Multiplexed p53 Mutation Detection by Microchip Electrophoresis with Laser-Induced Fluorescence Detector

A form of single‐strand DNA‐conformation polymorphism analysis (SSCP) employing nondenaturing slab gel electrophoresis is applicable to the genetic diagnosis of mutations at exons 7, 8 and 9 of the p53 gene. Recently, microchip electrophoresis (ME) systems have been used in SSCP analysis instead of conventional slab gel electrophoresis in terms of speed, sensitivity and automation. The aim of the present study was to investigate the application of SSCP and ME analysis as a rapid and effective method to the detection of mutations for exons 7, 8 and 9 of the p53 gene. It was found that using the electric field strength 260 V/cm and the sieving matrix of 4 mg/mL poly(ethylene oxide) was very useful to achieve better resolution and fast detection of mutations at exons 7, 8 and 9 of p53 gene. Under the optimized conditions, mutations at exons 7–9 of p53 gene were analyzed within 60 s and the relative standard deviation values of the migration times were less than 5.81% (n=5). The detection limit can be as low as 1 ng·L^?1.     

Capillary and microchip gel electrophoresis for simultaneous detection of Salmonella pullorum and Salmonella gallinarum by rfbS allele-specific PCR

We report the use of capillary gel electrophoresis (CGE) based on a rfbS allele-specific polymerase chain reaction (PCR) for the analysis and simultaneous detection of Salmonella pullorum and Salmonella gallinarum, which are the major bacterial pathogens in poultry. rfbS allele-specific PCR was used to concurrently amplify two specific 147- and 187-bp DNA fragments for the simultaneous detection of S. pullorum and S. gallinarum at an annealing temperature of 54 ± 1 °C and an MgCl₂ concentration of 2.8-5.6 mM. Under an electric field of 333.3 V/cm and a sieving matrix of 1.0% poly(ethyleneoxide) (M_r 600 000), the amplified PCR products were analyzed within 6 min by CGE separation. This CGE assay could be translated to microchip format using programmed field strength gradients (PFSG). In the microchip gel electrophoresis with PFSG, both of the Salmonella analyses were completed within 30 s, without decreasing the resolution efficiency. rfbS allele-specific PCR-microchip gel electrophoresis with the PFSG technique might be a new tool for the simultaneous detection of both S. pullorum and S. gallinarum, due to its ultra-speed and high efficiency.     

Ultra-fast simultaneous analysis of genetically modified organisms in maize by microchip electrophoresis with LIF detector

This study examined the potential of microchip electrophoresis (ME) with a LIF detector using a programmed field strength gradient (PFSG) in a conventional glass double-T microchip for the ultra-fast detection and simultaneous analysis of genetically modified (GM) maize. The separation efficiency and sensitivity at various sieving gels (poly(ethylene oxide) (PEO, M(r) 8,000,000) and 2-hydroxyethylcellulose (HEC) (M(r) 250,000)) and fluorescent dye concentrations were investigated. The PCR products of both the GM and non-GM maize were analyzed within 30 s under the PFSG (470.6 V/cm for 20 s, 117.6 V/cm for 12 s, and 470.6 V/cm for 30 s) with a 2.5% HEC sieving matrix in the running buffer, 1 x Tris-borate EDTA (TBE) (pH 8.30) and 0.5 ppm ethidium bromide. The five transgenic maize varieties (Event176, MON810, Bt11, GA21, and T25) examined in this study were also clearly differentiated by ME-PFSG within 30 s in a single run without any loss of resolution. The ME-PFSG technique is a powerful tool for the ultra-fast detection and simultaneous analysis of GMOs in a variety of foods including maize.     

