Integrated portable polymerase chain reaction-capillary electrophoresis microsystem for rapid forensic short tandem repeat typing

A portable forensic genetic analysis system consisting of a microfluidic device for amplification and separation of short tandem repeat (STR) fragments as well as an instrument for chip operation and four-color fluorescence detection has been developed. The microdevice performs polymerase chain reaction (PCR) in a 160-nL chamber and capillary electrophoresis (CE) in a 7-cm-long separation channel. The instrumental design integrates PCR thermal cycling, electrophoretic separation, pneumatic valve fluidic control, and four-color laser excited fluorescence detection. A quadruplex Y-chromosome STR typing system consisting of amelogenin and three Y STR loci (DYS390, DYS393, DYS439) was developed and used for validation studies. The multiplex amplification of these 4 loci with 35 PCR cycles followed by CE separation and 4-color fluorescence detection was completed in 1.5 h. All the amplicons can be detected with a limit of detection of 20 copies of male standard DNA in the reactor. Real-world forensic analyses of oral swab and human bone extracts from case evidence were also successfully performed. Mixture analysis demonstrated that a balanced profile can be obtained even at a male-to-female template ratio of 1:10. The successful development and operation of this portable PCR-CE system establishes the feasibility of rapid point-of-analysis DNA typing of forensic casework, of mass disaster samples or of individuals at a security checkpoint.     

Single-molecule DNA amplification and analysis in an integrated microfluidic device

Stochastic PCR amplification of single DNA template molecules followed by capillary electrophoretic (CE) analysis of the products is demonstrated in an integrated microfluidic device. The microdevice consists of submicroliter PCR chambers etched into a glass substrate that are directly connected to a microfabricated CE system. Valves and hydrophobic vents provide controlled and sensorless loading of the 280-nL PCR chambers; the low volume reactor, the low thermal mass, and the use of thin-film heaters permit cycle times as fast as 30 s. The amplified product, labeled with an intercalating fluorescent dye, is directly injected into the gel-filled capillary channel for electrophoretic analysis. Repetitive PCR analyses at the single DNA template molecule level exhibit quantized product peak areas; a histogram of the normalized peak areas reveals clusters of events caused by 0, 1, 2, and 3 viable template copies in the reactor and these event clusters are shown to fit a Poisson distribution. This device demonstrates the most sensitive PCR possible in a microfabricated device. The detection of single DNA molecules will also facilitate single-cell and single-molecule studies to expose the genetic variation underlying ensemble sequence and expression averages.     

Multichannel PCR-CE microdevice for genetic analysis

We have developed a fully integrated multichannel polymerase chain reaction-capillary electrophoresis (PCR-CE) microdevice with nanoliter reactor volumes for highly parallel genetic analyses. Resistance temperature detectors and heaters made out of Ti/Pt are integrated on the microchip using a scalable radial design to provide precise temperature control of the four parallel PCR-CE reactor systems. Heating rates of >15 degrees C s(-1) and cooling rates of >10 degrees C s(-1) allow cycle times of 50 s and 30 complete PCR cycles in <27 min. PDMS membrane valves control and localize PCR reagents in the 380-nL reactors. By directly integrating PCR reactors with the CE separation system, efficient coupling of amplification with separation is achieved. The microdevice demonstrates good amplification uniformity and sensitivity down to 10 initial template copies in the 380-nL reactor (approximately 43 aM) with signal-to-noise ratio greater than 10. Parallel PCR-CE multiplex amplification and genetic analyses of four different samples with (1) both M13mp18 control template and E. coli K12 cells, (2) only M13mp18 template, (3) only E. coli K12 cells, and (4) negative control are completed in less than 30 min in a single run.     

MPIC: A High-Throughput Analytical Method for Multiple DNA Targets

We describe the development of a novel combined approach for high-throughput analysis of multiple DNA targets based on multiplex Microdroplet PCR Implemented Capillary gel electrophoresis (MPIC), a two-step PCR amplification strategy. In the first step, the multiple target DNAs are preamplified using bipartite primers attached with universal tail sequences on their 5'-ends. Then, the preamplified templates are compartmentalized individually in the microdroplet of the PCR system, and multiple targets can be amplified in parallel, employing primers targeting their universal sequences. Subsequently, the resulting multiple products are analyzed by capillary gel electrophoresis (CGE). Using genetically modified organism (GMO) analysis as a model, 24 DNA targets can be simultaneously detected with a relative limit of detection of 0.1% (w/w) and absolute limit of detection of 39 target DNA copies. The described system provides a promising alternative for high-throughput analysis of multiple DNA targets.     

