微流控芯片操纵传输及实时监测单细胞量子释放

微流控芯片技术用于细胞生化分析已引起了广泛关注.Harrison等首次在微流控芯片上对细胞群体进行操纵、传输及反应.yang等在微流控芯片上操纵细胞群体的排列,并用荧光检测细胞群体摄取钙的反应.至今还未见到微流控芯片对单个细胞进行操纵传输、定位及实时监测的报道.单细胞受激释放的监测对探索生物体神经传导具有重要意义.     

微流控芯片荧光检测系统的研究进展北大核心CSCD

微流控芯片又称芯片实验室,具有检测高效、消耗试剂少、高通量、微型化和集成化等特点,许多检测方式(如光学检测、电化学检测)已经集成于微流控芯片上,而荧光检测是微流控芯片检测技术的常见手段之一。为此,在介绍了荧光检测技术的基本原理和光路结构的基础上,从激发光源、光传辅助手段和检测器等方面综述了微流控芯片荧光检测系统的研究进展,并对其发展进行了展望(引用文献55篇)。     

新型纳米荧光探针在微流控细菌芯片检测中的应用

微流控芯片分析技术可以集成不同的生物化学分析功能单元,广泛应用于生化分析领域,在细菌检测方面具有传统检测方法不可比拟的优越性。近来年,在微流控细菌芯片中引入高荧光强度、低背景荧光干扰和高选择性的纳米荧光探针为实现细菌高效检测分析提供了新的研究途径和技术手段。本文通过对细菌检测中的几类新型荧光标记探针的介绍和比较,分析其荧光效应和应用特点,尤其是在细菌检测中的应用特性,重点综述了新型高效的纳米荧光探针与微流控细菌芯片分析方法和技术结合,实现微尺度空间和荧光检测模式下的细菌高效检测。     

基于微流控芯片的72重单核苷酸多态性族群推断系统的构建

建立了常染色体单核苷酸多态性（SNPs）复合检测芯片体系,用于未知个体的族群来源推断。基于前期筛选的74-SNPs组合,采用竞争性等位基因特异性聚合酶链式反应（PCR）的原理构建SNPs的扩增体系,在微流控芯片的每个反应孔内完成一个SNP的检测,通过高通量PCR微流控芯片实现了其中72个SNPs的同步检测。芯片的扩增由平板PCR仪完成,反应孔的荧光信号通过激光共聚焦扫描仪检测,最终通过提取的荧光值进行结果分析。使用该芯片检测获得52份样本的SNPs分型,分型结果的准确率为100%。以57个人群的3628个样本为参考人群数据库,进行20份样本的族群来源推断,推断结果与样本的实际来源一致。本研究建立的常染色体72个SNPs微流控芯片体系可以有效地进行SNP多态性分析检测,基于参考数据库,20份检测样本族群推断的准确性为100%。     

微流控芯片电泳的研究进展

该文综述了微流控芯片电泳的制备、结构和应用,比较了不同材料微流控芯片电泳的制备机理、表面改性和性能特点,归纳和总结了不同结构微流控芯片电泳的进样、分离和检测系统以及不同类型微流控芯片电泳在荧光物质、金属离子、糖、药物、核酸、DNA、氨基酸、多肽和蛋白质分析中的应用,并对微流控芯片电泳的未来发展方向做了展望.     

微流控芯片-激光诱导荧光检测器的研制及核酸片段分离检测中应用

采用自行设计的共焦式激光诱导荧光检测器（激发波长635nm，发射波长670nm），以cy5染料对检测器的空间分辨能力和灵敏度进行了测试，检出限达到10^-9mol／L。建立了一种微流控芯片快速分离检测DNA片段的方法。以0．8％羟丙基甲基纤维素（HPMC）为筛分介质，以（噻唑橙单体）To-pro-3为荧光标记染料，在玻璃微流控芯片中实现了dsDNA片段的无胶筛分和激光诱导荧光检测，12条DNA片段在75s内得到分离。     

微流控芯片荧光检测系统研究进展

评述了1996～2010年以来微流控芯片荧光检测系统的研究进展,主要介绍微流控芯片中荧光检测系统,包括激光诱导荧光（LIF）、发光二极管（LED）诱导荧光和其他荧光检测装置的原理、光路结构及其应用（引用文献60篇）。     

