constant of the microcalorimeter, steady state response of the system, and the location of the sensor in the flow stream and provide essential guidelines for the optimization of single-stream thermopile systems. The calorimeter consists of a 100μm Y-shaped channel microfluidic device, which is made by sandwiching a microscope glass slide, Kapton tape cut in the form of channel and a microscope glass coverslip, and a bismuth (Bi)antimony (Sb) thin film thermopile (Seebeck coefficient of 5.95 mV/K) integrated on the outer wall of the microscope glass coverslip. The performance of the microcalorimeter was characterized by measuring the heat released during the mixing reaction between water and ethanol. The ratio of flow rates is adjusted to change the location of the reaction zone relative to the measuring or reference junctions of the thermopile. Results indicate as the flow velocity increases the time constant to reach steady state response is decreased.

Single-step intercalating dye strategies for DNA damage studies.

Many analytical protocols exist for the quantification of varied types of DNA damage, which span a range of complexity and sensitivity. As an alternative or companion to existing procedures, this article demonstrates the application of quantitative PCR (qPCR) and high-resolution DNA melting analysis (HRMA) to the detection and quantification of intramolecular DNA damage and/or strand breaks. These proven molecular biology methods are essentially single-step processes. When implemented with a third-generation saturating DNA dye, high sensitivity can be obtained. The experiments presented here demonstrate how DNA damage can inhibit amplification of the affected molecules. This corresponding decrease in the initial concentration of amplifiable DNA can be measured with qPCR. In addition, damage in the form of intramolecular dimerization and strand breaks alters the stored energy in the hydrogen bonds between the two strands in the dsDNA molecule. This significantly affects the thermal stability, which can be measured with extreme precision using HRMA. Simplified damage models were used in these experiments: UV-C irradiation to produce photoproducts, and restriction enzyme digestion to simulate double-strand breaks. The findings of this work, however, can be intuitively applied to the broad scope of DNA damage mechanisms.

A Hybrid CFD-Mathematical Model for Simulation of a MEMS Loop Heat Pipe

A Hybrid CFD-Mathematical (HyCoM) model was developed to predict the performance of a Micro Loop Heat Pipe (MLHP) as a function of input heat rate. A micro loop heat pipe is a passive two-phase heat transport device, consisting of microevaporator, microcondenser, micro-compensation chamber (CC), and liquid and vapor lines. A CFD model was incorporated into a loop solver code to identify heat leak to the CC. Two-phase pressure drop in the condenser was calculated by several two phase correlations and results were compared [2]. Capillary tube correlations [3] were used for pressure drop calculations in fluid lines. Effects of working fluid and change in geometry were studied. For a heat transport distance of 10 mm, the base model MLHP was 50mm long, 16mm wide and 1mm thick. In the base model, widths of the grooves, liquid and vapor lines, evaporator, and condenser were 55μm, 200μm, 750μm, 2mm, and 4mm respectively.Copyright