Multiplex quantitative competitive polymerase chain reaction based on a multianalyte hybridization assay performed on spectrally encoded microspheres

Quantitative polymerase chain reaction (PCR) may be performed by two general approaches, namely, real-time PCR and quantitative competitive PCR (QC-PCR). QC-PCR makes use of the concept of a DNA competitor, which is the "gold standard" approach to circumvent the problem of the variation of amplification efficiency. However, QC-PCR in its classical form is a low-throughput method since it requires titration of each sample with the competitor followed by electrophoresis. The throughput of QC-PCR has been improved by capillary electrophoresis and microtiter well-based hybridization assays. The present work introduces a multiplex QC-PCR method, which is based on a multianalyte hybridization assay that is performed on spectrally encoded microspheres. The DNA competitors use the same primers and have equal size with their corresponding target DNA sequences but differ in a short (24 bp) centrally located sequence. Following multiplex PCR, biotinylated amplification products from all DNA targets and competitors are heat-denatured and hybridized with oligonucleotide probes, which are attached to addressable sets of fluorescent microspheres. The hybrids react with a streptavidin-phycoerythrin conjugate. The microspheres are then analyzed by flow cytometry employing two lasers. A red laser line is used for classification of the microspheres, and a green line excites phycoerythrin, whose fluorescence is related to the concentration of the analyte DNA. As a model, we have developed a multiplex quantitative competitive PCR assay for four targets. The amplification products from targets and competitors (a total of 8 DNA fragments) are determined simultaneously by the multianalyte hybridization assay. The limits of quantification for the hybridization assay of all amplified DNA fragments are below 13 pM. The multiplex quantitative competitive PCR assay detects approximately 500 copies from each target DNA. To our knowledge, the proposed method is the only approach to quantitative PCR that offers such a high potential for multiplexing.     

Integrating polymerase chain reaction, valving, and electrophoresis in a plastic device for bacterial detection

An integrated plastic microfluidic device was designed and fabricated for bacterial detection and identification. The device, made from poly(cyclic olefin) with integrated graphite ink electrodes and photopatterned gel domains, accomplishes DNA amplification, microfluidic valving, sample injection, on-column labeling, and separation. Polymerase chain reaction (PCR) is conducted in a channel reactor containing a volume as small as 29 nL; thermal cycling utilizes screen-printed graphite ink resistors. In situ gel polymerization was employed to form local microfluidic valves that minimize convective flow of the PCR mixture into other regions. After PCR, amplicons (products) are electrokinetically injected through the gel valve, followed by on-chip electrophoretic separation. An intercalating dye is admixed to label the amplicons; they are detected using laser-induced fluorescence. Two model bacteria, Escherichia coli O157 and Salmonella typhimurium, were chosen to demonstrate bacterial detection and identification based on amplification of several of their unique DNA sequences. The limit of detection is about six copies of target DNA.     

Simple multiplex genotyping by surface-enhanced resonance Raman scattering

The accurate detection of DNA sequences is essential for a variety of post human genome projects including detection of specific gene variants for medical diagnostics and pharmacogenomics. A specific DNA sequence detection assay based on surface-enhanced resonance Raman scattering (SERRS) and an amplification refractory mutation system (ARMS) is reported. Initially, generation of PCR products was achieved by using specifically designed allele-specific SERRS active primers. Detection by SERRS of the PCR products confirmed the presence of the sequence tested for by the allele-specific oligonucleotides. This lead directly to the multiplex genotyping of human DNA samples for the deltaF508 mutational status of the cystic fibrosis transmembrane conductance regulator gene using SERRS active primers in an ARMS assay. Removal of the unincorporated primers allowed fast and accurate analysis of the three genotypes possible in this system in a multiplex format without any separation of amplicons. The results indicate that SERRS can be used in modern genetic analysis and offers an opportunity for the development of novel assays. This is the first demonstration of the use of SERRS in multiplex genotyping and shows potential advantages over fluorescence as a detection technique with considerable promise for future development.     

