Simultaneous Laser-Induced Fluorescence And Contactless-Conductivity Detection For Microfluidic Chip

A combined detection system involving simultaneous LIF and contactless-conductometric measurements at the same place of the microfluidic chip was described. The LIF measurement was designed according to the confocal principle and a moveable contactless-conductivity detector was used in (CD)-D-4. Both measurements were mutually independent and advantageous in analyses of mixtures. Various experimental parameters affecting the response were examined and optimized. The performances were demonstrated by simultaneous detection of Rhodamine B. And the results showed that the combined detection system could be used sensitively and reliably. (C) 2008 Yong Yu. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.

Experimental Characterization Of Electrical Current Leakage In Poly(Dimethylsiloxane) Microfluidic Devices

Poly(dimethylsiloxane) (PDMS) is usually considered as a dielectric material and the PDMS microchannel wall can be treated as an electrically insulated boundary in an applied electric field. However, in certain layouts of microfluidic networks, electrical leakage through the PDMS microfluidic channel walls may not be negligible, which must be carefully considered in the microfluidic circuit design. In this paper, we report on the experimental characterization of the electrical leakage current through PDMS microfluidic channel walls of different configurations. Our numerical and experimental studies indicate that for tens of microns thick PDMS channel walls, electrical leakage through the PDMS wall could significantly alter the electrical field in the main channel. We further show that we can use the electrical leakage through the PDMS microfluidic channel wall to control the electrolyte flow inside the microfluidic channel and manipulate the particle motion inside the microfluidic channel. More specifically, we can trap individual particles at different locations inside the microfluidic channel by balancing the electroosmotic flow and the electrophoretic migration of the particle.

Dual confocal laser-induced fluorescence/moveable contactless

A new dual simultaneous detector was developed for capillary electrophoresis microchip. Confocal laser-induced fluorescence (LIF) and moveable contactless conductivity detection (MCCD) were combined together for the first time. The two detection systems shared a common detection cell and could respond simultaneously. They were mutually independent and advantageous in analyses of mixtures containing organic and inorganic ions. The confocal LIF had high sensitivity and the MCCD could move along the separation channel and detect in different positions of the channel. The detection conditions of the dual detector were optimized. Rhodamine B was used to evaluate the performance of the dual detector. The limit of detection of the confocal LIF was <5 nM, and that of the MCCD was 0.1 μM. The dual detector had highly sensitivity and could offer response easily, rapidly and simultaneously.

Analytical strategies to solve problems in industrial and official laboratories

210 p.

Identification of Sex and Female’s Reproductive Stage in Commercial Fish Species through the Quantification of Ribosomal Transcripts in Gonads

The estimation of maturity and sex of fish stocks in European waters is a requirement of the EU Data Collection Framework as part of the policy to improve fisheries management. On the other hand, research on fish biology is increasingly focused in molecular approaches, researchers needing correct identification of fish sex and reproductive stage without necessarily having in house the histological know-how necessary for the task. Taking advantage of the differential gene transcription occurring during fish sex differentiation and gametogenesis, the utility of 5S ribosomal RNA (5S rRNA) and General transcription factor IIIA (gtf3a) in the molecular identification of sex and gametogenic stage was tested in different economically-relevant fish species from the Bay of Biscay. Gonads of 9 fish species (, Atlantic, Atlantic-chub and horse mackerel, blue whiting, bogue, European anchovy, hake and pilchard and megrim), collected from local commercial fishing vessels were histologically sexed and 5S and 18S rRNA concentrations were quantified by capillary electrophoresis to calculate a 5S/18S rRNA index. Degenerate primers permitted cloning and sequencing of gtf3a fragments in 7 of the studied species. 5S rRNA and gtf3a transcript levels, together with 5S/18S rRNA index, distinguished clearly ovaries from testis in all of the studied species. The values were always higher in females than in males. 5S/18S rRNA index values in females were always highest when fish were captured in early phases of ovary development whilst, in later vitellogenic stages, the values decreased significantly. In megrim and European anchovy, where gonads in different oogenesis stages were obtained, the 5S/18S rRNA index identified clearly gametogenic stage. This approach, to the sexing and the quantitative non-subjective identification of the maturity stage of female fish, could have multiple applications in the study of fish stock dynamics, fish reproduction and fecundity and fish biology in general.