微流控芯片分析及其在酶抑制剂筛选中的应用*

微流控芯片以其消耗少、易于微型化和集成化等优点在酶分析领域占有重要地位。近年来随着新检测技术的不断出现,酶抑制剂筛选芯片的结构也从简单的“混合-反应”和“分离-检测”,变得更加多样化和多功能化。微流控芯片上分子固定化酶、细胞培养等技术的进步为微流控芯片上实现酶抑制剂的高通量和高内涵筛选带来了巨大优势。本文对用于酶分析的微流控芯片的种类和构型进行简介和归纳总结,重点讨论和综述了其在酶抑制剂筛选中的应用及其最新研究进展。     

微流控芯片电化学检测单细胞分析

微流控芯片已被用于进行各种细胞分析的研究.最近,方肇伦等^[1]用十字型微流控芯片压力进样,激光诱导荧光检测进行了人单个血红细胞内谷胱甘肽的测定.用双T型微流控芯片电化学检测方法对小麦愈伤组织中抗坏血酸(AA)的单细胞分析进行了研究.     

微流控芯片酶分析及其在酶抑制剂筛选中的应用

微流控芯片以其消耗少、易于微型化和集成化等优点在酶分析领域占有重要地位。近年来随着新检测技术的不断出现,酶抑制剂筛选芯片的结构也从简单的"混合-反应"和"分离-检测"变得更加多样化和多功能化。微流控芯片上分子固定化酶、细胞培养等技术的进步为微流控芯片上实现酶抑制剂的高通量和高内涵筛选带来了巨大优势。本文对用于酶分析的微流控芯片的种类和构型进行简介和归纳总结,重点讨论和综述了其在酶抑制剂筛选中的应用及其最新研究进展。     

Microchip-based capillary electrophoresis for determination of lactate dehydrogenase isoenzymes

This article describes a novel microchip-based capillary electrophoresis and oncolumn enzymatic reaction analysis protocol for lactate dehydrogenase (LDH) isoenzymes with a home-made xenon lamp-induced fluorescence detection system. A microchip integrated with a temperature-control unit is designed and fabricated for low-temperature electrophoretic separation of LDH isoenzymes, optimal enzyme reaction temperature control, and product detection. A four-step operation and temperature control are employed for the determination of LDH activity by on-chip monitoring of the amount of incubation product of NADH during the fixed incubation period and at a fixed temperature. Experiments on the determination of LDH standard sample and serum LDH isoenzymes from a healthy adult donor are carried out. The results are comparable with those obtained by conventional CE. Shorter analysis times and a more stable and lower background baseline can be achieved. The efficient separation of different LDH forms indicates the potential of microfluidic devices for isoenzyme assay.     

Screening of drug metabolism by CE

The use of CE for rapid assessment of metabolic stability of drugs with cytochrome P450 (CYP) enzymes, based on relative rates of reduced nicotinamide adenine dinucleotide phosphate (NADPH) consumption and nicotinamide adenine dinucleotide phosphate (NADP) production, was investigated. The separation conditions were as follows: capillary, 80.5 cm (75 microm id, 72 cm effective length for UV detection, 58 cm effective length for fluorescence detection); 25 mM sodium phosphate buffer (pH 8.8); 28 kV (80 microA) applied voltage; UV, 260 nm; fluorescence detection, excitation wavelength, 310 nm, emission wavelength, 418 nm; capillary temperature, 25 degrees C. For UV detection, the incubation conditions were as follows: CYP3A4: 20 pmol/mL; NADPH: 1 mM; EDTA: 1 mM; concentration of the substrate: 5-10 times its reported literature K(m) value; temperature: 37 degrees C; incubation time: 15 min. For fluorescence detection, the concentrations were reduced to CYP3A4: 4 pmol/mL, NADPH: 20 microM, EDTA: 20 microM and substrate: 10 microM. Blank incubations were performed in the absence of substrate. Compared with the blank, significant differences were found for the consumption of NADPH and the production of NADP. The development of this assay system allows rapid assessment of metabolic stability relative to standard compounds, as well as potential identification of the major CYP involved in the metabolism. It would reduce the backlog of compounds that require LC/MS analysis, and thereby expedite the process of metabolic stability screening.     

