Solid composite electrodes for DNA enrichment and detection

New plastic composite electrodes with appliance in medical diagnostic are described. The new electrode material offers the possibility of specific electrical enrichment and electrochemical analysis of nucleic acid sequences. To facilitate selective enrichment of target nucleic acids, specific probe oligonucleotides were attached covalently to free carboxyl groups of conducting polycarbonate/carbon fiber electrodes. Complementary oligonucleotides were enriched from analyte solutions by electric field supported methods. The analysis of the PCR product shows the efficiency and selectivity of the electrical enrichment. We have also shown that inexpensive and robust solid electrodes made of polycarbonate and conductive carbon powder are suitable for electrochemical examination of nucleic acids. The combination of electrochemical enrichment of DNA and subsequent electrochemical detection is a promising approach towards an inexpensive molecular diagnosis kit.     

Use of nanoparticles in the electrochemical analysis of biological samples

The specific effects of various nanoparticles on processes occurring in the electrochemical analysis of biological samples are considered. The material classification is based on a structural principle of the organization of a sensing element. Nanoparticles can occur immediately on its surface or in the bulk of an analyte (in the latter case, they can be chemically bound to the surface); they can be incorporated into the analyte and serve as its labels. The use of nanoparticles significantly extends the capabilities of bioelectrochemical methods of analysis. In a number of cases, the use of nanoparticles provides an opportunity to abandon the use of enzymes; this is very promising for clinical analysis. Methods for the electrochemical analysis of biological samples with the use of nanoparticles can be a serious addition to classical biochemical methods.     

Theory and practice of enzyme bioaffinity electrodes. Direct electrochemical product detection

The use of enzyme labeling techniques to convert biorecognition events into high sensitivity electrochemical signals may follow two different strategies. One, in which the current is the electrocatalytic response of a redox couple serving as cosubstrate to a redox enzyme label and another that consists in the detection of an electrochemically active product of the enzyme label. The theoretical relationships that link, in the latter case, the electrochemical current response to the amount of recognized labeled target analyte are established for steady-state diffusion-convection chronoamperometric regimes. Two governing parameters thus emerge. One measures the Michaelis-Menten competition in the enzyme kinetics. The other characterizes the competition between the enzymatic kinetics and the diffusion of the substrate. The electrochemical response is finally related to the labeled target analyte concentration in solution through the recognition isotherm. The direct electrochemical product detection thus provides a route to the characteristics of the recognition isotherm, which serves as a calibration curve in analytical applications. The establishment of further theoretical relationships allows one to surmise the increase in sensitivity that may be obtained by using cyclic voltammetry instead of steady-state chronoamperometry in standard electrochemical cells or by accumulation of the enzyme-product in cells of small volume/surface ratios. The theoretical predictions are tested with the example of the avidin-biotin recognition process in a system that involves alkaline phosphatase as enzyme label and 4-amino-2,6-dichlorophenyl phosphate as substrate, generating 4-amino-2,6-dichlorophenol as electrochemically active product. The advantages of the dichloro-substitution are discussed. The theoretical analysis is a requisite for a rational and realistic discussion of the analytical performances of the steady-state chronoamperometric and cyclic voltammetric approaches. These are shown to compare favorably with the best heterogeneous bioaffinity assays so far reported.     

Theoretical models for electrodialytic sample treatment in trace analysis by liquid chromatography

Summary Basic considerations for analyte enrichment and recovery obtainable by electrodialysis as a sample treatment method are given. Equations are derived which describe the dependence of the concentration profiles of ionic compounds on the electric field strength in a set-up with stagnant donor and acceptor solutions. It is shown that analyte recovery increases when less ion-selective membranes are used in the electrodialysis cell. Computer models are used to estimate the analyte enrichment for a flowing donor (sample) and a stagnant acceptor phase. About 10-fold enrichment can be obtained in an electrodialytic sample treatment system within 20 min under maximum current conditions. A compromise has to be found between analyte recovery and the donor (sample) flow rate.     