Integrated portable genetic analysis microsystem for pathogen/infectious disease detection

An integrated portable genetic analysis microsystem including PCR amplification and capillary electrophoretic (CE) analysis coupled with a compact instrument for electrical control and laser-excited fluorescence detection has been developed. The microdevice contains microfabricated heaters, temperature sensors, and membrane valves to provide controlled sample positioning and immobilization in 200-nL PCR chambers. The instrument incorporates a solid-state laser and confocal fluorescence detection optics, electronics for sensing and powering the PCR reactor, and high-voltage power supplies for conducting CE separations. The fluorescein-labeled PCR products are amplified and electrophoretically analyzed in a gel-filled microchannel in <10 min. We demonstrate the utility of this instrument by performing pathogen detection and genotyping directly from whole Escherichia coli and Staphylococcus aureus cells. The E. coli detection assay consists of a triplex PCR amplification targeting genes that encode 16S ribosomal RNA, the fliC flagellar antigen, and the sltI shigatoxin. Serial dilution demonstrates a limit of detection of 2-3 bacterial cells. The S. aureus assay uses a femA marker to identify cells as S. aureus and a mecA marker to probe for methicillin resistance. This integrated portable genomic analysis microsystem demonstrates the feasibility of performing rapid high-quality detection of pathogens and their antimicrobial drug resistance.     

Fluorescence lifetime for on-the-fly multiplex detection of DNA restriction fragments in capillary electrophoresis

This paper describes the first use of frequency-domain fluorescence lifetime for multiplex detection of DNA restriction fragments in capillary electrophoresis (CE). The fragments were labeled with monomeric intercalating dyes that can be excited by either the 488- or 514-nm line of an argon ion laser and have lifetimes in the range of 0.5-2.5 ns. We were able to achieve multiplex lifetime detection in the CE separation of a restriction fragment digest and a DNA size ladder in the same run, for fragments shorter than 700 bp. Different gel buffer systems, including a modified polyacrylamide gel and several tris-borate-EDTA/hydroxyethylcellulose (TBE/HEC) gels, were investigated for separation and detection of the dye-labeled DNA fragments. Best results for both electrophoretic resolution and lifetime detection were obtained using a gel containing 1% high molecular weight (90,000-105,000) HEC and 0.3% low molecular weight (24,000-27,000) HEC in TBE buffer.     

Predicting viruses accurately by a multiplex microfluidic loop-mediated isothermal amplification chip

Multiplex gene assay is a valuable molecular tool not only in academic science but also in clinical diagnostics. Multiplex PCR assays, DNA microarrays, and various nanotechnology-based methods are examples of major techniques developed for analyzing multiple genes; none of these, however, are suitable for point-of-care diagnostics, especially in resource-limited settings. In this report, we describe an octopus-like multiplex microfluidic loop-mediated isothermal amplification (mμLAMP) assay for the rapid analysis of multiple genes in the point-of-care format and provide a robust approach for predicting viruses. This assay with the ability of analyzing multiple genes qualitatively and quantitatively is highly specific, operationally simple, and cost/time-effective with the detection limit of less than 10 copies/μL in 2 μL quantities of sample within 0.5 h. We successfully developed a mμLAMP chip for differentiating three human influenza A substrains and identifying eight important swine viruses.     

Calibration-free quantitative analysis of mRNA

Here we introduce a method for accurate and sensitive quantitative analysis of mRNA, which does not require calibration with mRNA. The method uses a fluorescently labeled hybridization probe as a reference standard. It involves the following: (i) annealing mRNA to the excess of the fluorescently labeled ssDNA hybridization probe, (ii) separation of the mRNA-probe hybrid from the excess of the probe by gel-free capillary electrophoresis mediated by ssDNA-binding protein, (iii) fluorescence detection of the hybrid and the excess probe, and (iv) quantification of mRNA using a simple algebraic formula. The method also overcomes a number of other limitations of conventional methods: the entire procedure currently takes only 2 h and accurately quantifies 10(5) copies of mRNA. With further improvements to the method, the procedure can be potentially shortened to 10 min, and the limit of quantification can be decreased to as few as 100 copies of mRNA. In this work, we prove the principle of the method by quantifying mRNA of green fluorescent protein in the matrix of total cellular RNA. The developed method is quantitative, simple, fast, and highly sensitive. It requires commercially available instrumentation only. The method will be an indispensable tool for molecular and cell biology studies.     