Microchip gel electrophoresis with programmed field strength gradients for ultra-fast detection of canine T-cell lymphoma in dogs

This paper describes the applicability of microchip gel electrophoresis using a programmed field strength gradients (MGE-PFSG) method coupled with a polymerase chain reaction (PCR) for the ultra-fast diagnosis of canine T-cell lymphoma. The variable region in the T-cell receptor gamma (TCRgamma) gene from a T-cell lymphoma was used in PCR amplification. The contributions of the various parameters, including the effects of the molecular weight, concentration of the sieving matrix and field strength in MGE, were examined. 0.5% poly (ethyleneoxide) (PEO, M(r) 8000000) was used as the sieving matrix for the ultra-rapid separation of the amplified-PCR products (90 and 130-bp DNA fragments) from the PFSG at an effective length of 20mm in a glass microchip. The PCR products (90 and 130-bp DNA) of the T-cell lymphoma were analyzed within 41.7+/-0.1s, 15.5+/-0.2s and only 7.0+/-0.1s using a low-constant field strength, high-constant field strength and the PFSG, respectively. When 11 clinical samples were analyzed using the MGE-PFSG method, there was a 100% correlation with those obtained using conventional slab gel electrophoresis. The ultra-fast detection and rapid separation capabilities of MGE-PFSG make it an efficient tool for diagnosing T-cell lymphoma in clinical samples with high sensitivity.     

Separation of DNA fragments for fast diagnosis by microchip electrophoresis using programmed field strength gradient

We evaluated a novel strategy for fast diagnosis by microchip electrophoresis (ME), using programmed field strength gradients (PFSG) in a conventional glass double-T microfluidic chip. The ME-PFSG allows for the ultrafast separation and enhanced resolving power for target DNA fragments. These results are based on electric field strength gradients (FSG) that use an ME separation step in a sieving gel matrix poly-(ethylene oxide). The gradient can develop staircase or programmed shapes FSG over the time. The PFSG method could be easily used to increase separation efficiency and resolution in ME separation of specific size DNA fragments. Compared to ME that uses a conventional and constantly applied electric field (isoelectrostatic) method, the ME-PFSG achieved about 15-fold faster analysis time during the separation of 100 bp DNA ladder. The ME-PFSG was also applied to the fast analysis of the PCR products, 591 and 1191 bp DNA fragments from the 18S rRNA of Babesia gibsoni and Babesia caballi.     

Simultaneous detection of 19 K‐ras mutations by free‐solution conjugate electrophoresis of ligase detection reaction products on glass microchips

We demonstrate here the power and flexibility of free‐solution conjugate electrophoresis (FSCE) as a method of separating DNA fragments by electrophoresis with no sieving polymer network. Previous work introduced the coupling of FSCE with ligase detection reaction (LDR) to detect point mutations, even at low abundance compared to the wild‐type DNA. Here, four large drag‐tags are used to achieve free‐solution electrophoretic separation of 19 LDR products ranging in size from 42 to 66 nt that correspond to mutations in the K‐ras oncogene. LDR‐FSCE enabled electrophoretic resolution of these 19 LDR‐FSCE products by CE in 13.5 min (E = 310 V/cm) and by microchip electrophoresis in 140 s (E = 350 V/cm). The power of FSCE is demonstrated in the unique characteristic of free‐solution separations where the separation resolution is constant no matter the electric field strength. By microchip electrophoresis, the electric field was increased to the maximum of the power supply (E = 700 V/cm), and the 19 LDR‐FSCE products were separated in less than 70 s with almost identical resolution to the separation at E = 350 V/cm. These results will aid the goal of screening K‐ras mutations on integrated “sample‐in/answer‐out” devices with amplification, LDR, and detection all on one platform.     

Blinded study determination of high sensitivity and specificity microchip electrophoresis-SSCP/HA to detect mutations in the p53 gene

Knowledge of the genetic changes that lead to disease has grown and continues to grow at a rapid pace. However, there is a need for clinical devices that can be used routinely to translate this knowledge into the treatment of patients. Use in a clinical setting requires high sensitivity and specificity (>97%) in order to prevent misdiagnoses. Single‐strand conformational polymorphism (SSCP) and heteroduplex analysis (HA) are two DNA‐based, complementary methods for mutation detection that are inexpensive and relatively easy to implement. However, both methods are most commonly detected by slab gel electrophoresis, which can be labor‐intensive, time‐consuming, and often the methods are unable to produce high sensitivity and specificity without the use of multiple analysis conditions. Here, we demonstrate the first blinded study using microchip electrophoresis (ME)‐SSCP/HA. We demonstrate the ability of ME‐SSCP/HA to detect with 98% sensitivity and specificity >100 samples from the p53 gene exons 5–9 in a blinded study in an analysis time of <10 min.     

