Practical integration of polymerase chain reaction amplification and electrophoretic analysis in microfluidic devices for genetic analysis

An integrated system of a silicon-based microfabricated polymerase chain reaction (microPCR) chamber and microfabricated electrophoretic glass chips have been developed. The PCR chamber was made of silicon and had aluminum heaters and temperature sensors integrated on the glass anodically bonded cover. Temperature uniformity in the reaction chamber was +/-0.3 degrees C using an improved novel "joint-heating" scheme. Thermal cycling was digitally controlled with a temperature accuracy of +/- 0.2 degrees C. Small operating volumes together with high thermal conductivity of silicon made the device well suited to rapid cycling; 16 s/cycle were demonstrated. For analysis of the PCR products, the chamber output was transferred to the glass microchip by pressure. Analysis time of PCR amplified genomic DNA was obtained in the microchip in less than 180 s. The analysis procedure employed was reproducible, simple and practical by using viscous sieving solutions of hydroxypropylmethylcellulose and dynamically coated microchip channels with poly(vinylpyrrolidone). DNA fragments that differ in size by 18 base pairs (bp) were resolved. Analysis of genomic male and female amplified DNA by microPCR was achieved in microchip, and application of the integrated microPCR-microchip for the identification of bird sex was tested. Genomic DNA samples from several bird species such as pigeon and chicken were analyzed. Hence, the system could be used as well to determine the sex of avian species.     

Integration of gene amplification and capillary gel electrophoresis on a polydimethylsiloxane-glass hybrid microchip

We report on the development of a hybrid polydimethylsiloxane (PDMS)-glass microchip for genetic analysis by functional integration of polymerase chain reaction (PCR) and capillary gel electrophoresis (CGE), and on related temperature control systems for PCR on a PDMS-glass hybrid microchip. The microchip was produced by molding PDMS against a microfabricated master with comparatively simple and inexpensive methods. PCR was successfully carried out on the PDMS-glass hybrid microchip with 500 bp target of lambdaDNA and the amplified gene was subsequently analyzed by CGE on the same PDMS-glass microchip. The chip could be considered as an inexpensive single-use apparatus compared to glass or silicon-made microchips for the same purpose.     

High speed single nucleotide polymorphism typing of a hereditary haemochromatosis mutation with capillary array electrophoresis microplates

A single nucleotide polymorphism (SNP) typing assay is developed and evaluated on a microfabricated capillary array electrophoresis system. Using fluorescently labeled allele-specific primers, the S65C (193A-->T) substitution associated with hereditary haemochromatosis in the HFE gene is genotyped. The covalently labeled polymerase chain reaction (PCR) products are separated on a microfabricated radial capillary array electrophoresis microplate using nondenaturing gel media in under two minutes. Detection is accomplished with a laser-excited rotary confocal scanner. The Rox-labeled A-allele specific amplicon (211 bp) is differentiated from the R110-labeled T-allele specific amplicon (201 bp) by both size and color. This study demonstrates the feasibility of using allele-specific PCR with covalently labeled primers for high speed fluorescent SNP typing on microfabricated radial capillary array electrophoresis microplates.     

Recent developments in amperometric detection for microchip capillary electrophoresis

The interest in microfluidic devices has increased considerably over the past decade due to the numerous advantages of working within a miniature, microfabricated format. This review focuses on recent advances in coupling amperometric detection with microchip capillary electrophoresis (CE). Advances in electrochemical cell design, isolation of the detector from the separation field, and integration of both pre- and postseparation reaction chambers are discussed. The use of microchip CE with amperometric detection for enzyme/immunoassays, clinical and environmental assays, and the determination of neurotransmitters is described.     

Modulated capillary titration calorimeter

A modulated capillary titration calorimeter has been developed. New software and optimization of the calorimetric unit CTD2156 are used as a basis of the modulated capillary titration calorimeter. The scanning mode of the calorimeter has been theoretically substantiated. The scanning of chambers temperature is provided due to the fact that the shield temperature is linearly varied at heating and cooling. The reversing and kinetic part of the total heat flow are measured at heating of a diluted collagen solution. The main advantage of the calorimeter is its ability to operate in a modulation mode, in an isothermal mode, in modes of linear heating and cooling of homogeneous and dispersoid liquid samples at an effective mixing of reagents in calorimetric chambers.     