A microdevice with integrated liquid junction for facile peptide and protein analysis by capillary electrophoresis/electrospray mass spectrometry

A novel microfabricated device was implemented for facile coupling of capillary electrophoresis with mass spectrometry (CE/MS). The device was constructed from glass wafers using standard photolithographic/wet chemical etching methods. The design integrated (a) sample inlet ports, (b) the separation channel, (c) a liquid junction, and (d) a guiding channel for the insertion of the electrospray capillary, which was enclosed in a miniaturized subatmospheric electrospray chamber of an ion trap MS. The replaceable electrospray capillary was precisely aligned with the exit of the separation channel by a microfabricated guiding channel. No glue was necessary to seal the electrospray capillary. This design allowed simple and fast replacement of either the microdevice or the electrospray capillary. The performance of the device was tested for CE/MS of peptides, proteins, and protein tryptic digests. On-line tandem mass spectrometry was used for the structure identification of the protein digest products. High-efficiency/high-resolution separations could be obtained on a longer channel (11 cm on-chip) microdevice, and fast separations (under 50 s) were achieved with a short (4.5 cm on-chip) separation channel. In the experiments, both electrokinetic and pressure injections were used. The separation efficiency was comparable to that obtained from conventional capillary electrophoresis.     

Multiplex dsDNA Fragment Sizing Using Dimeric Intercalation Dyes and Capillary Array Electrophoresis: Ionic Effects on the Stability and Electrophoretic Mobility of DNA-Dye Complexes

Methods have been developed for performing accurate, high-resolution, multiplex capillary electrophoresis separations of dsDNA using dimeric intercalation dyes as noncovalent labeling reagents. The quality of these separations is highly dependent on the cation present during electrophoresis. Using buffers that contain only one cation, we show that the tetrapentylammonium (NPe(4)(+)) ion results in high-resolution, high-sensitivity separations but that smaller ions such as sodium or the commonly used buffer ion tris produce low-resolution, low-intensity separations of DNA-dye complexes. Using an 80 mM taps-NPe(4), 1 mM H(2)EDTA, pH 8.4, 0.8% HEC separation buffer, high-quality multiplex separations were performed using TOTO and buTOTIN, YOYO and TOED2, and TO and buTOTIN labeled restriction digests. In the taps-NPe(4) buffer, there is no significant mobility shift when complexes are formed with DNA-dye ratios from 100 to 5 bp per dye and very little dye transfer was observed. This property permits accurate multiplex sizing of samples having a wide concentration range simply by mixing the DNA with a dye solution before electrophoresis. This capability is demonstrated by diluting unpurified PCR products 10-, 100-, and 1,000-fold before mixing with a 1 nM TOTO solution and separating these samples with a ΦX174 HAEIII sizing ladder complexed with buTOTIN. Sizing precisions of better than 1% were obtained at all concentrations of target DNA. The mechanism for the increased DNA-dye complex stability and electrophoretic resolution in the taps-NPe(4) buffer is discussed.     

Detection of G proteins by affinity probe capillary electrophoresis using a fluorescently labeled GTP analogue

An affinity probe capillary electrophoresis (APCE) assay for guanine-nucleotide-binding proteins (G proteins) was developed using BODIPY FL GTPgammaS (BGTPgammaS), a fluorescently labeled GTP analogue, as the affinity probe. In the assay, BGTPgammaS was incubated with samples containing G proteins and the resulting mixtures of BGTPgammaS-G protein complexes and free BGTPgammaS were separated by capillary electrophoresis and detected with laser-induced fluorescence detection. Separations were completed in less than 30 s using 25 mM Tris, 192 mM glycine at pH 8.5 as the electrophoresis buffer and applying 555 V/cm over a 4-cm separation distance. BGTPgammaS-Galpha(o) peak heights increased linearly with Galpha(o) up to approximately 200 nM using a 50 nM BGTPgammaS probe. The detection limit for Galpha(o) was 2 nM, corresponding to a mass detection limit of 3 amol. The high speed of the APCE assays allowed reaction kinetics and the dissociation constant (Kd) to be determined. The on-rate and off-rate of BGTPgammaS to Galpha(o) were 0.0068 +/- 0.0004 and 0.000 23 +/- 0.000 01 s(-1), respectively. The half-life of the BGTPgammaS-Galpha(o) complex was 3060 +/- 240 s and Kd was 8.6 +/- 0.7 nM. The estimates of these parameters are in good agreement with those obtained using established techniques, indicating the suitability of this method for such measurements. Lowering the temperature of the separation improved the detection of the complex, allowing the assay to be performed on a commercial instrument with longer separation times. Additionally, the capability of the technique to detect several G proteins based on their binding to BGTPgammaS was demonstrated with assays for Galpha and Galpha(i1) and for Ras and Rab3A.     