Electrokinetic trapping and concentration enrichment of DNA in a microfluidic channel

We report a simple and efficient method for enriching the concentration of charged analytes within microfluidic channels. The method relies on exerting spatial control over the electrokinetic velocity of an analyte. Specifically, the electroosmotic (eo) velocity of the buffer solution in one region of the microfluidic system opposes the electrophoretic (ep) velocity of the analyte in the other region. This results in ep transport of DNA to the location where the ep and eo velocities are equal and opposite. Accumulation of the analyte occurs at this location. This enrichment method is conceptually distinct from field-amplification stacking, isotachophoresis, micelle sweeping, size exclusion, and other methods that have been previously reported. The method requires no complex microfabricated structures, no special manipulation of the solvent, and the enriched analyte remains in solution rather than being captured on a solid support. A concentration enrichment factor of 800 can be achieved for 20mer DNA in a fluidic channel having dimensions of 100 mum x 25 mum x 5 mm. The time required to achieve this level of enrichment is 300 s, and the enriched zone has a minimum width of 100 mum.     

Development of plasticised PVC sensing films for the determination of BTEX compounds in aqueous samples

A novel plasticised PVC polymer membrane as a sensing film for the determination of BTEX compounds using ATR-FTIR spectroscopy is demonstrated. A range of 10 plasticised PVC phases have been investigated using toluene and tetrachloroethylene as test analytes. Both analyte enrichment rates and infrared absorbance values were considered when choosing a suitable polymer for sensing. An enhancement in analyte absorbance at the characteristic IR absorption bands was noted as the plasticiser concentration in the film was increased. 2% PVC with 75% diisooctyl azelate was found to show promising results for simultaneous determination of the BTEX compounds. All BTEX analytes can be measured in less than 8 min. A study of a multicomponent sample demonstrated that analyte enrichment times were influenced by the presence of even one additional analyte component in the sample.     

Single-cell analysis by electrochemical detection with a microfluidic device

A novel electrochemical method with a microfluidic device was developed for analysis of single cells. In this method, cell injection, loading and cell lysis, and electrokinetic transportation and detection of intracellular species were integrated in a microfluidic chip with a double-T injector coupled with an end-channel amperometric detector. A single cell was loaded at the double-T injector on the microfluidic chip by using electric field. Then, the docked cell was lysed by a direct current electric field strength of 220 V/cm. The analyte of interest inside the cell was electrokinetically transported to the detection end of separation channel and was electrochemically detected. External standardization was used to quantify the analyte of interest in individual cells. Ascorbic acid (AA) in single wheat callus cells was chosen as the model compound. AA could be directly detected at a carbon fiber disk bundle electrode. The selectivity of electrochemical detection made the electropherogram simple. The technique described here could, in principle, be applied to a variety of electroactive species within single cells.     

Highlights of 20?years of electrochemical measurements of exocytosis at cells and artificial cells

Advances in electrochemical methodology over the past 30?years have allowed chemical measurements to be made with decreasing amounts of analyte and at smaller spatial dimensions. This has allowed the investigation of single cells and single vesicles in cells either during release of chemical transmitter or separately. The cellular event called exocytosis can be measured with amperometry or cyclic voltammetry as discovered by Wightman and first published in 1990. In addition, the measurement of vesicle contents with electrochemistry is a new approach we have termed electrochemical cytometry. This involves isolation of intact vesicles, separation of the vesicles, and then lysing followed by coulometric analysis of the electroactive vesicle content. In this review, we will highlight work done by us and by others to discuss measurements of exocytosis at single cells and measurements at artificial cell models for studying the biophysical properties of vesicle membrane dynamics and lipid nanotubes connecting artificial cells using electrochemical methods.     

