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Rapid and quantitative detection of foodborne bacteria is of great significance to public health. In this work, an aptamer-mediated double strand displacement amplification (SDA) strategy was first explored to couple with microchip electrophoresis (MCE) for rapid and ultrasensitive detection of Salmonella typhimurium (S. Typhimurium). In double-SDA, a bacteria-identified probe consisting of the aptamer (Apt) and trigger sequence (Tr) was ingeniously designed. The aptamer showed high affinity to the S. Typhimurium , releasing the Tr sequence from the probe. The released Tr hybridized with template C1 chain, initiating the first SDA to produce numerous output strands (OS). The second SDA process was induced with the hybridization of the liberated OS and template C2 sequence, generating a large number of reporter strands (RS), which were separated and quantified through MCE. Cascade signal amplification and rapid separation of nucleic acids could be realized by the proposed double-SDA method with MCE, achieving the limit of detection for S. typhimurium down to 6 CFU/mL under the optimal conditions. Based on the elaborate design of the probes, the double-SDA assisted MCE strategy achieved better amplification performance, showing high separation efficiency and simple operation, which has satisfactory expectation for bacterial disease diagnosis. [Display omitted] • An aptamer-mediated double SDA-MCE method was first explored for rapid and ultrasensitive detection of S. typhimurium. • The proposed method shows high sensitivity and the limit of detection for S. typhimurium down to 6 CFU/mL. • This method has satisfactory expectation for bacterial disease diagnosis. [ABSTRACT FROM AUTHOR]

T4 polynucleotide kinase (T4 PNK) may catalyze the phosphorylation of 5′-hydroxyl termini in nucleic acids, which play a crucial role in DNA recombination, replication and damage repair. Here, a microchip electrophoresis laser induced fluorescence (MCE-LIF) method based on biochemical reaction was developed for the detection of T4 PNK activity and inhibitors. In this method, the single strand DNA (ssDNA) was hybridized with the 5-carboxyfluorescein (FAM) labeled single strand DNA (ssDNA-FAM) to form FAM labeled double-stranded DNA (dsDNA-FAM). In the presence of T4 PNK and adenosine triphosphate (ATP), T4 PNK catalyzes the transfer of γ-phosphate residues from ATP to the 5-hydroxyl terminal of dsDNA-FAM. The phosphorylated dsDNA-FAM can be gradually hydrolyzed by λexo to produce a FAM labeled single nucleotide fragment. Then the FAM labeled single nucleotide fragment and the unhydrolyzed dsDNA-FAM were separated by MCE, and two electrophoresis peaks appeared in the electrophoretogram. The detection of T4 PNK activity and inhibitors was realized by measuring the peak height of the FAM labeled single nucleotide fragment in electrophoretogram. This assay is very sensitive with a limit of detection of 0.002 U/mL, and it can be further used to screen the T4 PNK inhibitors. Image 1 • A new MCE-LIF method was developed for the assay of T4 polynucleotide kinase activity and inhibitors. • The MCE-LIF method is simple and does not need complex liquid handling procedures. • The as-proposed method provides a wide linear range and a low detection limit for T4 PNK activity analysis. [ABSTRACT FROM AUTHOR]

The number of cases in which scopolamine (SCO) was used for both recreational and predatory purposes has increased dramatically in recent decades. Linked to this, there is a concern about obtaining SCO through thermal degradation of butylscopolamine (BSCO) – an active ingredient of Buscopan® – a drug sold without a medical prescription. In this study, mixtures containing SCO and BSCO were separated and detected on a microchip electrophoresis (ME) device with integrated capacitively coupled contactless conductivity detection (C4D) using a running buffer composed of 40 mmol L−1 of butyric acid and 25 mmol L−1 of sodium hydroxide (pH 5.0). The separation was performed within ca. 115 s with a resolution of 1.3 and separation efficiency ranging from 1.4 × 105 to 1.5 × 105 theoretical plates m−1. A detection limit of 1.1 μmol L−1 was achieved for both species and the developed method revealed satisfactory repeatability with relative standard deviation (RSD) values for forty-eight injections between 4.8 and 9.4% for peak areas and lower than 3.3% for migration times. Furthermore, inter-day precision was evaluated for sixteen injections (a sequence of four injections performed over four days), and RSD values were less than 6.6% for peak areas and 2.2% for migration times. Satisfactory recovery values (95–114%) were obtained for all evaluated beverage samples (cachaça, vodka, whiskey, beer, Coca-Cola, and grape juice) as well as for artificial urine samples (95–107%). Finally, the conversion of BSCO into SCO was observed after simple heating procedure of Buscopan® sample (not subject to medical prescription), which was successfully confirmed through analysis by capillary electrophoresis coupled to the mass spectrometry (CE-MS). Based on the reported results, the use of ME-C4D devices has demonstrated a huge potential for applications in the forensic chemistry field. [Display omitted] • Scopolamine and butylscopolamine were analyzed using electrophoresis microchips. • The proposed method was applied to beverages, urine and Buscopan® samples. • The conversion of butylscopolamine into scopolamine was investigated and confirmed. • The proposed approach provided fast, portable and low-cost drugs analysis. • Electrophoresis chips are promising tools for forensic and clinical applications. [ABSTRACT FROM AUTHOR]

