Label-free fluorescence detection in capillary and microchip electrophoresis

Herein, we summarize the current status of native fluorescence detection in microchannel electrophoresis, with a strong focus on chip-based systems. Fluorescence detection is a powerful technique with unsurpassed sensitivity down to the single-molecule level. Accordingly fluorescence detection is attractive in combination with miniaturised separation techniques. A drawback is, however, the need to derivatize most analytes prior to analysis. This can often be circumvented by utilising excitation light in the UV spectral range in order to excite intrinsic fluorescence. As sensitive absorbance detection is challenging in chip-based systems, deep-UV fluorescence detection is currently one of the most general optical detection techniques in microchip electrophoresis, which is especially attractive for the detection of unlabelled proteins. This review gives an overview of research on native fluorescence detection in capillary (CE) and microchip electrophoresis (MCE) between 1998 and 2008. It discusses material aspects of native fluorescence detection and the instrumentation used, with particular focus on the detector design. Newer developments, featured techniques, and their prospects in the future are also included. In the last section, applications in bioanalysis, drug determination, and environmental analysis are reviewed with regard to limits of detection.     

Fluorescence-based nucleic acid detection and microarrays

Recent analytical innovations for nucleic acid detection have revolutionized the biological sciences. Single nucleic acid sequence detection methods have been expanded to incorporate multiplexed detection strategies. A variety of nucleic acid detection formats are now available that can address high throughput genomic interrogation. Many of these parallel detection platforms or arrays, employ fluorescence as the signaling method. Fluorescence-based assays offer many advantages, including increased sensitivity, safety and multiplexing capabilities, as well as the ability to measure multiple fluorescence properties. Multiplexed microarray platforms provide parallel detection capabilities capable of measuring thousands of simultaneous responses. This review will discuss both single target detection and microarray applications with a focus on gene expression and pathogenic microorganism (PM) detection.     

HPLC/UV-spectrometric and electrochemical detection of lignin-decomposition products in alcoholic beverages

Summary The determination of compounds responsible for the flavour of alcoholic beverages, which were separated by HPLC-RP-chromatography, was performed with a combination of UV-diode-array and electrochemical detection. The recording and comparing of diode-array spectra on the rising and descending flanks of each peak was very suitable for identifying peak impurities. The selectivity of the detection depends strongly on the detection wavelength, detection potential and electrode material. For higher sensitivity the electrochemical detection is favoured. The lowest detection limit is 30 pg (absolute) for syringic acid.     

检出限的涵义和计算方法

介绍仪器检出限、方法检出限及样品检出限的基本定义及涵义,以气相色谱法测定海水中的十氯酮为例,进行不同方法的检出限计算,讨论了不同方法计算检出限引起的差异及计算方法中所选取参数的合理性,对如何科学使用检出限进行实验室质量控制提出了建议.     

Light-scattering and turbidimetric detection of silica colloids in size-exclusion chromatography

Silica colloids were separated by size-exclusion chromatography and monitored by fluorimetric and UV detection. In the former means of detection, silica colloids were visualized by light-scattering. The signal intensity based on the light scattering increased with increasing size of the silica colloids. The maximum intensity was observed at excitation wavelengths around 270–290nm. In UV detection, silica colloids were visualized based on turbidimetry, and the signal intensity also increased with increasing size of the silica colloids and with decreasing detection wavelength. The signal intensities for both light-scattering and turbidimetric detection were a linear function of the concentration of the silica colloids. The detection limit at S/N = 3 for 78-nm colloids was 0.06 ppm for light-scattering detection whereas the LOD was 2.3 ppm for UV detection. Effects of mobile phase conditions and flow rate on resolution and peak shape were examined. Use of phosphate buffer allowed the separation of silica colloids of different sizes in size-exclusion chromatography.     

基于氧化还原反应界面可视化定量检测辣根过氧化物酶

该文建立了一种可视化的、基于氧化还原反应界面移动距离定量检测辣根过氧化物酶（HRP）的方法。比较了隐色结晶紫显色体系和3,3',5,5'-四甲基联苯胺显色体系对HRP的显色效率,并构建了基于3,3',5,5'-四甲基联苯胺显色体系的氧化还原反应电泳滴定模型。同时,文中还设计了适用于该模型的小型化、便携式滴定检测芯片,并对滴定通道凝胶中组分进行了优化。结果表明,界面移动距离与HRP浓度存在对数线性关系,检测灵敏度可达0.002 mg/L,且可在10 min内完成HRP的裸眼检测。该方法不需要配备信号读取装置,用户只需要读取有色界面移动的距离即可实现对待测物的可视化定量检测,对于即时检测具有潜在的应用价值。     

High-performance liquid chromatographic separation and detection methods for anabolic compounds.

The role of high-performance liquid chromatography (HPLC) in methods of analysis for anabolic compounds in biological samples is reviewed. Special attention is given to both the separation and detection of anabolic compounds. A distinction is made between on-line detection systems, such as ultraviolet detection and diode-array detection, and off-line detection methods with special emphasis on immunochemical detection methods using non-isotopic labels. A number of applications are given to elucidate the possibilities of HPLC in the analysis of anabolic compounds.     