Marcadores Inserção/Deleção (Indel): estudo de ancestralidade e identificação humana na população brasileira

Os polimorfismos denominados Indels são variações de comprimento geradas por inserção ou deleção de um ou mais nucleotídeos em uma sequência de DNA. Estes marcadores genéticos vêm apresentando um grande potencial para fins forenses e populacionais por combinar características dos marcadores SNPs, tais como a capacidade de analisar fragmentos curtos (menores que 250pb) e baixas taxas de mutação, com a facilidade da genotipagem dos STR em uma única PCR, seguida de detecção dos fragmentos amplificados por eletroforese. Com o objetivo de avaliar a eficiência dos Indels em aplicações forenses e esclarecer os detalhes da formação de diferentes populações brasileiras através de dados genéticos, amostras populacionais de diferentes estados brasileiros foram genotipadas através de dois sistemas multiplex. O primeiro (indelplex-HID) foi otimizado para fins de Identificação Humana (HID) e inclui um grupo de 38 marcadores Indels selecionados por apresentarem altos valores de diversidade genética dentro das principais populações continentais. Já o segundo (46-AI-indels), foi selecionado para estudos de ancestralidade e é composto por um conjunto de 46 marcadores informativos de ancestralidade (AIMs). Nesse último caso, ao contrário do anterior, o sistema multiplex inclui marcadores com alta divergência nas frequências alélicas entre populações continentais. Na primeira etapa, o multiplex HID foi aplicado em uma amostra populacional do Rio de Janeiro e em uma amostra populacional dos índios Terena. Um banco de dados de frequências alélicas foi construído para essas duas amostras populacionais. Os valores das frequências alélicas foram utilizados nas comparações estatísticas e parâmetros de vínculos genéticos e forenses foram calculados. O Poder de Discriminação acumulado na população do Rio de Janeiro para os 38 loci testados foi de 0,9999999999999990 e na população dos índios Terena de 0,9999999999997, validando o uso desse sistema numa população heterogênea como a brasileira. A eficiência do indelplex-HID também mostrou-se elevada nas amostras de casos forenses comprometidas, apresentando melhor resultados que marcadores STR em termos de número de loci genotipados e de qualidade de amplificação. Na segunda etapa, o multiplex 46-AI-indels foi aplicado com objetivo de avaliar a ancestralidade em amostras de diferentes estados do Brasil por permitir a identificação de diferenças entre frequências alélicas de grupos populacionais separados geograficamente. A maioria das populações analisadas apresentou elevada herança européia. As populações do Rio de Janeiro, Pernambuco, Mato Grosso do Sul, Amazonas, Alagoas, Minas Gerais e São Paulo apresentaram cerca de 50% de ancestralidade européia, enquanto que nas populações que formam o sul do país e o Espírito Santo este percentual girou em torno de 70%. De uma maneira geral, as contribuições ameríndias e africanas variaram um pouco de acordo com a região. As amostras de Santa Isabel do Rio Negro e dos índios Terena (amostras indicadas como ameríndio-descendentes) de fato mostraram majoritariamente ancestralidade ameríndia (>70%). Os resultados obtidos indicaram que os dados gerados a partir da tipagem dos AIMs estão em estreita concordância com os registros históricos e com outros estudos genéticos acerca da formação da população brasileira e os loci do sistema HID evidenciaram que os são altamente informativos, constituindo uma ferramenta importante em estudos de identificação humana e de relações de parentesco.

毛细管电泳电化学检测联用的应用研究

本论文探讨了毛细管电泳电化学检测器的应用。主要包括以下六个方面的内容：1．毛细管电泳与电化学检测联用测定别嗓呤醇及其代谢产物氧化镖吟醇。该方法简单、快速、灵敏。并且应用于加标尿样的分析时也取得了令人满意的结果。2．采用毛细管电泳一安培检测来定量测定中草药中的活性组分姜黄素，用自制的磷酸三丁酷树脂萃取柱来预处理样品，同时实现对姜黄素的浓缩。经毛细管电泳分离后，姜黄素可以用碳纤维电极检测。3．毛细管电泳电化学发光对尿液中的丙环定的检测。用离子交换柱尽可能除去尿中的离子的干扰，电化学发光试剂印比淀钉采用柱后加入的方式，具有三级胺结构的丙环定可被定量检测到。4．溶胶一凝胶一碳复合材料电极作为毛细管电泳安培检测器的表征。我们报道了这种电极的制作，探讨了不同尺寸的电极在毛细管电泳中的应用。当电极直径100μm，一些分析指标如：检测限、线性范围以及重现性都较好，可以做为安培检测器与毛细管电泳联用。5，毛细管电泳脉冲安培检测生物胺。分离并定量检测了四种生物胺，腐胺、尸胺、亚精胺及精胺。并用该方法测定了牛奶中的生物胺。该方法比已报道的毛细管电泳间接紫外检测的检测限低，比气相色谱及液相色谱需样品体积小，不需要烦琐的衍生步骤。6．溶胶一凝胶一碳复合材料电极作为电泳芯片的安培检测器。我们报道了一种简单，重现性好的芯片上的盘电极的制作方法。由于该安培检测器最大的特点就是稳定性好，重现性高所以我们在其上又沉积了一层铜，以扩大该芯片的应用范围。用集成了该安培检测器的芯片，我们分别测定了肾上腺素和葡萄糖。