The analysis of exosomes is significant as they can be used for various pathophysiological processes, especially cancer related intercellular communication. Therefore, a convenient, reliable, and sensitive detection method is urgently needed. Strand displacement amplification (SDA) and catalytic hairpin assembly (CHA) are two kinds of effective isothermal nucleic acid amplification methods. In this article, an efficient quantitative MCE method for detecting human breast cancer cell (MCF-7) exosomes assisted by triple amplification strategies combining cholesterol probe (Chol-probe) with SDA-CHA was first developed. CD63 aptamer was immobilized on the avidin magnetic beads to specifically capture exosomes and then Chol-probe with high affinity was spontaneously inserted into the exosome membrane, which was the first step of amplification strategy to improve detection sensitivity. After magnetic separation, Chol-probe could complement ssDNA and trigger SDA, producing a large number of DNA sequences (Ta) to trigger CHA, achieving SDA-CHA amplification. Under optimal conditions, the detection limit (LOD) for MCF-7 exosomes was as low as 26 particle/μL (S/N = 3). This method provides an effective approach for sensitive and accurate quantification of tumor exosomes, and can be expected to detect exosomes in clinical samples. [Display omitted] • A quantitative MCE method for detecting MCF-7 exosomes combining triple amplification strategies was first developed. • This method effectively decreases the detection limit (LOD) of exosomes and the LOD was as low as 26 particle/μL. • This method provides an effective approach for exosomes detection and can be expected to apply in clinical samples. [ABSTRACT FROM AUTHOR]

Excessive intake of kanamycin (KANA) can cause some serious drug-resistant diseases, so it is urgent to develop some accurate and rapid analytical methods for monitor KANA residues in foodstuffs with complex matrix. Recently, many ratiometric assays were reported to be capable of overcoming matrix interference. Herein, a ratiometric and homogeneous assay for KANA detection based on microchip electrophoresis (MCE) was developed. First, by one single strand DNA (S-DNA) and one hairpin DNA (H-DNA), a novel R shape DNA probe (R-DNA) was prepared. After the probe was incubated with KANA, the S-DNA-KANA complex was formed, and H-DNA was released. Moreover, in the presence of exonuclease I (Exo-I), S-DNA-KANA complex would be digested to release the captured KANA for triggering target recycling and signal amplification. With the reaction going on, the fluorescence intensity of H-DNA (I H ) increased and that of R-DNA (I R ) decreased. They can be separated at different voltage intensities and converted to fluorescent signals for signal readout by MCE. The signal ratio of I H /I R was found to be linear toward target from 0.5 pg mL −1 to 10 ng mL −1 , and the limit of detection was 150 fg mL −1 . Moreover, it was successfully employed for KANA detection in milk and fish samples with consistent results of enzyme linked immune sorbent assay (ELISA). The R-DNA probe can quantitatively convert the amount of target to the intensity of DNA without label by MCE, and achieved exonuclease assisted signal amplification in homogenous solution. It was valuable to detect antibiotics residues in foodstuff with complex matrix. This approach broadened the application field of MCE to detect antibiotics without derivatization, which provided a promising platform for rapid screening of antibiotic residues in food. [ABSTRACT FROM AUTHOR]

A multiplex fluorescence signal amplification method based on microchip electrophoresis (MCE) platform was developed for the improvements in the separation and detection of microRNAs. The method used two kinds of fluorescein amidite labeled DNA signal probes to hybridize with its target microRNAs, utilizing T7 exonuclease assisting target circling realized the fluorescence signal amplification. Then, two kinds of fluorescein amidite labeled DNA segments with different size were separated and detected on the MCE-laser induced fluorescence detection platform. The microRNAs-126 and microRNAs-141 were used as model analytes in the proof-of-concept experiments. Two calibration curves between the fluorescence intensity and microRNAs concentration all showed good linearity in the range of 0.025–20 nM. The correlation coefficients obtained were 0.9975 and 0.9925, respectively. The limits of detection for two kinds of microRNAs were estimated to all be 15 pM. By spiking T24 cell lysate samples with varying amounts of miRNA-126 and miRNA-141, the recovery of analytes ranged from 96.0% to 115%, and the relative standard deviations are lower than 5.5%. The present method showed high sensitivity and selectivity, which has a promising application in biomedical research. [ABSTRACT FROM AUTHOR]

Almost four decades of investigations have opened up many avenues to explore the production and utilization of planar (i.e., microchip) gas chromatographic columns. However, there remain many practical constraints that limit their widespread commercialization and use. The main challenges arise from non-ideal column geometries, dead volume issues and inadequate interfacing technologies, which all affect both column performance and range of applications. This review reflects back over the years on the extensive developments in the field, with the goal to stimulate future creative approaches and increased efforts to accelerate microchip gas chromatography development toward reaching its full potential. [ABSTRACT FROM AUTHOR]

Glutathione (GSH) is a major endogenous antioxidant that has a central role in cellular defense against toxins and free radicals. Rapid and accurate detection of GSH content in single cells is important to the early diagnosis of disease and biomedical research. In this work, a novel method based on microchip electrophoresis chemiluminescence (MCE-CL) detection was developed for the quantification of glutathione (GSH) in single cells from rat liver. The detection of GSH is based on the strong sensitization of mercapto compound to luminol-H 2 O 2 CL system. The injection, localization, and membrane dissolution of single cell were simply and rapidly carried out on the microchip by direct electric field force, which did not require any additional membrane dissolution reagent. Under optimized experimental conditions, single cell assay was achieved within 2 min. The peak area of the GSH was taken as quantification of GSH, and a good linear relationship of GSH concentration to peak area in the range of 3.0 × 10 −6 M to 6.0 × 10 −4 M was obtained. The detection limit for GSH is 9.6 × 10 −7 M, calculated by S/N = 3. The measured GSH content in single cells from rat liver (n = 10) ranged from 7.8 fmol to 13. fmol with a mean value of 10.8 fmol. [ABSTRACT FROM AUTHOR]