Polymerase Chain Reaction-based Detection of Total and Specific Vibrio Species

The polymerase chain reaction (PCR) technique is widely used for efficient detection of food-borne pathogens because of speed and specificity. However, PCR methods have focused mostly on species-specific detection. In the present work, we describe a PCR-based method for the simultaneous detection of all Vibrio species because lots of them are notorious food-borne human pathogens. We then combined this total detection method with specific detection of Vibrio cholerae pathogen. Using a degenerate primer set based on the sequence of the potassium uptake gene, trkA, we were able to successfully detect all Vibrio species. Specific detection of V. cholerae was also possible using primer sets based on putative flagellin sequence. Importantly, simultaneous total and species-specific Vibrio detection was possible using all two primer sets in a multiplexed PCR strategy. Thus, the PCR method we have developed is applicable to both simultaneous and two-step detection of total and specific Vibrio species.     

Coupling Capillary Electrophoresis and Pulsed Electrochemical Detection

Pulsed electrochemical detection (PED) is an excellent method for detection of analytes that normally foul electrodes. In PED, the detection electrode is first cleaned at a high positive potential, then reactivated at a negative potential dissolving the surface oxide, and finally used to oxidize the analyte at a moderate positive potential. Due to the advantages and versatility of PED, many different variations of the detection waveform can be found in literature. This review focuses on application of PED to CE and in particular, the most commonly used modes: pulsed amperometric detection (PAD) and integrated pulsed amperometric detection (iPAD).     

MOFs传感器在癌症及其生物标志物检测中的应用

癌症是世界上最致命的疾病之一,因此癌细胞的有效捕获和敏感检测对基础研究以及临床诊断和治疗都具有重要意义。基于金属有机骨架(MOFs)的催化活性和固有的发光性能等特点,MOFs已被成功地开发为传感平台实现对癌症及其标志物的检测。综述了基于MOFs的电化学、荧光、电化学发光、比色传感器在癌细胞及核酸、蛋白质等生物标志物检测中近3年的研究进展,从MOFs材料,检测物类型,检测方法、检测能力等方面进行了综述,并对各自的特点进行了讨论,对以后MOFs纳米材料在癌症检测中的应用进行了展望。     

Analytical potential of mid-infrared detection in capillary electrophoresis and liquid chromatography: a review

Literature published in the last decade concerning the use of mid-infrared spectrometry as a detection system in separation techniques employing a liquid mobile phase is reviewed. In addition to the continued use of isocratic liquid chromatographic (LC) techniques, advances in chemometric data evaluation techniques now allow the use of gradient techniques on a routine basis, thus significantly broadening the range of possible applications of LC-IR. The general trend towards miniaturized separation systems was also followed for mid-IR detection where two key developments are of special importance. Firstly, concerning on-line detection the advent of micro-fabricated flow-cells with inner volumes of only a few nL for transmission as well as attenuated total reflection measurements enabled on-line mid-IR detection in capillary LC and opened the path for the first successful realization of on-line mid-IR detection in capillary zone electrophoresis as well as micellar electrokinetic chromatography. Secondly, concerning off-line detection the use of micro-flow through dispensers now enables to concentrate eluting analytes on dried spots sized a few tens of micrometers, thus matching the dimensions for sensitive detection by mid-IR microscopy. Finally in an attempt to increase detection sensitivity of on-line mid-IR detection, mid-IR quantum cascade lasers have been used. Applications cover the field of food analysis, environmental analysis and the characterization of explosives among others. Best detection sensitivities for on-line and off-line detection have been achieved in miniaturized systems and are in the order of 50 ng and 2 ng on column, respectively.     

Analysis of delta-L-alpha-aminoadipyl-L-cysteinyl-D-valine by ion chromatography and pulsed amperometric detection

A novel high-performance liquid chromatography (HPLC) method is presented for the detection and trace level determination of the tripeptide delta-L-alpha-aminoadipyl-L-cysteinyl-D-valine (ACV). The tripeptide, an intermediate in penicillin production, is derived from fungal fermentation. The technique relies on ion-exchange separation of the tripeptide on an anion-exchange column followed by detection by reduction on a gold electrode using pulsed amperometry. The sensitivity of direct determination of ACV is increased by employing pulsed amperometric detection (PAD) over direct ultraviolet detection. Choice of the working electrode and optimisation of electrode potentials was based on cyclic voltammograms recorded for the tripeptide in the mobile phase. A linear regression equation was obtained over the range 0-100 micrograms ml-1. The detection limit in fermentation broths was found to be 0.1 micrograms ml-1 whereas in buffer the detection limit was found to be 10 ng ml-1. A good correlation coefficient was observed when ACV concentrations determined by ion chromatography-PAD were compared with measurements obtained by pre-column derivatisation with fluoromethylorthochloroformate followed by HPLC separation on a reversed-phase C18 silica column with UV detection. The procedure has been applied to the measurement of natural levels of ACV in fermentation broths of selected strains of Aspergillus nidulans and Penicillin chrysogenum.     