ITO导电玻璃和PDMS微芯片毛细管电泳电化学发光检测的研究

微全分析系统是目前很前沿的研究领域，尽管现在还没有真正意义的微全分析系统出现，但它代表了分析科学的发展趋势。本文主要研究了ITO导电玻璃和PDMS微芯片毛细管电泳和电化学发光检测方法。微芯片毛细管电泳对与其联用的检测器有相当高的要求，一些传统的检测方法很难适应于微芯片毛细管电泳。电化学发光检测是一种新兴的检测技术，在化学、生物、医学诊断以及免疫分析中展现出良好的应用前景。如何实现和完善微芯片毛细管电泳与电化学发光检测联用技术是本论文的重点。我们采用聚二甲基硅氧烷（poly（dimethylsiloxone），简称PDMS）和玻璃作为芯片材料，以锢锡氧化物（indium桩n oXide，简称工T0）导电玻璃为工作电极设计了一种集成化的微芯片毛细管电泳电化学发光检测器。其中，芯片的底片由工TO导电玻璃经光刻、化学腐蚀等方法处理后得到。ITO是一种透明的导电材料，作为工作电极集成到芯片的底片上，PDMS层与芯片底片采用可逆键合的方式键合，大大简化了操作并提高了电化学发光信号的采集效率。我们采用脯氨酸作为被测物对检测器进行了表征。在实验过程中，微芯片毛细管电泳及工T0工作电极都表现出良好的稳定性。我们还提出了电化学和电化学发光同时检测技术，应用于微芯片毛细管电泳和常规毛细管电泳。在这种电化学和电化学发光双检测模式中，三联吡陡钉（Ru（bpy）32+既作为电化学发光检测所需的发光试剂与被分析物反应生成激发态的Ru（bpy）32+＊产生电化学发光信号，又在电极表面平行催化电化学反应得到增强的电流响应，提高电化学检测的灵敏度。电化学信号与电化学发光信号同时产生并分别记录，从而实现了电化学和电化学发光同时检测。我们将这种检测技术与芯片或常规毛细管电泳结合，以多巴胺及三种药物分子山蓖若碱、氧氟沙星和利多卡因作为被测物对其进行了表征。这种同时检测方法与其它多检测模式相比更为简单、方便，比单一的电化学或电化学发光检测可以获得更多的被分析物信息，扩大单一检测方式的应用范围。

Determination of SARS-coronavirus by a microfluidic chip system

We have developed a new experimental system based on a microfluidic chip to determine severe acute respiratory syndrome coronavirus (SARS-Cov). The system includes a laser-induced fluorescence microfluidic chip analyzer, a glass microchip for both polymerase chain reaction (PCR) and capillary electrophoresis, a chip thermal cycler based on dual Peltier thermoelectric elements, a reverse transcription-polymerase chain reaction (RT-PCR) SARS diagnostic kit, and a DNA electrophoretic sizing kit. The system allows efficient cDNA amplification of SARS-CoV followed by electrophoretic sizing and detection on the same chip. To enhance the reliability of RT-PCR on SARS-CoV detection, duplex PCR was developed on the microchip. The assay was carried out on a home-made microfluidic chip system. The positive and the negative control were cDNA fragments of SARS-CoV and parainfluenza virus, respectively. The test results showed that 17 positive samples were obtained among 18 samples of nasopharyngeal swabs from clinically diagnosed SARS patients. However, 12 positive results from the same 18 samples were obtained by the conventional RT-PCR with agarose gel electrophoresis detection. The SARS virus species can be analyzed with high positive rate and rapidity on the microfluidic chip system.