A novel label-free, universal, and high throughput aptasensor was developed based on a microchip electrophoresis (MCE) platform for automatic detection of antibiotic residues in food. Firstly, chloramphenicol (CAP) was employed as a model to be captured by its aptamer probe (Apt). Then, the partial complementary oligonucleotide of CAP's aptamer (C-DNA) was introduced into the reaction system. Because the Apt-CAP complex can’t further hybrid with free C-DNA, the amount of hybrid Apt-C-DNA double strand DNA (dsDNA) was less than that without adding the target. Finally, the above mixture was introduced into the microchip electrophoresis (MCE) platform for detection, both dsDNA and Apt-CAP can be separated and produce different fluorescence signals in the MCE. In a certain concentration range, the ratio of signal between dsDNA and Apt-CAP (I dsDNA /I Apt-CAP ) was proportional to the concentration of targets. Under the optimum conditions, the ratio showed a satisfactory linearity range from 0.008 to 1 ng/mL of CAP with a detection limit of 0.003 ng/mL. Thus, a universal MCE-based assay was developed for quantifying CAP automatically. The method was also successfully applied in the different food samples for CAP detection, which showed a good recovery (Milk: 91.1–108%, Fish: 86.1–114%) and the results were consistent with that of ELISA. This method owned many merits as follows: firstly, MCE was a high throughput screening platform and the detection time is limited to 3 min for each sample. Secondly, the aptamer probes can be directly used for detection without labeling any signal tag which can facilitate the preparation procedures of probes. Thirdly, the operation was easy just by the following steps: firstly, the mixture of aptamer probes were incubated followed adding C-DNA; then measurement was performed. Moreover, the assay with MCE platform can be used to detect other targets just by changing the corresponding aptamer probe; it can even realize simultaneous detection when the targets have aptamers with different number of base pairs. Above all, it's a high- throughput and prospective method which can be applied in high throughput screening of antibiotics in food safety. [ABSTRACT FROM AUTHOR]

A sensitive immunoassay method based on microchip electrophoresis chemiluminescence (MCE-CL) detection technology was developed for the detection of tumor marker alpha-fetoprotein (AFP). This method adopts the non-competitive immunoassay mode, and was conducted after AFP reacted with excessive horseradish peroxidase (HRP) labeled monoclonal antibody. The extreme pH value was adopted in the electrophoresis buffer solution. The use of brij 35 as an additive of electrophoresis buffer increased dramatically the resolution (Rs) and the reproducibility of the analysis. Under the optimized experimental conditions, effective separation of the immune complex Ag-Ab* and free Ab* was achieved within 60 s. The peak height of the immune complex Ag-Ab* was taken as quantification of AFP. Good linearity was observed within AFP concentrations ranging from 10 ng/mL to 150 ng/mL, and the detection limit was found to be 7.2 ng/mL (1.0×10 −10 M). The present method was successfully applied for the detection of AFP in human serum from both healthy and cancer patients, and the AFP levels in the both were found be in the range of 16.5–23.4 ng/mL and 416.2–825.4 ng/mL, respectively. [ABSTRACT FROM AUTHOR]

This work presents a simple hot embossing method with a shrinking procedure to produce cross-shape microchannels on poly(methyl methacrylate) (PMMA) substrate for the fabrication of an electrophoresis chip. The proposed method employed a simple two-step hot embossing technique, carried out consecutively on the same piece of substrate to make the crossing channels. Studies of embossing conditions, i.e. temperature, pressure and time, were carried out to investigate their effects on the dimension of the microchannels. Applying a simple shrinking procedure reduced the size of the channels from 700±20 µm wide×150±5 µm deep to 250±10 µm wide×30±2 µm deep, i.e . 80% and 64% reduction in the depth and width, respectively. Thermal fusion was employed to bond the PMMA substrate with a PMMA cover plate to produce the microfluidic device. Replication of microchip was achieved by precise control of conditions in the fabrication process (pressure, temperature and time), resulting in lower than 7% RSD of channel dimension, width and depth ( n =10 devices). The method was simple and robust without the use of expensive equipment to construct the microstructure on a thermoplastic substrate. The PMMA microchip was used for demonstration of amine functionalization on the PMMA surface, measurement of electroosmotic flow and for electrophoretic separation of amino acids in functional drink samples. The precision of migration time and peak area of the amino acids, Lys, Ile and Phe at 125 μM to 500 μM, were in the range 3.2–4.2% RSD ( n =9 devices) and 4.5–5.3% RSD ( n =9 devices), respectively. [ABSTRACT FROM AUTHOR]

This report describes the development of an analytical methodology on microchip electrophoresis (ME) devices coupled with capacitively coupled contactless conductivity detection (C 4 D) to monitor inorganic anions in environmental samples. The buffer composition as well as detection operating parameters were optimized to achieve the best separation selectivity and detector sensitivity, respectively. Electrophoretic separations of Cl − , NO 3 − , SO 4 2− and NO 2 − were successfully performed within 60 s using a running buffer composed of 30 mmol L −1 latic acid and 15 mmol L −1 l -histidine (His). The best detectability levels were found applying a sinusoidal wave with 1100-kHz-frequency and 60- V pp amplitude. Quantitative analyzes of inorganic anions were carried out in the presence of Cr 2 O 7 2− ion as internal standard (IS), which ensured great repeatability in terms of migration times (<1%) and peak areas (6.2–7.6%) for thirty consecutive injections. The analytical performance revealed a linear behavior for concentration ranges between 0–120 μmol L −1 (Cl − , NO 2 − and NO 3 − ) and 0–60 μmol L −1 (SO 4 2− ) and limits of detection (LODs) varying from 2.0 to 4.9 μmol L −1 . The concentration levels of anionic species were determined in aquarium, river and biofertilizer samples with recovery values between 91% and 105%. The nitrification steps associated with conversion of ammonium to nitrite followed by the conversion of nitrite to nitrate were successfully monitored in a simulated environment without fishes during a period of twelve weeks. Lastly, the monitoring of anionic species was carried out during eight weeks in an aquarium environment containing ten fishes from Danio rerio (Ciprynidae) . The recorded data revealed the absence of nitrite and a gradual increase on the ammonium and nitrate concentration levels during eight weeks, thus suggesting the direct conversion of ammonium to nitrate. Based on the data herein reported, the proposed analytical methodology can be used for routine environmental analysis. [ABSTRACT FROM AUTHOR]