Fast parallel detection of feline panleukopenia virus DNA by multi‐channel microchip electrophoresis with programmed step electric field strength

A multi‐channel microchip electrophoresis using a programmed step electric field strength (PSEFS) method was investigated for fast parallel detection of feline panleukopenia virus (FPV) DNA. An expanded laser beam, a 10× objective lens, and a charge‐coupled device camera were used to simultaneously detect the separations in three parallel channels using laser‐induced fluorescence detection. The parallel separations of a 100‐bp DNA ladder were demonstrated on the system using a sieving gel matrix of 0.5% poly(ethylene oxide) (M_r = 8 000 000) in the individual channels. In addition, the PSEFS method was also applied for faster DNA separation without loss of resolving power. A DNA size marker, FPV DNA sample, and a negative control were simultaneously analyzed with single‐run and one‐step detection. The FPV DNA was clearly distinguished within 30 s, which was more than 100 times faster than with conventional slab gel electrophoresis. The proposed multi‐channel microchip electrophoresis with PSEFS was demonstrated to be a simple and powerful diagnostic method to analyze multiple disease‐related DNA fragments in parallel with high speed, throughput, and accuracy.     

Open microfluidic gel electrophoresis: Rapid and low cost separation and analysis of DNA at the nanoliter scale

Gel electrophoresis is one of the most applied and standardized tools for separation and analysis of macromolecules and their fragments in academic research and in industry. In this work we present a novel approach for conducting on‐demand electrophoretic separations of DNA molecules in open microfluidic (OM) systems on planar polymer substrates. The approach combines advantages of slab gel, capillary‐ and chip‐based methods offering low consumable costs (<0.1$) circumventing cost‐intensive microfluidic chip fabrication, short process times (5 min per analysis) and high sensitivity (4 ng/μL dsDNA) combined with reasonable resolution (17 bases). The open microfluidic separation system comprises two opposing reservoirs of 2–4 μL in volume, a semi‐contact written gel line acting as separation channel interconnecting the reservoirs and sample injected into the line via non‐contact droplet dispensing and thus enabling the precise control of the injection plug and sample concentration. Evaporation is prevented by covering aqueous structures with PCR‐grade mineral oil while maintaining surface temperature at 15°C. The liquid gel line exhibits a semi‐circular cross section of adaptable width (∼200–600 μm) and height (∼30–80 μm) as well as a typical length of 15–55 mm. Layout of such liquid structures is adaptable on‐demand not requiring time consuming and repetitive fabrication steps. The approach was successfully demonstrated by the separation of a standard label‐free DNA ladder (100–1000 bp) at 100 V/cm via in‐line staining and laser induced fluorescent end‐point detection using an automated prototype.     

Development of a PDMS‐based microchip electrophoresis device for continuous online in vivo monitoring of microdialysis samples

A PDMS‐based microfluidic system for online coupling of microdialysis sampling to microchip electrophoresis with fluorescence detection for in vivo analysis of amino acid neurotransmitters using naphthalene‐2,3‐dicarboxaldehyde and sodium cyanide as the derivatization reagents is described. Fabricating chips from PDMS rather than glass was found to be simpler and more reproducible, especially for chips with complex designs. The microchip incorporated a 20‐cm serpentine channel in which sample plugs were introduced using a “simple” injection scheme; this made fluid handling and injection on‐chip easier for the online system compared with gated or valve‐based injection. The microchip was evaluated offline for the analysis of amino acid standards and rat brain microdialysis samples. Next, precolumn derivatization was incorporated into the chip and in vivo online microdialysis‐microchip electrophoresis studies were performed. The system was employed for the continuous monitoring of amino acid neurotransmitters in the extracellular fluid of the brain of an anesthetized rat. Fluorescein was dosed intravenously and monitored simultaneously online as a marker of in vivo blood–brain barrier permeability. The microdialysis‐microchip electrophoresis system described here will be employed in the future for simultaneous monitoring of changes in blood–brain barrier permeability and levels of amino acid neurotransmitters in the rat stroke model.     