Screening of inhibitors of uridine diphosphate glucuronosyltransferase with a miniaturized on-line drug-metabolism system

Inhibition of uridine diphosphate glucuronosyltransferase (UGT), a major drug-metabolyzing enzyme, has been studied using an on-line drug-metabolism system integrated into capillary electrophoresis. Microsomes isolated from rat liver were encapsulated in tetramethoxysilane (TMOS)-based silica matrices within a capillary in a single step under mild conditions. This microsome-immobilized capillary column allows both the metabolism of drugs and determination of the metabolites in a single capillary simultaneously, just by injecting the substrate-coenzyme mixture onto the column. Glucuronidation of acetaminophen, a widely used pharmaceutical analgesic and antipyretic agent, was investigated using this system. The glucuronidation was inhibited by 4-nitrophenol (4NP) or probenecid that was injected onto a column along with the substrate-coenzyme mixture. On the other hand, valproate did not inhibit the metabolizing reaction. The extents of inhibition using encapsulated UGT were almost the same as those obtained using free UGT. On the other hand, this electrophoretic enzyme-inhibitor assay in microfabricated devices consumes 10(4) less sample and 10(3) less microsome per experiment compared to the conventional reaction schemes. These results demonstrate that this on-line system can circumvent laborious procedures for the isolation and determination of drug metabolites from the reaction mixtures required in the conventional schemes and can provide an attractive alternative technique for the analysis of drug interactions in the metabolic pathways.     

DNA sequencing and genotyping in miniaturized electrophoresis systems

Advances in microchannel electrophoretic separation systems for DNA analyses have had important impacts on biological and biomedical sciences, as exemplified by the successes of the Human Genome Project (HGP). As we enter a new era in genomic science, further technological innovations promise to provide other far-reaching benefits, many of which will require continual increases in sequencing and genotyping efficiency and throughput, as well as major decreases in the cost per analysis. Since the high-resolution size- and/or conformation-based electrophoretic separation of DNA is the most critical step in many genetic analyses, continual advances in the development of materials and methods for microchannel electrophoretic separations will be needed to meet the massive demand for high-quality, low-cost genomic data. In particular, the development (and commercialization) of miniaturized genotyping platforms is needed to support and enable the future breakthroughs of biomedical science. In this review, we briefly discuss the major sequencing and genotyping techniques in which high-throughput and high-resolution electrophoretic separations of DNA play a significant role. We review recent advances in the development of technology for capillary electrophoresis (CE), including capillary array electrophoresis (CAE) systems. Most of these CE/CAE innovations are equally applicable to implementation on microfabricated electrophoresis chips. Major effort is devoted to discussing various key elements needed for the development of integrated and practical microfluidic sequencing and genotyping platforms, including chip substrate selection, microchannel design and fabrication, microchannel surface modification, sample preparation, analyte detection, DNA sieving matrices, and device integration. Finally, we identify some of the remaining challenges, and some of the possible routes to further advances in high-throughput DNA sequencing and genotyping technologies.     

Recent advances in DNA sequencing by capillary and microdevice electrophoresis

A number of significant improvements in the electrophoretic performance and design of DNA sequencing devices have culminated in the introduction of truly industrial grade production scale instruments. These instruments have been the workhorses behind the massive increase in genomic sequencing data available in public and private databases. We highlight the recent progress in aspects of capillary electrophoresis (CE) that has enabled these achievements. In addition, we summarize recent developments in the use of microfabricated devices for DNA sequencing that promise to bring the next leap in productivity.     

DNA sequencing by capillary array electrophoresis and microfabricated array systems

To comply with the current needs for high-speed DNA sequencing analysis, several instruments and innovative technologies have been introduced by several groups in recent years. This review article discusses and compares the issues regarding high-throughput DNA sequencing by electrophoretic methods in miniaturized systems, such as capillaries, capillary arrays, and microchannels. Initially, general features of several capillary array designs (including commercial ones) will be considered, followed by similar analyses with microfabricated array electrophoretic devices and how they can contribute to the success of large sequencing projects.     

Rapid microvolume PCR of DNA confirmed by microchip electrophoresis.

PCR is an indispensable technique used in DNA analysis. However, with the traditional methods, the time spent on amplification and the subsequent analysis of PCR products is generally long. Therefore, it is essential to improve these two steps so that the whole procedure can be made faster. In the present work, with lambda-DNA as the control template, the amplification of 300-bp fragment could be completed within 37 s with capillary reaction chambers of LightCycler, and the following analysis of PCR products could be completed within 120 s with microchip electrophoresis as the detector. Since the high detection sensitivity of microchip electrophoresis, PCR products with template concentration as low as 5 fg/microL could be detected only after 435 s of amplification. In addition, based on additional optimized conditions simulated by CoventorWare, PCR microchips with distinct structure of the reaction chambers have been designed and successfully applied to the amplification of 300-bp fragment. By comparison, those chambers with ellipse and racket shapes were found to offer very high amplification efficiency. All of these results demonstrate the promise of integrating PCR and electrophoresis on microchip for developing easy-carrying instruments for the fast in situ detection of DNA.     