Bladder cancer (BC) cells spontaneously exfoliated in the urine of patients with BC. Detection of exfoliated tumor cells has clinical significance in cancer therapy because it would enable earlier non-invasive screening, diagnosis, or prognosis of BC. In this research, a method for analyzing genetic abnormalities of BC cells collected from urine samples was developed. Target BC cells were isolated by filtration. To find conditions that achieve high cell recovery, we investigated the effects of filter type, concentration of fixative, and flow rate. Cells captured on the filter membrane were completely retrieved within 15 s. Selected genes for genomic analysis, mutated genes (FGFR3, TERT and HRAS) and methylated genes (ALX4, RALL3, MT1A, and RUNX3) were amplified by polymerase chain reaction (PCR), and subsequently, were identified by microchip electrophoresis (MCE). Analysis by MCE reduces the risk of contamination, sample consumption, and analysis time. Our developed approach is economical, effectively isolates cancer cells, and permits flexible molecular characterization, all of which make this approach a promising method for non-invasive BC detection. [ABSTRACT FROM AUTHOR]

A new method has been developed for separation of chelerythrine and sanguinarine in medicinal plants used in traditional Chinese medicine (TCM). The separation is achieved by microchip electrophoresis (CE) using laser-induced fluorescence detection. The CE separation is achieved by using a hydro-organic medium as the electrolyte buffer. The experimental results are consistent with the prediction by theory in terms of resolution and migration speed because of the low Joule heat generated in microchip CE. In addition, formamide was found to have a potential for separation of molecules with similar chemical structures. Based on these findings, a run buffer containing 50% formamide was used to separate chelerythrine (CHE) and sanguinarine (SAN). The influencing factors, such as solvent of run buffer, pH of buffer, separation distance, and separation voltage, were optimized. Baseline separation of chelerythrine and sanguinarine was achieved within 120 s under an electrical voltage of 1.8 kV. Good linearity was observed in the concentration range of 0.15–550 μg mL −1 ( r =0.9993) for CHE and in the range of 0.3–600 μg mL −1 ( r =0.9998) for SAN. A low limit of detection (LOD) was achieved because of the high sensitivity achieved by laser-induced fluorescence detection (i.e. 5.0 ng mL −1 and 2.0 ng mL −1 for CHE and SAN, respectively). The contents of CHE are found to be 641.8±7.5 and 134.0±2.3 mg/kg in extracts of Macleaya cordata and Chelidonium majus , respectively, with good recovery of above 99%. The corresponding values for SAN found in these Chinese herbal extracts are 681.8±7.9 mg/kg and 890.5±8.9 mg/kg, respectively. [ABSTRACT FROM AUTHOR]

An on-line preconcentration strategy combining field-amplified stacking and reversed-field stacking was developed for efficient and sensitive analysis of amino acids and vitamin B 3 including lysine (Lys), taurine (Tau), and niacinamide (NA) by microchip electrophoresis with LIF detection. In this technique, the addition of a reversed-polarity step termed reversed-field stacking could enhance the preconcentration effect of field-amplified stacking and push most of the sample matrix out of the separation channel, thus greatly improving the sensitivity enhancement by 1–2 orders of magnitude over the classical MCE–LIF methods. The related mechanism as well as important parameters governing preconcentration and separation have been investigated in order to obtain strongest sensitivity amplification and maximum resolution. Under optimal conditions, all analytes were successfully focused and completely separated within 4 min. The limits of detection for Lys, Tau, and NA were 0.25, 0.50, and 0.20 nM (S/N=3), respectively, and enhancement factors of 165-, 285-, and 236-fold were obtained for Lys, Tau, and NA as compared to using the no concentration step. Other validation parameters such as linearity and precision were considered as satisfactory. The proposed method also gave accurate and reliable results in the analysis of these functional ingredients in eight functional drink samples. [ABSTRACT FROM AUTHOR]

Abstract: In this work, a simple and convenient method for the detection of mini-short tandem repeat (miniSTR) loci has been developed by the combination of polymerase chain reaction (PCR) and microchip electrophoresis (MCE). Degraded or inhibitor DNA greatly limited STR loci analysis. Therefore, The proper primers was designed as close as possible to the STRs region to produce smaller size STRs, and made the assay suitable for the destroyed samples. Two annealing temperatures were applied in one PCR procedure and the corresponding cycle numbers were studied to improve the sensitivity of PCR reaction. Under optimal conditions, 0.001ng DNA templates were enough to generate miniSTRs. The relative standard deviations (n=3) of the size fifteen miniSTRs from DNA9947A ranged from 0.49% to 4.41%. The RSDs of concentrations were between 0.94% and 4.95%. Fifteen miniSTRs were also well produced from human hair, indicating that the method has great potential application in criminal identification and paternity testing. [Copyright &y& Elsevier]