Solid-phase extraction based on multi-walled carbon nanotube (MWCNT) sorbents combined with bio-coacervation extraction for the determination of atrazine from water samples followed by HPLC analysis

In this study, combined technique of solid-phase extraction based on multi-walled carbon nanotubes with bio-coacervation extraction (SPE-MWCNT-BCAE) has been developed as a new sample preparation method for the determination of atrazine from water samples. The proposed method involves two steps: analyte enrichment on the solid sorbent and subsequently elution of the analyte by an appropriate solvent. Multi-walled carbon nanotubes (MWCNTs) were used as the sorbent. They have high specific surface area, nano-scale structure and high diffusion rate. The second step is based on the use of bioaggregates for analyte re-enrichment, which consists of biosurfactants and ionic liquid. This method follows the principles of green chemistry. Parameters affecting the extraction efficiency were optimized. Under optimum conditions, the enrichment factor was 176. The linear dynamic range (LDR) and limit of detection (LOD) were 2–100 µg L^?1 and 0.66 µg L^?1, respectively. The relative standard deviation (RSD) for six replicate measurements was 3.8%. The method was applied to the determination of ultratrace levels of atrazine in environmental water samples with satisfactory results.     

Liquid-liquid extraction procedures for sample enrichment in capillary zone electrophoresis

This review article presents an overview of applications of liquid-liquid extraction (LLE) for analyte enrichment and clean-up of samples prior to capillary zone electrophoresis (CZE). The basic principles of LLE are discussed with special emphasis on analyte enrichment. In addition, attention is focused on the requirements for the final extract to be compatible with CZE. The paper discusses selected examples from the literature with special emphasis on detection limits in drug analysis and in environmental chemistry. Finally, the paper focus on alternative liquid-phase extraction concepts based on electroextraction, supported liquid membranes, and liquid-phase microextraction.     

Electrochemical detection on electrowetting-on-dielectric digital microfluidic chip

In this work, the use of three-electrode electrochemical sensing system with an electrowetting-on-dielectric (EWOD) digital microfluidic device is reported for quantitative analysis of iodide. T-junction EWOD mixer device was designed using arrays of 50-μm spaced square electrodes for mixing buffer reagent and analyte droplets. For fabrication of EWOD chips, 5-μm thick silver EWOD electrodes were formed on a glass substrate by means of sputtering and lift-off process. PDMS and Teflon thin films were then coated on the electrodes by spin coating to yield hydrophobic surface. An external three-electrode system consisting of Au working, Ag reference and Pt auxiliary wires were installed over EWOD electrodes at the end of T-junction mixer. In experiment, a few-microliter droplets of Tris buffer and iodide solutions were moved toward the mixing junction and transported toward electrochemical electrodes by EWOD process. A short processing time within seconds was achieved at EWOD applied voltage of 300 V. The analyte droplets mixed with different concentrations were successfully analyzed by cyclic voltametry. Therefore, the combination of EWOD digital microfluidic and electrochemical sensing system has successfully been demonstrated for rapid chemical analysis with minimal reagent consumption.     

Molecularly Imprinted Polymers Combined with Electrochemical Sensors for Food Contaminants Analysis

Detection of relevant contaminants using screening approaches is a key issue to ensure food safety and respect for the regulatory limits established. Electrochemical sensors present several advantages such as rapidity; ease of use; possibility of on-site analysis and low cost. The lack of selectivity for electrochemical sensors working in complex samples as food may be overcome by coupling them with molecularly imprinted polymers (MIPs). MIPs are synthetic materials that mimic biological receptors and are produced by the polymerization of functional monomers in presence of a target analyte. This paper critically reviews and discusses the recent progress in MIP-based electrochemical sensors for food safety. A brief introduction on MIPs and electrochemical sensors is given; followed by a discussion of the recent achievements for various MIPs-based electrochemical sensors for food contaminants analysis. Both electropolymerization and chemical synthesis of MIP-based electrochemical sensing are discussed as well as the relevant applications of MIPs used in sample preparation and then coupled to electrochemical analysis. Future perspectives and challenges have been eventually given.     

