A versatile oscillating-flow microfluidic PCR system utilizing a thermal gradient for nucleic acid analysis.

We report the development of a versatile system based on the oscillating-flow methodology in a thermal gradient system for nucleic acid analysis. Analysis of DNA and RNA samples were performed in the device, without additional temperature control and complexity. The technique reported in this study eliminates the need for predetermined fluidic channels for thermocycles, and complexity involved with additional incubation steps required for RNA amplification. A microfluidic device was fabricated using rapid prototyping by simply sandwiching dual side adhesive Kapton tape and a polydimethylsiloxane spacer between glass microscope slides. Amplification of the 181-bp segment of a viral phage DNA (ΦX174) and B2M gene in human RNA samples was demonstrated using the system. The developed system enables simultaneous acquisition of amplification and melt curves, eliminating the need for postprocessing. A direct comparison between the oscillating-flow system and a commercial real-time polymerase chain reaction (PCR) instrument showed complete agreement in PCR data and improved sample-to-result time by eliminating an additional 30 min melt curve step required in commercial PCR systems.

Lab-on-a-chip mRNA purification and reverse transcription via a solid-phase gene extraction technique.

Extraction and purification of high quality RNA is a crucial initial step required for a variety of genomic assays. We report a solid phase gene extraction (SPGE) method for automated extraction, purification and reverse transcription of mRNA in a microfluidic device. This is performed using a 130 µm diameter stainless steel needle that is amino-linked to dT(15) oligonucleotides for selective hybridization of mRNA. By inserting this probe into the biological sample for only 30 seconds, mRNA is captured with high selectivity and a yield greater than 10 pg per mm of probe length. The probe is then inserted into a lab-on-a-chip device, where the bound poly-adenylated RNA is thermally released and immediately reverse transcribed for subsequent PCR amplification. The insertion of the probe into the microfluidic device is straightforward: the microchannel is formed with an elastomer (PDMS) that, when punctured, will seal around the probe. The specificity and RNA loading capacity of the probes were evaluated using conventional qPCR. This procedure was successfully used to extract, purify, and transcribe mRNA from rat glioblastoma cell spheroids in less than seven minutes. Analysis of the product confirmed that the SPGE technique selectively captures and inherently purifies high-quality mRNA directly from biological material with no need for additional pre-processing steps. Integrating this elegant sample preparation method into a complete lab-on-a-chip system will substantially enhance the speed and automation of mRNA assays for research and clinical diagnostics.

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.

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.

Real-time damage monitoring of irradiated DNA.

This article presents a microfluidic technique for the real-time analysis of DNA damage due to radiation exposure. A continuous-flow spatial melting analysis was performed every three seconds on a sample of isolated DNA while it was being irradiated. The formation of photoproducts being caused by the UV-C radiation was monitored during the process. Cumulative damage produced distinct changes in the DNA melting curves, characterized by a shifting and broadening of the melting peaks. The design of the microfluidic device, the experimental procedure, and the analysis algorithm and interactive GUI are discussed herein. In addition, the advantages of this system are correlated to specific needs of related scientific studies, such as the investigation of sequence-specific damage susceptibility and the characterization of exposure-damage nonlinearities.

Glass-composite prototyping for flow PCR with in situ DNA analysis.

In this article, low cost microfluidic devices have been used for simultaneous amplification and analysis of DNA. Temperature gradient flow PCR was performed, during which the unique fluorescence signature of the amplifying product was determined. The devices were fabricated using xurography, a fast and highly flexible prototype manufacturing method. Each complete iterative design cycle, from concept to prototype, was completed in less than 1 h. The resulting devices were of a 96% glass composition, thereby possessing a high thermal stability during continuous-flow PCR. Volumetric flow rates up to 4 microl/min induced no measurable change in the temperature distribution within the microchannel. By incorporating a preliminary channel passivation protocol, even the first microliters through the system exhibited a high amplification efficiency, thereby demonstrating the biocompatibility of this fabrication technique for DNA amplification microfluidics. The serpentine microchannel induced 23 temperature gradient cycles in 15 min at a 2 microl/min flow rate. Fluorescent images of the device were acquired while and/or after the PCR mixture filled the microchannel. Because of the relatively high initial concentration of the phage DNA template (PhiX174), images taken after 10 min (less than 15 PCR cycles) could be used to positively identify the PCR product. A single fluorescent image of a full device provided the amplification curve for the entire reaction as well as multiple high resolution melting curves of the amplifying sample. In addition, the signal-to-noise ratio associated with the spatial fluorescence was characterized as a function of spatial redundancy and acquisition time.

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.

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.

Plastic versus glass capillaries for rapid-cycle PCR.

Rapid-cycle PCR uses fast temperature transitions and minimal denaturation and annealing times of "0" s to complete 30 cycles in 10 to 30 min. The most popular platform amplifies samples in glass capillaries arranged around a carousel with circulating air for temperature control. Recently, plastic capillary replacements for glass capillaries became available. We compared the performance of plastic and glass capillaries for rapid-cycle PCR. Heat transfer into plastic capillaries was slowed by thicker walls, lower thermal conductivity, and a lower surface area-to-volume ratio than glass capillaries. Whereas the denaturation and annealing target temperatures were reached by samples in glass capillaries, samples in plastic capillaries fell short of these target temperatures by 6 degrees -7 degrees C. Rapid-cycle PCR was performed on two human genomic targets (APOE and ACVRL1) and one plasmid (pBR322) to amplify fragments of 225-300 bp in length with melting temperatures of 90.3 degrees -93.1 degrees C. Real-time amplification data, end-point melting curves, and end-point gel analysis revealed strong, specific amplification of samples in glass and complete amplification failure in plastic. Only the APOE target was successfully amplified by extending the denaturation and annealing times to 5 or 10 s. A 20 s holding period was necessary to reach target temperatures in plastic capillaries.

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.

Continuous-flow thermal gradient PCR.

Continuous-flow thermal gradient PCR is a new DNA amplification technique that is characterized by periodic temperature ramping with no cyclic hold times. The device reported in this article represents the first demonstration of hold-less thermocycling within continuous-flow PCR microfluidics. This is also the first design in which continuous-flow PCR is performed within a single steady-state temperature zone. This allows for straightforward miniaturization of the channel footprint, shown in this device which has a cycle length of just 2.1 cm. With a linear thermal gradient established across the glass device, the heating and cooling ramp rates are dictated by the fluid velocity relative to the temperature gradient. Local channel orientation and cross-sectional area regulate this velocity. Thus, rapid thermocycling occurs while the PCR chip is maintained at steady state temperatures and flow rates. Glass PCR chips (25 x 75 x 2 mm) of both 30 and 40 serpentine cycles have been fabricated, and were used to amplify a variety of targets, including a 181-bp segment of a viral phage DNA (PhiX174) and a 108-bp segment of the Y-chromosome, amplified from human genomic DNA. With this unique combination of hold-less cycling and gradient temperature ramping, a 40-cycle PCR requires less than 9 min, with the resulting amplicon having high yield and specificity.