Effect of Joule heating on efficiency and performance for microchip-based and capillary-based electrophoretic separation systems: a closer look

An attempt is made to revisit the main theoretical considerations concerning temperature effects ("Joule heating") in electro-driven separation systems, in particular lab-on-a-chip systems. Measurements of efficiencies in microfabricated devices under different Joule heating conditions are evaluated and compared to both theoretical models and measurements performed on conventional capillary systems. The widely accepted notion that planar microdevices are less susceptible to Joule heating effects is largely confirmed. The heat dissipation from a nonthermostatically controlled glass microdevice was found to be comparable to that from a liquid-cooled-fused silica capillary. Using typically dimensioned glass and glass/silicon microdevices, the experimental results indicate that 5-10 times higher electric field strengths can be applied than on conventional capillaries, before detrimental effects on the separation efficiency occur. The main influence of Joule heating on efficiency is via the establishment of a radial temperature profile across the lumen of the capillary or channel. An overall temperature increase of the buffer solution has only little influence on the quality of the separation. Still, active temperature control (cooling, thermostatting) can help prevent boiling of the buffer and increase the reproducibility of the results.     

Human genomic DNA analysis using a semi-automated sample preparation, amplification, and electrophoresis separation platform

The growing importance of analyzing the human genome to detect hereditary and infectious diseases associated with specific DNA sequences has motivated us to develop automated devices to integrate sample preparation, real-time PCR, and microchannel electrophoresis (MCE). In this report, we present results from an optimized compact system capable of processing a raw sample of blood, extracting the DNA, and performing a multiplexed PCR reaction. Finally, an innovative electrophoretic separation was performed on the post-PCR products using a unique MCE system. The sample preparation system extracted and lysed white blood cells (WBC) from whole blood, producing DNA of sufficient quantity and quality for a polymerase chain reaction (PCR). Separation of multiple amplicons was achieved in a microfabricated channel 30 microm x 100 microm in cross section and 85 mm in length filled with a replaceable methyl cellulose matrix operated under denaturing conditions at 50 degrees C. By incorporating fluorescent-labeled primers in the PCR, the amplicons were identified by a two-color (multiplexed) fluorescence detection system. Two base-pair resolution of single-stranded DNA (PCR products) was achieved. We believe that this integrated system provides a unique solution for DNA analysis.     

Heating and convection associated with alkaline electrophoresis

Alkaline version of the single-cell gel electrophoresis (Comet assay) is widely used in toxicological, environmental, and monitoring studies to assess the DNA damage levels in individual cells. The change in the temperature of the electrophoretic solution is one of the reasons leading to interlaboratory variation of Comet assay results. In this work, changes of surface temperature of the solution during electrophoresis were studied using technique of real-time thermal imaging. It has been found that the electrophoresis is accompanied by nonuniform temperature rise in different areas of the electrophoretic chamber. The maximum of heating was observed in the central region of the chamber, where temperature increased by an average of 7°C. The minimum temperature rise in other parts of the chamber was about 5°C. After removing the solution, the temperature on the surface of slides was higher than that on the surface of the solution. We believe that (1) nonuniform heating of the electrophoretic solution and convection could be the reasons responsible for the variability of results both in inter- and intralaboratory studies; (2) the spatial distribution of heating of the solution depends on the size and configuration of the electrophoretic chambers used.     

High-throughput polymerase chain reaction analysis of clinical samples by capillary electrophoresis with UV detection

Routine genetic analysis of large numbers of individuals by polymerase chain reaction (PCR) using capillary electrophoresis is often restricted by the low throughput of standard protocols and the tedious sample preparation process. Here, we demonstrate that capillary electrophoresis with UV detection can be used in PCR-based DNA analysis starting from clinical samples without purification or complicated sample manipulation. After PCR reaction using cheek cells, blood, or HIV-1 gag DNA, the reaction mixtures were injected into a capillary array either on-line or off-line by base stacking. The use of multiplexed absorption detection and the elimination of any purification steps both before and after PCR reaction can potentially provide significant benefits compared to current methods for DNA analysis with regard to time, cost, and labor.     

Analytical study of Joule heating effects on electrokinetic transportation in capillary electrophoresis

Electric fields are often used to transport fluids (by electroosmosis) and separate charged samples (by electrophoresis) in microfluidic devices. However, there exists inevitable Joule heating when electric currents are passing through electrolyte solutions. Joule heating not only increases the fluid temperature, but also produces temperature gradients in cross-stream and axial directions. These temperature effects make fluid properties non-uniform, and hence alter the applied electric potential field and the flow field. The mass species transport is also influenced. In this paper we develop an analytical model to study Joule heating effects on the transport of heat, electricity, momentum and mass species in capillary-based electrophoresis. Close-form formulae are derived for the temperature, applied electrical potential, velocity, and pressure fields at steady state, and the transient concentration field as well. Also available are the compact formulae for the electric current and the volume flow rate through the capillary. It is shown that, due to the thermal end effect, sharp temperature drops appear close to capillary ends, where sharp rises of electric field are required to meet the current continuity. In order to satisfy the mass continuity, pressure gradients have to be induced along the capillary. The resultant curved fluid velocity profile and the increase of molecular diffusion both contribute to the dispersion of samples. However, Joule heating effects enhance the sample transport velocity, reducing the analysis time in capillary electrophoretic separations.     