基于激光介导富集的表面增强拉曼分析

基于具有优异表面增强拉曼散射(SERS)性能的石墨烯隔离的金纳米晶(GIAN)能够在水-有机相界面自组装, 待测物分子在有机相中的分配系数较大以及GIAN能够通过π?π相互作用与待测物分子结合的优势, 构建了激光介导的待测物分子的高效富集策略, 进而实现了9,10-双苯乙炔基蒽(BPEA)分子的痕量SERS分析. 所构建的新型待测物分子高效富集策略在一定程度上避免了因“咖啡环效应”带来的信号波动, 有望为复杂体系中痕量待测物的SERS分析提供可靠的平台.     

Understanding mechanisms of pressure-assisted electrokinetic injection: application to analysis of bromate, arsenic and selenium species in drinking water by capillary electrophoresis-mass spectrometry

The mechanism underlying the enrichment power by pressure-assisted electrokinetic injection (PAEKI) in capillary electrophoresis (CE) was investigated for on-line pre-concentration of arsenic [As(III) and As(V)], selenium [Se(IV) and Se(VI)] and bromate (BrO(3)(-)). Analyte diffusion behaviour from PAEKI sample plugs were evaluated by monitoring peak broadening as a function of stagnant time and position in the capillary. During PAEKI, anionic analytes accumulate at the sample-separation buffer boundary. We proposed that a counter-ion layer formed in PAEKI, where a cation layer was formed at the separation buffer side of boundary. The cation layer served as a soft boundary which impeded zone broadening via electrostatic attraction between layers. This effect likely played an important role in maintaining focused analyte bands by suppressing diffusion. Comparison of analyte behaviour in PAEKI injected sample plugs to behaviour in hydrodynamically injected ones proved the existence of a counter-ion layer. The dependence of analyte diffusion in PAEKI plugs on electrochemical properties (viscosity, conductivity, electrophoretic mobility) further supported the hypothesis. Additionally, it was noted that analytes with low electrophoretic mobility were more efficiently pre-concentrated by PAEKI and were less subject to forces of dispersion than analytes with greater electrophoretic mobility. PAEKI-CE coupled to electrospray tandem mass spectroscopy (ESI-MS/MS) was then optimized and validated for detection of arsenic, selenium and bromate in water samples. On-line enrichment of the target analytes was achieved with 1-3 ng mL(-1) detection limits, which was below the maximum contaminant levels in drinking water for all five anions studied. Noteworthy, the potential of the method for unbiased detection of molecular species in untreated water was demonstrated. No contamination was detected in the water samples tested; however, recovery was 90-118% for spiked samples. The method was demonstrated be comparable to current methods for detection of inorganic contaminants in drinking water and is a good alternative method to ion chromatography/liquid chromatography-MS.     

Electrokinetic concentration enrichment within a microfluidic device using a hydrogel microplug

A simple and efficient approach for concentration of charged molecules in microfluidic devices is described. The functional component of the system is a hydrogel microplug photopolymerized within the main channel of a microfluidic device. When an appropriately biased voltage is applied across the hydrogel, charged analyte molecules move from the source well toward the hydrogel. Transport of the analyte through the hydrogel is slow compared to its velocity in the microfluidic channel, however, and therefore it concentrates at the hydrogel/solution interface. For an uncharged hydrogel, a bias of 100 V leads to a approximately 500-fold enrichment of the DNA concentration within 150 s, while the same conditions result in an enrichment of only 50-fold for fluorescein. Somewhat lower enrichment factors are observed when a negatively charged hydrogel is used. A qualitative model is proposed to account for the observed behavior.     