Determination of urinary oxidative DNA damage marker 8-hydroxy-2 '-deoxyguanosine and the association with cigarette smoking

8-hydroxy-2'-deoxyguanosine (8OHdG) has been widely used as a biomarker of oxidative DNA damage in both animal models and human studies. To evaluate the effect of cigarette smoking on oxidative stress, we studied the levels of urinary 8OHdG from smokers and non-smokers and investigated the association with cigarette smoking. The urinary 8OHdG concentrations were determinated by capillary electrophoresis with end-column amprometric detection (CE-AD) after a single-step solid phase extraction (SPE), and then quantitatively expressed as a function of creatinine excretion. To increase the concentration sensitivity, a dynamic pH junction was used and the focusing effect was obvious when using 30 mM phosphate (pH 6.50) as sample matrix. The limit of detection is 4.3 nM (signal-to-noise ratio S/N = 3). The relative standard deviation (R.S.D.) was 1.1% for peak current, and 2.3% for migration time. Based on the selected CE-AD method, it was found that the mean value of urinary 8OHdG levels in the smokers significantly higher than that in non-smokers (31.4 +/- 18.9 nM versus 14.4 +/- 7.6 nM, P = 0.0004; 23.5 +/- 21.3 mug g(-1) creatinine versus 12.6 +/- 13.2 mug g(-1) creatinine, P = 0.028). (C) 2004 Elsevier B.V. All rights reserved.

Study of urinary 8-hydroxydeoxyguanosine as a biomarker of oxidative DNA damage in diabetic nephropathy patients

Increased oxidative stress induced by hyperglycemia may contribute to the pathogenesis of diabetic complications. Urinary 8-hydroxydeoxyguanosine (8-OHdG) has been reported to serve as a sensitive biomarker of oxidative DNA damage and also of oxidative stress. This article studied oxidative DNA damage in patients with diabetic nephropathy and in healthy control subjects by urinary 8-OHdG evaluations. Contents of 8-OHdG in urine were analyzed by capillary electrophoresis with end-column amperometric detection (CE-AD) after a single-step solid-phase extraction (SPE). Levels of urinary 8-OHdG in diabetic nephropathy patients with macroalbuminuria was significant higher than in control subjects (5.72 +/- 6.89 mumol/mol creatinine versus 2.33 +/- 2.83 mumol/mol creatinine, P = 0.018). A significant difference of 24 h urinary 8-OHdG excretions exists between the patients with macroalbuminuria and the patients with nonnoalbuminuria (19.2 +/- 16.8 mug/24 h versus 8.1 +/- 1.7 mug/24 h, P = 0.015). There was a positive correlation between urinary excretion of 8-OHdG and glycosylated hemoglobin (HbA(1)c) (r = 0.287, P = 0.022). A weak correlation exists between the levels of 8-OHdG and triglyceride (r = 0.230, P = 0.074). However, the urinary 8-OHdG contents are not correlated with blood pressure and total cholesterol. The increased excretion of urinary 8-OHdG is seen as indicating an increased systemic level of oxidative DNA damage in diabetic nephropathy patients. (C) 2004 Elsevier B.V. All rights reserved.

ELECTROANALYTICAL FLOW MEASUREMENTS

A review based on 94 cited original papers describes recent achievements in application of different electrochemical detection in flow analysis, injection techniques of flow analysis, liquid chromatography and capillary electrophoresis.

Detection of neurotransmitters by a light scattering technique based on seed-mediated growth of gold nanoparticles

A simple light scattering detection method for neurotransmitters has been developed, based on the growth of gold nanoparticles. Neurotransmitters (dopamine, L-dopa, noradrenaline and adrenaline) can effectively function as active reducing agents for generating gold nanoparticles, which result in enhanced light scattering signals. The strong light scattering of gold nanoparticles then allows the quantitative detection of the neurotransmitters simply by using a common spectrofluorometer.

Solid-state electrochemiluminescence of tris(2,2 '-bipyridyl) ruthenium

Electrochemiluminescence (ECL) of tris(2,2'-bipyridyl) ruthenium [Ru(bpy)(3)(2+)] has received considerable attention. By immobilizing Ru(bpy)(3)(2+) on an e electrode surface, solid-state ECL provides several advantages over solution-phase ECL, such as reducing consumption of expensive reagent, simplifying experimental design and enhancing the ECL signal.This review presents the state of the art in solid-state ECL of Ru(bpy)(3)(2+).

[Ru(bpy)(3)](2+)-doped silica nanoparticles within layer-by-layer biomolecular coatings and their application as a biocompatible electrochemiluminescent tag material

[Ru(bpy)(3)](2+)-doped silica (RuSi) nanoparticles were synthesized by using a water/oil microemulsion method. Stable electrochemiluminescence (ECL) was obtained when the RuSi nanoparticles were immobilized on a glassy carbon electrode by using tripropylamine (TPA) as a coreactant. Furthermore, the ECL of the RuSi nanoparticles with layer-by-layer biomolecular coatings was investigated. Squential self-assembly of the polyelectrolytes and biomolecules on the RuSi nanoparticles gave nanocomposite suspensions, the ECL of which decreased on increasing the number of bilayers.