Capillary electrophoresis chips with a sheath-flow supported electrochemical detection system

Microfabricated capillary electrophoresis chips containing an integrated sheath-flow electrochemical detector are developed with the goal of minimizing the influence of separation voltages on end-column detection while maintaining optimum performance. The microdevice consists of an upper glass wafer carrying the etched separation, injection, and sheath-flow channels and a lower glass wafer on which gold- and silver-plated electrodes have been fabricated. The sheath-flow channels join the end of the separation channel from each side, and gravity-driven flow carries the analytes to the electrochemical detector placed at working distances of 100, 150, 200, and 250 microm from the separation channel exit. The performance of this detector is evaluated using catechol and a detection limit of 4.1 microM obtained at a working distance of 250 microm. Detection of DNA restriction fragments and PCR product sizing is demonstrated using the electroactive intercalating dye, iron phenanthroline. Additionally, an allele-specific, PCR-based single-nucleotide polymorphism typing assay for the C282Y substitution diagnostic for hereditary hemochromatosis is developed and evaluated using ferrocene-labeled primers. This study advances the feasibility of high-speed, high-throughput chemical and genetic analysis using microchip electrochemical detection.     

Integrated microfluidic electrophoresis system for analysis of genetic materials using signal amplification methods

An isothermal signal amplification technique for specific DNA sequences, known as cycling probe technology (CPT), was performed within a microfluidic chip. The presence of DNA from methicillin-resistant Staphylococcus aureus was determined by signal amplification of a specific DNA sequence. The microfluidic device consisted of four channels intersecting to mix the sample and reagents within 55 s, as they were directed toward the reactor coil by electrokinetic pumping. The 160-nL CPT reactor occupied approximately 220 mm2. Gel-free capillary electrophoresis separation of the biotin- and fluorescein-labeled probe from the probe fragments was performed on-chip following the on-chip reaction. An off-chip CPT reaction, with on-chip separation gave a detection limit of 2 fM (0.03 amol) target DNA and an amplification factor of 85,000. Calibration curves, linear at <5% probe fragmentation, obeyed a power law relationship with an argument of 0.5 [target] at higher target DNA concentrations for both on-chip and off-chip CPT reaction and analysis. An amplification factor of 42,000 at 250 fM target (25,000 target molecules) was observed on-chip, but the reaction was approximately 4 times less sensitive than off-chip under the conditions used. Relative SD values for on-chip CPT were 0.8% for the peak migration times, 9% for the area of intact probe peak, and 8% for the fragment/probe peak area ratio.     

High-throughput single-molecule DNA screening based on electrophoresis

In electrophoresis, the migration velocity is used for sizing DNA and proteins or for distinguishing molecules based on charge and hydrodynamic radius. Many protein and DNA assays relevant to disease diagnosis are based on such separations. However, standard protocols are not only slow (minutes to hours) but also insensitive (many molecules in a detectable band). We successfully demonstrated a high-throughput imaging approach that allows determination of the individual electrophoretic mobilities of many molecules at a time. Each measurement only requires a few milliseconds to complete. This opens up the possibility of screening single copies of DNA or proteins within single biological cells for disease markers without performing polymerase chain reaction or other biological amplification. The purpose is not to separate the DNA molecules but to identify each one on the basis of the measured electrophoretic mobility. We developed three different procedures to measure the individual molecular mobilities. The results correlate well with capillary electrophoresis (CE) experiments for the same samples (2-49 kb dsDNA) under identical separation conditions. The implication is that any electrophoresis protocols from slab gels to CE should be adaptable to single-molecule screening for disease diagnosis.     

Pd nanowires as new biosensing materials for magnified fluorescent detection of nucleic acid

The designed synthesis of new nanomaterials with controlled shape, composition, and structure is critical for tuning their physical and chemical properties, and further developing interesting analytical sensing devices. Herein, we presented that Pd nanowires (NWs) can be used as a new biosensing platform for high-sensitivity nucleic acid detection. The general sensing concept is based on the fact that Pd NWs can adsorb the fluorescently labeled single-stranded DNA probe and lead to substantial fluorescence quenching of dye, followed by specific hybridization with the complementary region of the target DNA sequence. This results in desorption of double-stranded DNA from Pd NWs surface and subsequent recovery of fluorescence. Furthermore, an amplification strategy based on Pd NWs for nucleic acid detection by using exonuclease III (Exo III) was demonstrated. The present dual-magnification sensing system combined Pd NWs with Exo III has a detection range of 1.0 nM to 2.0 μM with the detection limit of 0.3 nM (S/N = 3), which is about 20-fold higher than that of traditional unamplified homogeneous assays.