Abstract: An ultra-fast and precise microchip electrophoresis (ME) method was developed for the separation of infectious disease-related small DNA molecules. As a model of infectious disease-related small DNA molecules, the spike glycoprotein (S) gene of the Feline infectious peritonitis (FIP) virus was amplified using reverse transcript polymerase chain reaction. The amplified product of the FIP virus (223-bp) was analyzed within 10s by single-channel ME under a sieving gel of 0.3% poly(ethylene oxide) (M r=8,000,000) in 1x TBE buffer (pH 8.33) and a short effective channel length of 1.3cm with a programmed step electric field strength (PSEFS) condition as follows: 470.6V/cm for 9s, 294.1V/cm 1.5s, and 470.6V/cm for 9.5s. The single-channel ME/PSEFS method was 50 times faster than that obtained with conventional slab gel electrophoresis. When the single-channel ME method was applied to a multi-channel ME for high-throughput screening, the precision of migration time and peak area showed standard deviations of less than 1.0% without any loss of resolving power. The ME assay technique provides a simple, precise and accurate method for ultra-fast analysis of infectious disease-related DNA under 400-bp. [Copyright &y& Elsevier]

Abstract: A multi-channel microchip electrophoresis (MC-ME) system with a laser-induced fluorescence detector was developed for the fast simultaneous detection of rice knockout mutants in genetically modified (GM) rice. In addition, three parallel separation channels were fabricated on a glass microchip to investigate the possibility of high-throughput screening of amplified-polymerase chain reaction products representing wild-type rice and mutants. The MC-ME system was developed to simultaneously record data on all channels using specifically designed electrodes for an even distribution of electric fields, an expanded laser beam for excitation, a 10× objective lens to capture emissions, and a charge coupled device camera for detection. Under a programmed electric field strength and a sieving gel matrix of 0.7% poly(ethylene oxide) (M r=8,000,000), T-DNA-inserted rice mutants, two standard wild-type rice lines, and six rice knockout mutants were analyzed within 4min using three parallel channels on the microchip. Compared to conventional microchip electrophoresis, the MC-ME method is a valid and practical way to effectively analyze multiple samples in parallel for the identification of GM rice without any loss of resolving power or reproducibility. The MC-ME method was more than 15 times faster than traditional slab gel electrophoresis and proved to be a powerful tool for high-throughput screening of GM rice with high sensitivity, efficiency, and reproducibility. [Copyright &y& Elsevier]

Abstract: A new and fast method for haptoglobin phenotyping was developed based on microchip electrophoresis with laser induced fluorescence detection. Haptoglobin phenotypes 1-1 and 2-2 were labeled with fluorescein isothiocyanate. The analyses were performed on glass microchip which was simply treated with sodium dodecyl sulfate. After the optimization of the separation conditions, Hp 1-1 and Hp 2-2 could be differentiated in 150s and the detection limits for Hp 1-1 and Hp 2-2 were 0.39 and 0.62μg/mL, respectively. Finally, the method was applied to human serum samples from healthy people and liver cancer patients. A decrease in Hp concentration for liver cancer patients was confirmed. Featuring high efficiency, speed, simplicity, the method reveals great potentials for the diagnosis of diseases and proteome research. [Copyright &y& Elsevier]

Abstract: In this work, the rapid detection of cholesterol using poly(dimethylsiloxane) microchip capillary electrophoresis, based on the coupling of enzymatic assays and electrochemical detection, was developed. Direct amperometric detection for poly(dimethylsiloxane) (PDMS) microchip capillary electrophoresis was successfully applied to quantify cholesterol levels. Factors influencing the performance of the method (such as the concentration and pH value of buffer electrolyte, concentration of cholesterol oxidase enzyme (ChOx), effect of solvent on the cholesterol solubility, and interferences) were carefully investigated and optimized. The migration time of hydrogen peroxide, product of the reaction, was less than 100s when using 40mM phosphate buffer at pH 7.0 as the running buffer, a concentration of 0.68U/mL of the ChOx, a separation voltage of +1.6kV, an injection time of 20s, and a detection potential of +0.5V. PDMS microchip capillary electrophoresis showed linearity between 38.7μg/dL (1μM) and 270.6mg/dL (7mM) for the cholesterol standard; the detection limit was determined as 38.7ng/dL (1nM). To demonstrate the potential of this assay, the proposed method was applied to quantify cholesterol in bovine serum. The percentages of recoveries were assessed over the range of 98.9–101.8%. The sample throughput was found to be 60 samples per hour. Therefore, PDMS microchip capillary electrophoresis, based on the coupling of enzymatic assays and electrochemical detection, is very rapid, accurate and sensitive method for the determination of cholesterol levels. [Copyright &y& Elsevier]

Abstract: In this paper, a compact and inexpensive light emitting diode induced fluorescence (LED-IF) detector with simplified optical configuration was developed and assembled in an integrated microfluidic device for microscale electrophoresis. The facile detector mainly consisted of an LED, a focusing pinhole, an emission filter and a photodiode, and was encapsulated in the upper layer of an aluminum alloy device with two layers. At the bottom layer, integrated circuit (IC) was assembled to manipulate the voltage for sample injection and separation, LED emission and signal amplifying. A high-power LED with fan-shaped heat sink was used as excitation source. The excitation light was focused by a 1.1mm diameter pinhole fabricated in a thin piece of silver foil, and the obtained sensitivity was about 3 times as high as that using electrode plate. Other important parameters including LED driven current, fluorescence collection angle and detection distance have also been investigated. Under optimal conditions, considerable high-response of 0.09fmol and 0.18fmol mass detection limits at 0.37nL injection volume for sodium fluorescein (SF) and FITC was achieved, respectively. This device has been successfully employed to separate penicillamine (PA) enantiomers. Due to such significant features as low-cost, integration, miniaturization, and ease of commercialization, the presented microfluidic device may hold great promise for clinical diagnostics and bioanalytical applications. [Copyright &y& Elsevier]