Competitive immunoassay of phenobarbital by microchip electrophoresis with laser induced fluorescence detection

A microchip electrophoresis method with laser induced fluorescence detection was developed for the immunoassay of phenobarbital. The detection was based on the competitive immunoreaction between analyte phenobarbital and fluorescein isothiocyanate (FITC) labeled phenobarbital with a limited amount of antibody. The assay was developed by varying the borate concentration, buffer pH, separation voltage, and incubation time. A running buffer system containing 35 mM borate and 15 mM sodium dodecyl sulfate (pH 9.5), and 2800 V separation voltage provided analysis conditions for a high-resolution, sensitive, and repeatable assay of phenobarbital. Free FITC-labeled phenobarbital and immunocomplex were separated within 30s. The calibration curve for phenobarbital had a detection limit of 3.4 nM and a range of 8.6-860.0 nM. The assay could be used to determine the phenobarbital plasma concentration in clinical plasma sample.     

On-line post-column fluorescence detection for N-terminal tyrosine-containing peptides in high-performance liquid chromatography

A detection system based on on-line post-column fluorescence derivatization is described for the determination of N-terminal tyrosine-containing peptides by reversed-phase high-performance liquid chromatography. The peptides are automatically converted into fluorescent derivatives by reaction with hydroxylamine, cobalt (II) and borate after peptide separation on a reversed-phase column (TSKgel ODS-120T) followed by passage through an ultraviolet absorbance detector. The reaction system permits the fluorescence detection at 435 nm (emission) with excitation at 335 nm for N-terminal tyrosine-containing synthetic peptides in as little as picomole amounts. The facile fluorescence detection of N-terminal tyrosine-containing fragments produced from methionine-enkephalin by enzymatic degradation using a rat brain homogenate was achieved by comparison with the ultraviolet absorption detection at 215 nm.     

Rapid, sensitive and fully automated high-performance liquid chromatographic assay with fluorescence detection for sulmazole and metabolites

Sulmazole (2-[(2-methoxy-4-methylsulfinyl)phenyl]-3H-imidazo [4,5-b] pyridine; AR-L 115 BS) and two metabolites (sulfide, sulfone) were quantified from directly injected body fluids (plasma, urine, bile) after high-performance liquid chromatographic separation. No internal standard is needed, which is particularly advantageous when fluorescence detection is established. After automated pre-column enrichment on Corasil C18 (37-50 microns), the parent compound and biotransformation products could be backflushed and chromatographed on ODS-Hypersil (5 microns) with a mixture of 0.075 mol/l phosphate buffer-acetonitrile (2:1), an elution rate of 2.0 ml/min and fluorimetric detection (lambda ex = 330 nm; lambda em = 370 nm). A hydroxylated metabolite of sulmazole which occurs preferentially in urine (and bile) can be quantified in the above-mentioned solvent system diluted 1:1 with water, but with different fluorescence characteristics (lambda ex = 345 nm; lambda em = 515 nm). The assay was linear in the range 8-1000 ng/ml. The lower limit of detection was about 8 ng/ml or 80 pg with coefficients of variation between 0.4 and 5.8% for sulmazole.     

Automated high-performance liquid chromatographic assay for the determination of 7-ethoxycoumarin and umbelliferone.

An improved high-performance liquid chromatographic assay is presented for the determination of 7-ethoxycoumarin O-deethylase activity. Following a 30-min microsomal incubation, 7-ethoxycoumarin, 4-methylumbelliferone (internal standard), and the metabolite umbelliferone were extracted with chloroform. Separation was achieved with an isocratic mobile phase using a microBondapak phenyl (300 mm x 3.9 mm I.D.) analytical column. The effluent was monitored by fluorescence detection with an excitation wavelength of 360 nm and an emission wavelength of 470 nm. The intra- and inter-assay coefficients of variation were 10 and 6%, respectively. A detection limit of 0.07 micrograms/ml was achieved, making this method suitable for characterizing P-450 activity of human livers.     