Portable capillary electrophoresis system for identification of cattle breeds based on DNA mobility

A portable CE system was developed for the identification of cattle breeds. The system had a width of 44 cm, depth of 27 cm, height of 13 cm, and a weight of only ∼8 kg and included an LIF detector, with everything integrated into a small box. The specific sizes of genes were quickly separated and detected with a high sensitivity based on the difference in the DNA mobility using a diode‐pumped solid‐state LIF detector. Using this system, the 100‐bp DNA ladder was analyzed under a 1.0% PVP (M_r=300 000) sieving gel matrix in a fused silica capillary with LODs of 4.4–13.0 pg/μL (S/N=3) for 100–3000 bp DNAs, which indicates ten times improved value than other commercialized portable CE system. The migration times and the peak areas showed good reproducibilities with relative standard deviations that were less than 0.49 and 1.3% (n=5), respectively. Based on the difference in the DNA mobility of the microsatellite and SNP markers, Korean cattle and Holstein were exactly identified as the model cattle breeds within 32 and 3.5 min, respectively.     

Screening of seized cocaine samples using electrophoresis microchips with integrated contactless conductivity detection

This study describes the development of a new analytical method for the separation and detection of cocaine (COC) and its adulterants, or cutting agents, using microchip electrophoresis (ME) devices coupled with capacitively coupled contactless conductivity detection (C⁴D). All the experiments were carried out using a glass commercial ME device containing two pairs of integrated sensing electrodes. The running buffer composed of 20 mmol/L amino‐2‐(hydroxymethyl) propane‐1,3‐diol and 10 mmol/L 3,4‐dimethoxycinnamic acid provided the best separation conditions for COC and its adulterants with baseline resolution (R > 1.6), separation efficiencies ranging from (2.9 ± 0.1) to (3.2 ± 0.2) × 10⁵ plates/m, and estimated LOD values between 40 and 150 μmol/L. The quantification of COC was successfully performed in four samples seized by the Brazilian Federal Police Department and all predicted values agree with values estimated by the reference method. Some other interfering species were detected in the seized samples during the screening procedure on ME–C⁴D devices. While lidocaine was detected in sample 3, the presence of levamisole was observed in samples 2 and 4. However, their concentrations were estimated to be below the LOQ. ME–C⁴D devices have proved to be quite efficient for the identification and quantification of COC with errors lower than 10% when compared to the data obtained by a reference method. The approach herein reported offers great potential to be used for on‐site COC screening in seized samples.     

Concurrent determination and separation of inorganic cations and anions in microchip electrophoresis with precisely controlled high‐voltage

An improved method for the concurrent determination and separation of cations and anions by microchip electrophoresis with capacitively coupled contactless conductivity detection (ME‐C⁴D) is described. Two kinds of microchip structures were designed. The first microchip has a long bent separation channel. And for the defects of the first microchip, the second microchip with a Y‐type separation channel has been proposed. The background electrolyte (BGE) composed of 20 mm His/MES and 0.01 mm CTAB was optimized for inhibiting the electroosmotic flow (EOF). Due to the low electroosmotic flow, the cations and anions migrate in opposite directions and can be separated from each other. With the precisely controlled high‐voltage, cations and anions can be migrated in microchannels according to our requirements and sequentially detected by a C⁴D detector built in‐house. Samples containing K⁺, Na⁺, Li⁺, Cl⁻, F⁻ and PO₄³⁻ were analyzed simultaneously in a single run (within 140 s) by both methods. The reproducibility obtained by both methods remained below 5% for migration time and within 3.5–9.1% for peak areas. The proposed concurrent determination methods are inexpensive, simple, fast, ease of operation, high degree of integration.     