On-line electrothermal atomic absorption spectrometry configurations: recent developments and trends

In the present mini-review, an account of the actual state-of-the-art and future possibilities offered by on-line ET-AAS is presented. Topics such as: (1) on-line analyte preconcentration (by means of precipitation, sorption, solvent extraction, and solid phase extraction); (2) analyte separation by means of chromatography, and electrochemical, microdialysis and chemical vapor generation processes; and (3) sample treatment (by microwave sample digestion, sample emulsification and dilution processes) are used to illustrate the versatility of flow injection, sequential injection analysis, stop flow and continuous flow, when coupled to a graphite furnace. The use of some of the on-line systems for speciation and the simultaneous determination of different analytes is underlined.     

火焰原子吸收分析中表面活性剂对锰吸收讯号的增感效应及机理

提出了一种新的增感机理:在表面活性剂的cmc前,气溶腔形成过程中被测离子在小液滴上富集产生增惑;cmc后富集作用减小,但被测离子和表面活性剂形成胶团化物产生新的增感,火焰部分表面活性剂的不完全燃烧增强了火焰的还原性是增感因素之一。     

Acoustically levitated droplets — a new tool for micro and trace analysis

First experiences with the technique of acoustical levitation of droplets in the field of analytical and atmospheric chemistry are reported. Acoustical levitation enables the contactless handling of solid and liquid microsamples. This avoids adsorption of analyte at and desorption of contaminants from container walls especially for liquid samples. Common experiments of sample preparation procedures were conducted in levitated drops like liquid/liquid extractions, solvent exchange, and analyte enrichment by evaporation of the solvent. A first approach was made to use acoustical levitation for the simulation of atmospheric chemistry situations. Received: 10 October 1996 / Revised: 25 October 1996 / Accepted: 25 October 1996     

Overview of significant examples of electrochemical sensor arrays designed for detection of nitric oxide and relevant species in a biological environment

Ultramicroelectrode sensor arrays in which each electrode, or groups of electrodes, are individually addressable are of particular interest for detection of several species concomitantly, by using specific sensing chemistry for each analyte, or for mapping of one analyte to achieve spatio–temporal analysis. Microfabrication technology, for example photolitography, is usually used for fabrication of these arrays. The most widespread geometries produced by photolithography are thin-film microdisc electrode arrays with different electrode distributions (square, hexagonal, or random). In this paper we review different electrochemical sensor arrays developed to monitor, in vivo, NO levels produced by cultured cells or sliced tissues. Simultaneous detection of NO and analytes interacting with or released at the same time as NO is also discussed.     

Electrochemically controlled solid-phase microextraction and preconcentration using polypyrrole coated microarray electrodes in a flow system

Polypyrrole coated microarray electrodes have been used for electrochemically controlled solid-phase microextraction and preconcentration on individually addressable gold microband electrodes. In this study, a flow of analyte solution was maintained over the band electrodes by positioning a capillary in a vertical position over the electrode array during both the extraction and the detection of the desorbed compounds. This experimental set-up was used to evaluate the possibilities of using electrochemically controlled solid-phase microextraction with conducting polymers as a preconcentration step in miniaturised flow systems. The performance of the polymer, which was prepared by electrochemical polymerisation using a solution of 0.05 M pyrrole and 0.1 M LiClO4, was investigated using chloride as a model analyte employing different extraction times and analyte concentrations. It was found that significant preconcentration was possible using extraction times of only a few minutes and that a good linearity between the extraction time and detection response was present both for mM and microM chloride concentrations. Compared to a recent study (Liljegren et al., Analyst, 2002, 127, 591-597), using a more traditional solid-phase microextraction technique under electrochemical control, the preconcentration factor could be increased by a factor of about 210 by using the present flow system based approach. This increase in the preconcentration factor can be explained by the significant decrease in the desorption volume (i.e. reduced dilution of the desorbed analyte) associated with the use of the present flow system. With the present approach, the detection limit for the model analyte chloride could be decreased from 10 microM to 625 nM employing an extraction time of 180 s.