Abstract: A new SU-8 based microchip capillary electrophoresis (MCE) device has been developed for the first time with integrated electrochemical detection. Embedded electrophoretic microchannels have been fabricated with a multilayer technology based on bonding and releasing steps of stacked SU-8 films. This technology has allowed the monolithic integration in the device of the electrochemical detection system based on platinum electrodes. The fabrication of the chips presented in this work is totally compatible with reel-to-reel techniques, which guarantee a low cost and high reliability production. The influence of relevant experimental variables, such as the separation voltage and detection potential, has been studied on the SU-8 microchip with an attractive analytical performance. Thus, the effective electrical isolation of the end-channel amperometric detector has been also demonstrated. The good performance of the SU-8 device has been proven for separation and detection of the neurotransmitters, dopamine (DA) and epinephrine (EP). High efficiency (30,000–80,000N/m), excellent precision, good detection limit (450nM) and resolution (0.90–1.30) has been achieved on the SU-8 microchip. These SU-8 devices have shown a better performance than commercial Topas (thermoplastic olefin polymer of amorphous structure) microchips. The low cost and versatile SU-8 microchip with integrated platinum film electrochemical detector holds great promise for high-volume production of disposable microfluidic analytical devices. [Copyright &y& Elsevier]

Abstract: 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 γ (TCRγ) 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. [Copyright &y& Elsevier]

Abstract: Different methods for construction of contactless conductivity detectors (CCD) for microchip electrophoresis device are described in this review. This includes three main schemes of CCD for microchips, such as (i) the detection electrodes are placed along the microchannel from outside of the microchip and they are insulated from the channel by the cover lid of microchip device; (ii) the electrodes are placed across of the microchannel in the same plane and they are insulated by thin separation channel walls and (iii) electrodes are buried in widened part of microchannel and they are insulated from solution by ultrathin layer of silicon carbide. Specific issues related to the CCD on microfluidics are discussed, such as an influence of shape and magnitude of ac voltage and placement of electrodes and their insulation. Various applications for security, pharmacological, bioassays and food analysis purposes are described. [Copyright &y& Elsevier]

Abstract: 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 2.8–5.6mM. Under an electric field of 333.3V/cm and a sieving matrix of 1.0% poly(ethyleneoxide) (M r 600000), the amplified PCR products were analyzed within 6min 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 30s, 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. [Copyright &y& Elsevier]

Abstract: An easy and universal wall-jet configuration for microchip CE-ECL detection system was constructed and investigated in this work. Two detection modes of pre-column and post-column were applied to the above system. TPA, tramadol and lidocaine were chosen as model analytes to estimate the system in both modes. The important operational parameters such as the concentration of luminescent reagent and the distance between the separation outlet and the working electrode were optimally obtained and compared for the first time. [Copyright &y& Elsevier]

Abstract: Poly(dimethylsiloxane) (PDMS) microfluidic channels modified by citrate-stabilized gold nanoparticles after coating a layer of linear polyethylenimine (LPEI) were successfully used to separate dopamine and epinephrine, which were difficult to be separated from baseline in native and hybrid PDMS microchannels. In-channel amperometric detection with a single carbon fibre cylindrical electrode was employed. Experimental parameters of separation and detection processes were optimized in detail. The analytes were well separated within 100s in a 3.7cm long separation channel at a separation voltage of +800V using a 30mM phosphate buffer solution (PBS, pH 7.0). Linear responses of them were obtained both from 25 to 600μM with detection limits of 2μM for dopamine and 5μM for epinephrine, respectively. The modified PDMS channels have a long-term stability and an excellent reproducibility within 2 weeks. [Copyright &y& Elsevier]

Abstract: Microchip electrophoresis is a promising technique for analysis of bio-molecules. It has the advantages of fast analysis, high sensitivity, high resolution and low-cost of samples. Plastic chip has the potential of mass production for clinical use for its advantages in biocompatibility and low cost. In this work, the method for fabrication of poly(methyl methacrylate) (PMMA) chip was described, and conditions for DNA separation were investigated with the chip. The PMMA microchip was used for detection of multiplex PCR products of 18 and 36 cases with SARS and hepatitis B virus infection under optimized separation conditions. Microchip electrophoresis showed higher sensitivity, higher resolution and less time consumption when compared with gel electrophoresis. The microchip electrophoresis with PMMA chip provided a rapid, sensitive and reliable method for analysis of multiplex PCR products. [Copyright &y& Elsevier]

A glass electrophoresis microchip integrated a flow-type chemiluminescence (CL) detection cell has been developed and evaluated. The chip pattern is a double-T-type electrophoretic sample injection and separation combining with a Y-type chemiluminecent detector. The double-T geometry allows for high-efficiency sample injection and geometric definition of sample plug size. The branch of Y was used as CL reagent channel, and the CL reagent was delivered by a lab-made micropump. Bis[(2,4,6-trichlorophenyl)]oxalate-H2O2 CL system was employed to detect dansyl amino acids. On this microchip, dansyl-phenylalanine and -sarcosine were successfully separated by electrophoresis and detected within 250 s. The detection limits (S/N=3) of dansyl-phenylalanine and -sarcosine could reach to 2.8 and 3.2 μM, respectively, due to the vigorous dilution of sample with CL reagent and timely removal of the waste solution from reaction area. [Copyright &y& Elsevier]