A microfluidic device integrated with multichamber polymerase chain reaction and multichannel separation for genetic analysis

This work describes a microfluidic device integrated with multichamber polymerase chain reaction (PCR) and multichannel separation for parallel genetic analysis. The microdevice consists of three functional units: temperature control, multiple PCR (four chambers PCR), and multiple channel separation (four separation channels, each channel connected to a PCR chamber). Platinum (Pt)/titanium (Ti) microheater was used to ensure homogeneous temperature field, and Pt-chip sensor was used for temperature monitoring. The interface between chip-PCR and chip separation was simplified by connecting the PCR chamber with separation channel directly. After chip-PCR, PCR products were introduced into parallel separation channels for subsequent separation/detection by applying an electric field automatically. This microdevice was successfully applied for detection of pathogens including hepatitis B virus (HBV) and Mycobacterium tuberculosis (MTB), and genotyping of human leucocyte antigen (HLA)-B27 as well, demonstrating the feasibility of the integrated microdevice for parallel genetic analysis.     

Investigation of the electroosmotic flow effect on the efficiency of capillary electrophoresis.

The influence of the electroosmotic flow profile on the efficiency and resolution of capillary electrophoresis is studied. The mathematical model is formulated and the set of equations is solved numerically. The results of the analysis are applicable to a wide range of buffer concentrations and capillary diameters. The temperature dependence of electrophoretic mobility, viscosity and thermal conductivity and the dependence of electrical conductivity on temperature and ion concentration in the double layer are taken into account. It is shown that there exists a region of buffer concentrations and capillary diameters where the influence of the electroosmotic flow profile on the efficiency and resolution is much greater than that of the temperature dependence of the electrophoretic mobility. The results are especially essential for small buffer concentrations or capillary diameters comparable with the double electrical layer thickness.     

Microchip capillary electrophoresis/electrochemistry

Microfabricated fluidic devices have generated considerable interest over the past ten years due to the fact that sample preparation, injection, separation, derivatization, and detection can be integrated into one miniaturized device. This review reports progress in the development of microfabricated analytical systems based on microchip capillary electrophoresis (CE) with electrochemical (EC) detection. Electrochemical detection has several advantages for use with microchip electrophoresis systems, for example, ease of miniaturization, sensitivity, and selectivity. In this review, the basic components necessary for microchip CEEC are described, including several examples of different detector configurations. Lastly, details of the application of this technique to the determination of catechols and phenols, amino acids, peptides, carbohydrates, nitroaromatics, polymerase chain reaction (PCR) products, organophosphates, and hydrazines are described.     

A simple method for assessment and minimization of errors in determination of electrophoretic or electroosmotic mobilities and velocities associated with the axial electric field distortion

It is commonly accepted that the modern CE instruments equipped with efficient cooling system enable accurate determination of electrophoretic or electroosmotic mobilities. It is also often assumed that velocity of migration in a given buffer is constant throughout the capillary length. It is simultaneously neglected that the noncooled parts of capillary produce extensive Joule heating leading to an axial electric field distortion, which contributes to a difference between the effective and nominal electric field potentials and between velocities in the cooled and noncooled parts of capillary. This simplification introduces systematic errors, which so far were however not investigated experimentally. There was also no method proposed for their elimination. We show a simple and fast method allowing for estimation and elimination of these errors that is based on combination of a long‐end and short‐end injections. We use it to study the effects caused by variation of temperature, electric field, capillary length, and pH.     

Fast separation of oligonucleotide and triplet repeat DNA on a microfabricated capillary electrophoresis device and capillary electrophoresis

A laser-induced fluorescence detection system coupled with a highly sensitive silicon-intensified target (SIT) camera is successfully applied to the imaging of a band for DNA fragment labeling by fluorescence dye in a microchannel, and to the visualizing of the separation process on a microfabricated chip. We demonstrated that an only 6 mm separation channel is sufficient for the separation of triplet repeat DNA fragment and DNA molecular marker within only 12 s. The separation using the microfabricated capillary electrophoresis device is confirmed to be at least 18 times faster than the same separation carried out by conventional capillary electrophoresis with 24.5 cm effective length. The use of a short capillary with 8.5 cm effective length is also efficient for fast separation of DNA; however, the microchip technology is even faster than capillary electrophoresis using a short capillary.