Preparation of Fritless Packed Silica Columns for Capillary Electrochromatography

Fritless packed silica gel columns were prepared using sol‐gel technology. A part of a 75 μm i.d. fused silica capillary was filled with a mixture of tetramethoxysilane and poly(ethylene glycol). After gelling at 40°C and heating at 300°C, the resultant silica gel was derivatized with dimethyloctadecylchlorosilane. A scanning electron micrograph of a cross‐section of the capillary column showed that the gel took the form of a spherical particle aggregate and adhered to the column inner wall. The column performance was evaluated for electrochromatography using acetonitrile–50 mM HEPES buffer (pH 6.6) (60/40 or 40/60, v/v) as the mobile phase. An electroosmotic flow of 1.0 mm/s was generated with (60/40, v/v) acetonitrile/HEPES buffer at a field strength of 546 V/cm. Using a sol‐gel‐derived packed column at an electroosmotic flow of 0.5 mm/s, efficiencies of up to 1.1×10⁵ plates/m were obtained for retained solutes.     

Precise determination of N‐acetylcysteine in pharmaceuticals by microchip electrophoresis

A novel microchip electrophoresis method for the rapid and high‐precision determination of N‐acetylcysteine, a pharmaceutically active ingredient, in mucolytics has been developed. Isotachophoresis separations were carried out at pH 6.0 on a microchip with conductivity detection. The methods of external calibration and internal standard were used to evaluate the results. The internal standard method effectively eliminated variations in various working parameters, mainly run‐to‐run fluctuations of an injected volume. The repeatability and accuracy of N‐acetylcysteine determination in all mucolytic preparations tested (Solmucol 90 and 200, and ACC Long 600) were more than satisfactory with the relative standard deviation and relative error values <0.7 and <1.9%, respectively. A recovery range of 99–101% of N‐acetylcysteine in the analyzed pharmaceuticals predetermines the proposed method for accurate analysis as well. This work, in general, indicates analytical possibilities of microchip isotachophoresis for the quantitative analysis of simplified samples such as pharmaceuticals that contain the analyte(s) at relatively high concentrations.     

Microchip electrophoresis of Alu elements for gender determination and inference of human ethnic origin

We performed a series of multi‐locus PCRs followed by the rapid and efficient microchip electrophoretic sorting of Alu products with LIF detection. Five polymorphic human‐specific Alu insertions (RC5, A1, PV92, TPA and ACE) were used for inference of human ethnicity and two monomorphic Alu insertions for sex typing, one fixed on the X chromosome (AluSTXa) and the other on the Y chromosome (AluSTYa). These markers were used to generate unique DNA profiles for five different DNA samples. The PCR‐based assays used primers that flank the insertion point to determine genotypes based on the presence or absence of the Alu element. A1, RC5, PV92, TPA and ACE were used for ethnicity determinations and have two alleles, each indicating the presence (+) or absence (?) of the Alu element on the paired chromosomes, which results in three genotypes (+/+, +/? or ?/?). RC5 and A1 did not show ethnic heterogeneity resulting in a homozygous (?/?) genotype, which correctly inferred that DNA samples originating from a Caucasian male and an Asian male were not of African ancestry. The results from the five Alu markers indicated that these Alu loci could assist in identifying the individual's ethnicity using microchip electrophoresis in under 15 min of separation time. Using microchip electrophoresis and mixed genotype ratios, male DNA‐to‐female DNA of 1:9, corresponding to a ratio of Y‐to‐X chromosomes of 1:19, was also detected for both AluSTXa and AluSTYa to provide gender identification without requiring separation of female from male cells prior to the assay.     