A method to quantitatively perform reverse transcription-competitive PCR (RT-cPCR) of hepatitis C virus followed by both microchip and capillary electrophoretic separation and detection was described. In this method, HCV wild-type (WT) RNA extracted from serum was coretrotranscribed and coamplified with a constant amount of recombinant internal standard (IS) RNA which had the same primer binding region as the target RNA and was constructed by removing a centrally located 25-bp segment from the target template. A linear calibration curve was constructed by adding IS RNA at a constant concentration of 8000 copies μl−1 into a series of RNA target standards ranging from 400 to 106 copies μl−1. The amplified IS and target DNA were detected by both capillary and microchip electrophoresis via laser-induced fluorescence (LIF) using Cy5-labelled primer as the fluorescence probe. The method was further demonstrated for the quantitation of clinical patients with low, medium, and high viral titer and the results were found to be comparable to those determined by the commercial bDNA assay. [Copyright &y& Elsevier]

Microchip capillary electrophoresis (MCE) is a powerful technique for rapid separation; however, its acceptance in routine laboratories is still limited. Compromises caused by the efforts for solving different problems, such as reducing its cost of fabrication and ensuring high separation efficiency, undermine the competitiveness of this technology compared to other separation techniques. Contrary to the conventional pursuit of narrow microchannels, this study investigated the suitability of microchips with channels at the sub-millimeter level, targeting the simplification of the overall operation, cost reduction, and robustness improvement. To this effect, we considered the influence of pressurized flow and Joule heating on the separation. The suppression of pressurized flow with viscous solutions was confirmed through a combination of simulations and experimental results, indicating that the buffer viscosity was enough for successful separation. We fabricated channels of 200 μm × 230 μm using computer numerical controlled (CNC) machining and obtained theoretical plate numbers of 4.8 × 105 m−1 and 5.3 × 105 m−1 for fluorescein isothiocyanate (FITC) labeled small molecules and DNA fragments, respectively, with a buffer viscosity of 168 mPa s (0.5 % hydroxypropyl methylcellulose, HPMC). These values are comparable with that of narrow-bore microchips. Furthermore, we did not observe any deleterious effects with low-conductivity buffers. We investigated the rapid and highly sensitive detection of mycoplasma contamination and the real samples of circulating cell-free DNA (cfDNA), which gave a limit of detection (LOD) as low as 2.3 ng mL−1. Owing to the significant reduction in cost, ease of operation, and fast separation capabilities demonstrated in this work, MCE can be a viable alternative to the usual slab gel electrophoresis running in most biological laboratories. [Display omitted] • Sub-millimeter channels were adopted to enable easy-to-use microchip electrophoresis. • The use of large-bore channels can effectively reduce the cost of microchips. • The operation can be simplified with improved tolerance of airborne particles. • The circulating cell-free DNA extracted from serum can be easily detected. • The work is an attempt to prompt the acceptance of MCE in routine analyses. [ABSTRACT FROM AUTHOR]

This report describes the use of electrophoresis microchips integrated with contactless conductivity detection for the determination of organic acids and inorganic anions in wine samples and the subsequent classification based on the grape varieties. The best separation was achieved using a buffer composed of 30 mmol L−1 2-(N-morpholino)ethanesulfonic acid, 15 mmol L−1 l -histidine and 0.05 mmol L−1 cetyltrimethylammonium bromide (pH 5.8), allowing the determination of chloride, nitrate, sulfate, oxalate, tartrate, maleate, succinate, citrate, acetate, lactate, pyroglutamate and phosphate within ca. 100 s. The relative standard deviations obtained for the migration times were lower than 2%, while the obtained values for peak areas ranged from 2.5 to 8.4%. The limits of detection achieved for all compounds ranged between 3.0 and 12.6 μmol L−1. A total of 18 wines from Brazil and Chile were successfully investigated, including red, white and rosé, and the anionic species were quantified with recovery values between 92 and 117%. A statistical difference has not been observed between the data obtained by using electrophoresis microchips integrated with contactless conductivity detection (ME-C4D) and capillary electrophoresis with ultra-violet detection (CE-UV) and thus the results from newly developed method is validated. Finally, similarities among the anionic profile of wines were investigated by using a multivariate approach, and it was possible to discriminate samples mainly by grapes varieties. Furthermore, the proposed methodology has provided instrumental simplicity and good analytical performance, demonstrating to be useful for routine quality control of wines. [Display omitted] • Organic and inorganic anions were separated within 100 s using electrophoresis chips.. • Sulfate, tartrate, maleate, succinate, acetate, lactate and phosphate were quantified. • The methodology was applied to analyze 18 wine samples from Chile and Brazil. • Red, white and rose wines were discriminated through multivariate analysis.. • Microfluidic chips emerge as fast and portable platform for food science research. [ABSTRACT FROM AUTHOR]

This review article summarises aspects of the determination of amino acids using capillary and chip electrophoresis in combination with contactless conductivity detection from their historical beginnings to the present time. Discussion is included of the theory of conductivity detection in electromigration techniques, the design of contactless conductivity cells for detection in capillaries and on microchips, including the use of computer programs for simulation of the conductivity response and the process of the electrophoretic separation of amino acids. Emphasis is placed on optimisation of the background electrolyte composition, chiral separation, multidimensional separation, stacking techniques and the use of multidetection systems. There is also a description of clinical applications, the determination of amino acids in foodstuffs, waters, soils and composts with emphasis on modern techniques of sample treatment, such as microdialysis, liquid membrane extraction and many other techniques. Image 1 • Contactless conductivity detection in capillary and chip electrophoresis for analysis of amino acids. • Theory, design of detection cells, simulation of the conductivity response. • BGE, chiral separation, multidimensional separation, stacking and multidetection systems. • Clinical applications, determination of amino acids in foodstuffs, waters, and soils. • Emphasis on sample treatment - microdialysis, liquid membrane extraction. [ABSTRACT FROM AUTHOR]