An integrated enzyme-linked immunosorbent assay system with an organic light-emitting diode and a charge-coupled device for fluorescence detection

A fluorescence detection system for a microfluidic device using an organic light-emitting diode (OLED) as the excitation light source and a charge-coupled device (CCD) as the photo detector was developed. The OLED was fabricated on a glass plate by photolithography and a vacuum deposition technique. The OLED produced a green luminescence with a peak emission at 512 nm and a half bandwidth of 55 nm. The maximum external quantum efficiency of the OLED was 7.2%. The emission intensity of the OLED at 10 mA/cm(2) was 13 μW (1.7 mW/cm(2)). The fluorescence detection system consisted of the OLED device, two band-pass filters, a five microchannel poly(dimethylsiloxane) (PDMS) microfluidic device and a linear CCD. The fluorescence detection system was successfully used in a flow-based enzyme-linked immunosorbent assay on a PDMS microfluidic device for the rapid determination of immunoglobulin A (IgA), a marker for human stress. The detection limit (S/N=3) for IgA was 16.5 ng/mL, and the sensitivity was sufficient for evaluating stress. Compared with the conventional 96-well microtiter plate assay, the analysis time and the amounts of reagent and sample solutions could all be reduced.     

Determination of tertatolol enantiomers in biological fluids by high-performance liquid chromatography.

A stereospecific high-performance liquid chromatographic method for the quantification of (-)- and (+)-tertatolol in plasma and urine is described. The method involves solid-phase extraction followed by derivatization with S(+)-naphthylethylisocyanate to form the urea derivative, which is more sensitive to fluorescence detection. The separation of the diastereomeric derivatives was performed by reversed-phase high-performance liquid chromatography. Fluorimetric detection (lambda excitation = 220 nm, lambda emission = 320 nm) allows the quantification of tertatolol enantiomers down to 6 ng/ml. The assay was used to study the pharmacokinetic profile of tertatolol enantiomers following oral administration of racemic tertatolol; preliminary results suggest enantioselective absorption and/or disposition of tertatolol.     

Development of an online p38α mitogen-activated protein kinase binding assay and integration of LC–HR-MS

A high-resolution screening method was developed for the p38α mitogen-activated protein kinase to detect and identify small-molecule binders. Its central role in inflammatory diseases makes this enzyme a very important drug target. The setup integrates separation by high-performance liquid chromatography with two parallel detection techniques. High-resolution mass spectrometry gives structural information to identify small molecules while an online enzyme binding detection method provides data on p38α binding. The separation step allows the individual assessment of compounds in a mixture and links affinity and structure information via the retention time. Enzyme binding detection was achieved with a competitive binding assay based on fluorescence enhancement which has a simple principle, is inexpensive, and is easy to interpret. The concentrations of p38α and the fluorescence tracer SK&F86002 were optimized as well as incubation temperature, formic acid content of the LC eluents, and the material of the incubation tubing. The latter notably improved the screening of highly lipophilic compounds. For optimization and validation purposes, the known kinase inhibitors BIRB796, TAK715, and MAPKI1 were used among others. The result is a high-quality assay with Z′ factors around 0.8, which is suitable for semi-quantitative affinity measurements and applicable to various binding modes. Furthermore, the integrated approach gives affinity data on individual compounds instead of averaged ones for mixtures.     

An automated analyzer for methylated arginines in rat plasma by high-performance liquid chromatography with post-column fluorescence reaction

A fully automated analyzer for methylated L-arginine metabolites [N,N-dimethyl-L-arginine (ADMA), N-methylarginine (NMMA) and N,N'-dimethyl-L-arginine (SDMA)] by high-performance liquid chromatography with post-column fluorescence derivatization was developed. This system consists of an on-line extraction, a separation on a reversed phase ion-pair chromatograph, a post-column derivatization by o-phthaladehyde (OPA) and thiol reaction, and fluorescence detection. NMMA, ADMA and SDMA were separated in 40 min with isocratic elution by a combination of octanoate and cyclohexane carboxylate as ion-pair reagents. The eluate was monitored at 450 nm with excitation at 337 nm. The calibration curves for NMMA, ADMA and SDMA showed linearity over the range from 0.05 micromol l(-1) (0.5 pmol on column) to 5.0 micromol l(-1) (50 pmol on column). This method does not require any time-consuming pre-treatment and requires only 10 microl of plasma sample for assay.