Development of a microchip‐pulsed electrochemical method for rapid determination of L‐DOPA and tyrosine in Mucuna pruriens

L ‐3,4‐dihydroxyphenylalanine (L‐DOPA) is a well‐recognized therapeutic compound to Parkinson's disease. Tyrosine is a precursor for the biosynthesis of L‐DOPA, both of which are widely found in traditional medicinal material, Mucuna pruriens. In this paper, we described a validated novel analytical method based on microchip capillary electrophoresis with pulsed electrochemical detection for the simultaneous measurement of L‐DOPA and tyrosine in M. pruriens. This protocol adopted end‐channel amperometric detection using platinum disk electrode on a homemade glass/polydimethylsiloxane electrophoresis microchip. The background buffer consisted of 10 mM borate (pH 9.5) and 0.02 mM cetyltrimethylammonium bromide, which can produce an effective resolution for the two analytes. In the optimal condition, sufficient electrophoretic separation and sensitive detection for the target analytes can be realized within 60 s. Both tyrosine and L‐DOPA yielded linear response in the concentration range of 5.0–400 μM (R² > 0.99), and the LOD were 0.79 and 1.1 μM, respectively. The accuracy and precision of the established method were favorable. The present method shows several merits such as facile apparatus, high speed, low cost and minimal pollution, and provides a means for the pharmacologically active ingredients assay in M. pruriens.     

Separation of carbohydrates on electrophoresis microchips with controlled electrolysis

This study reports the separation of fructose, galactose, glucose, lactose and sucrose on glass microchip electrophoresis (ME) devices using a microfluidic platform adapted with external reservoirs for controlling the electrolysis phenomenon. The connections between external reservoirs and microfluidic platform were performed by saline bridges created using silicone tubing filled with BGE. The separation conditions were optimized and the best results were achieved using a BGE containing 75 mmol/L NaOH and 15 mmol/L trisodium phosphate. Electrophoretic separations were monitored using a capacitively coupled contactless conductivity detection system. The controlled electrolysis has successfully allowed the application of a higher voltage on the separation channel promoting the baseline separation of five carbohydrates within 180 s with great run‐to‐run repeatability (RSD < 1%). The achieved efficiencies ranged from 45 000 ± 6000 to 70 000 ± 3000 plates/m demonstrating a performance better than ME devices without controlled electrolysis. The proposed system offered good linearity from 1 to 10 mmol/L and LODs between 150 and 740 μmol/L. The use of external tubes for controlling the electrolysis phenomenon on ME devices has solved common problems associated to run‐to‐run repeatability and analytical reliability required for routine and quantitative analysis.     

Rapid separation of post‐blast explosive residues on glass electrophoresis microchips

This study describes the development of an analytical methodology based on the use of microchip electrophoresis (ME) devices integrated with capacitively coupled contactless conductivity detection (C⁴D) for the separation and detection of inorganic anions in post‐blast explosive residues. The best separation condition was achieved using a running buffer composed of 35 mmol/L lactic acid, 10 mmol/L histidine and 0.070 mmol/L cetyl(trimethyl ammonium) bromide. For C⁴D measurements, the highest sensitivity was obtained applying a 700 kHz sinusoidal wave with excitation voltage of 20 V_pp. The separation of Cl^?, NO₃^?, NO₂^?, SO₄^2?, ClO₄^? and ClO₃^? was performed within ca. 150 s with baseline resolution and efficiencies between 4.4 × 10⁴ and 1.7 × 10⁵ plates/m. The found limits of detection ranged between 2.5 and 9.5 μmol/L. Last, real samples of post‐blast explosive residues were analyzed on the ME‐C⁴D devices obtaining successfully the determination of Cl^?, NO₃^? and SO₄^2?. The achieved concentration values varied between 12.8–72.5 mg/L for Cl^?, 1.7–293.1 mg/L for NO₃^? and 1.3–201.3 mg/L for SO₄^2?. The data obtained using ME‐C⁴D devices were in good agreement with the concentrations found by ion chromatography. The approach reported herein has provided short analysis time, instrumental simplicity, good analytical performance and low cost. Furthermore, the ME‐C⁴D devices emerge as a powerful and portable analytical platform for on‐site analysis demonstrating to be a promising tool for the crime scene investigation.