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the main pathogens involved in hospital and community infection. To rapidly and sensitively detect the mec A gene, which is relevant to methicillin-resistant strains, microchip electrophoresis (MCE) integrated with isothermal strand-displacement polymerase reaction (ISDPR) was developed. In the ISDPR signal recycle amplification, the target DNA opened the DNA hairpin structure by specifically binding with the hairpin probe (HP), and then the primer hybridized with the probe and released the target DNA in the presence of Klenow Fragment exo− (KF exo−) polymerase. The released target DNA hybridized with the next HP and then was displaced by the primer again, consequently achieving target recycling and amplification. The amplified products of the HP-cDNA duplex were separated rapidly from other DNAs by MCE. Under optimal conditions, the limit of detection of the target DNA was as low as 12.3 pM (S/N = 3). The proposed ISDPR with MCE method was also successfully applied to detect methicillin-resistant S. aureus , and the experimental results showed that it had some advantages such as being label free, ultrasensitive, rapid and well separated. Image 1 • ISDPR was combined with microchip electrophoresis to achieve target recycling amplification and rapid separation. • The proposed method shows high specificity and gets low limit of detection. • This strategy can successfully detect the mec A gene in MRSA and other nucleic acids. [ABSTRACT FROM AUTHOR]

The separation of chiral amino acids using microchip electrophoresis (MCE) was investigated using chiral nematic mesoporous silica (CNMS) as the chiral stationary phase, with hydroxypropyl-β-cyclodextrin (HP-β-CD) as the chiral selector. Individually, neither CNMS nor HP-β-CD achieved separation, so they were combined. Ten chiral amino acids (phenylalanine, tryptophan, glutamic, alanine, serine, aspartic acid, cysteine, methionine, tyrosine, and histidine) were selected as the model analytes. Under optimized conditions, we achieved baseline separation of six chiral amino acids, and the other four chiral amino acids displayed improved resolution. These results indicate the presence of a synergistic effect between CNMS and HP-β-CD, showing that the combination of a chiral stationary phase and a chiral additive is a promising approach for enantioseparation using MCE. The chiral nematic mesoporous silica (CNMS) was applied for the first time in MCE to evaluate the potential synergistic effects with hydroxypropyl-β-cyclodextrin (HP-β-CD) for enantiomeric separations. The results showed that six chiral amino acids were achieved baseline separations and other four chiral amino acids were successfully improved resolutions although baseline separations were not achieved. The results displayed a significant synergistic effect. Hence, the combination of synergistic effect using CNMS with HP-β-CD in MCE is a promising approach to enantioseparation. Image 1 • The CNMS was applied in MCE to evaluate the potential synergistic effects with HP-β-CD for enantiomeric separations. • This paper showed that the results displayed a significant synergistic effect between CNMS and HP-β-CD. • The combination of synergistic effect using CNMS with HP-β-CD in MCE is a promising approach to enantioseparation. [ABSTRACT FROM AUTHOR]

Electrohydrodynamic migration, which is based on hydrodynamic actuation with an opposing electrophoretic force, enables the separation of DNA molecules of 3–100 kbp in glass capillary within 1 h. Here, we wish to enhance these performances using microchip technologies. This study starts with the fabrication of microchips with uniform surfaces, as motivated by our observation that band splitting occurs in microchannels made out of heterogeneous materials such as glass and silicon. The resulting glass-adhesive-glass microchips feature the highest reported bonding strength of 11 MPa for such materials (115 kgf/cm2), a high lateral resolution of critical dimension 5 μm, and minimal auto-fluorescence. These devices enable us to report the separation of 13 DNA bands in the size range of 1–150 kbp in one experiment of 5 min, i.e. 13 times faster than with capillary. In turn, we observe that bands split during electrohydrodynamic migration in heterogeneous glass-silicon but not in homogeneous glass-adhesive-glass microchips. We suggest that this effect arises from differential Electro-Osmotic Flow (EOF) in between the upper and lower walls of heterogeneous channels, and provide evidence that this phenomenon of differential EOF causes band broadening in electrophoresis during microchip electrophoresis. We finally prove that our electrohydrodynamic separation compares very favorably to microchip technologies in terms of resolution length and features the broadest analytical range reported so far. Image 1 • Single run separation of DNA from 1 to 150 kbp in 4 min. • Report of a fabrication technology for perfectly homogeneous glass-glass microchips. • Demonstration that separation performances are improved with glass-glass vs. Silicon-glass microchips. • Evidence that differential electro-osmotic flow in silicon-glass chips enhances band broadening. [ABSTRACT FROM AUTHOR]

Rapid and accurate detection of microRNA content in cells is of great significance. Here, an ultrasensitive microchip electrophoresis (MCE) method based on cascade chemiluminescence (CL) signal amplification was developed for the detection of microRNA-21 in cells. In this method, horseradish peroxidase labeled DNA was used as a signal probe, which could induce CL signal by the reaction of luminol and H 2 O 2. Combining with two cyclic enzyme digestion reactions by T7 exonuclease, a large number of signal probes were degraded. By using MCE-CL as a separation and detection platform, an amplified CL signal peak was achieved. The developed MCE-CL method can detect miR-21 at a concentration as low as 1.0 × 10−15 M, which was enhanced by six orders of magnitude compared with those of conventional MCE-CL assay. This method has been applied for the detection of microRNA-21 in cell lysate, which show that there were significant differences of miR-21 among different types of cells, and the content in cancer cells was much higher than that in normal cells, which can be used for the identification of cancer cells. Therefore, the proposed method held great application potential in early diagnosis of tumor and biomedical research. Image 1 • An ultrasensitive MCE-CL method was developed for the detection of microRNA-21 in cells. • The MCE-CL method is simple and does not need complex liquid handling procedures. • The as-proposed method provides a very low detection limit for the identification of cancer cells. [ABSTRACT FROM AUTHOR]