Integrated capillary electrophoresis amperometric detection microchip with replaceable microdisk working electrode. II. Influence of channel cross-sectional area on the separation and detection of dopamine and catechol

The interference of separation high voltage with the electrochemical detection is a major challenge to the microchip capillary electrophoresis-electrochemical detection systems with end-channel detection mode. Using dopamine and catechol as model analytes, the influences of channel cross-sectional area and channel-to-electrode distance on the high-voltage interference, accordingly on the separation and detection performances of the microchip capillary electrophoresis-electrochemical detection system were investigated. With the increase of the channel cross-sectional area from 312 through 450-615 microm2, the apparent half-wave potentials of hydrodynamic voltammetry for dopamine at the field strength of 288 V/cm shifted positively from 285 through 330-400 mV. By using a chip with the smallest channel cross-section (312 microm2 with top width of 37.3 microm and depth of 8.9 microm) the residual high-voltage field in the detection cell was small, so that detection was conducted at a channel-to-electrode distance of 20 microm to achieve better performances of separation and detection.     

Study of Bare and Mercury‐Coated Vibrated Carbon,Gold and Silver Microwire Electrodes for the Determination of Lead and Cadmium in Seawater by Anodic Stripping Voltammetry

Carbon, gold and silver microwires are revisited under vibrated conditions for detection of trace lead and cadmium in seawater. The Pb and Cd peaks fully overlapped on the bare gold and carbon electrodes and partially on the silver electrode. The sensitivity of all three was insufficient for detection in uncontaminated waters. Peak separation was obtained after coating with mercury (Hg). Only the Hg‐coated silver electrode is suitable when preplated. Limits of detection for Pb using the Hg/C and Hg/Ag electrodes (20–40 pM), and Cd (70 pM), are sufficiently low for Pb and Cd detection in seawater.     

Advances in amperometric and conductometric detection in capillary and chip-based electrophoresis

Amperometric and conductometric detection are currently the two major electrochemical detection modes in capillary and chip electrophoresis. The ease of miniaturization and integration of electrochemical detection elements offers a high potential for the development of portable analytical devices based on electromigrative separations. The challenges and basic concepts of both detection principles in the context of capillary/chip electrophoresis are shortly introduced and milestones of the methodical developments are summarized from a historical perspective. Recent advances and applications are discussed with more detail. Particular attention is paid to new trends in this area of research such as measurements in short capillaries and the enormous progress and increased popularity of contactless conductivity detection. Correspondence: Frank-Michael Matysik, Institute of Analytical Chemistry, University of Leipzig, Linnéstr. 3, D-04103 Leipzig, Germany     

Enhancement of detection sensitivity for indirect photometric detection of anions and cations in capillary electrophoresis

This review focuses on the indirect photometric detection of anions and cations by capillary electrophoresis. Special emphasis has been placed on the sensitivity of the technique and approaches taken to enhance detection limits. Theoretical considerations and requirements have been discussed, including buffering, detection sensitivity, separation of cations, and detector linearity. A series of tables detailing highly absorbing probes and the conditions of their use for indirect photometric detection are included.     

固相萃取富集-高效液相色谱分离和测定邻甲苯胺和邻硝基甲苯

建立了以固相萃取技术进行富集 ,高效液相色谱进行分离和检测邻甲苯胺和邻硝基甲苯的方法。污染水中的邻甲苯胺和邻硝基甲苯采用Sep pakC1 8萃取柱进行固相萃取。色谱分离条件是 :Shim PackCLCODS(1 5 0mm× 4 .6mmid ,5 μm)柱为分析柱 ,甲醇 水 =60∶4 0 (V V)为流动相 ,流速为 1 .0mL min,邻甲苯胺和邻硝基甲苯的紫外检测波长分别为 2 3 0nm和 2 5 4nm ,本法具有良好的灵敏度和重现性。     

Amplified detection of DNA and analysis of single-base mismatches by the catalyzed deposition of gold on Au-nanoparticles

A novel amplification route for DNA detection based on the deposition of gold on a 10 nm Au-colloid/avidin conjugate label acting as a 'seeding' catalyst, is described. Microgravimetric quartz-crystal-microbalance measurements are employed to transduce the catalyzed deposition of gold on the piezoelectric crystals. Three different DNA detection schemes are described: (i) analysis of a 27-base nucleic acid fragment; (ii) analysis of the entire M13phi DNA (7229 bases); and (iii) detection of a single-base mismatch in a DNA. Ultrasensitive detection of DNA is accomplished by the catalyzed deposition of gold, detection limit approximately 1 x 10(-15) M.     

基因传感技术及目前存在的问题和发展对策

对最近几年报道的比较有特色的光学、压电、电化学等基因(DNA)传感器方面的研究工作进行介绍,并就目前基因传感器研究中存在的问题进行了分析讨论,提出了相应的发展对策.