Electrochemical immunoassays

Immunoassays (IA) use the specific antigen antibody complexation for analytical purposes. Radioimmunoassays (RIA), fluorescence immunoassays (FIA) and enzyme immunoassays (EIA) are well established in clinical diagnostics. For the development of hand-held devices which can be used for point of care measurements, electrochemical immunoassays are promising alternatives to existing immunochemical tests. Moreover, for opaque or optically dense matrices electrochemical methods are superior. Potentiometric, capacitive and amperometric transducers have been applied for direct and indirect electrochemical immunoassays. However, due to their fast detection, broad linear range and low detection limit, amperometric transducers are preferred. Competitive and non-competitive amperometric immunoassays have been developed with redox compounds or enzymes as labels. This review will give an overview of the most frequently applied principles in electrochemical immunoassays. The potential of an indirect competitive amperometric immunoassay for the determination of creatinine within nanomolar range and the circumvention of the most serious problem in electrochemical immunoassays, namely regeneration, will be discussed.     

4-Amino-1-naphthylphosphate as a substrate for the amperometric detection of alkaline phosphatase activity and its application for immunoassay

Immunosensors and biochemical array detection systems based on electrochemical transducers have many advantages such as low detection limit, fast response, simple design and ease of miniaturization. However, further development of such sensors will depend on the availability of suitable substrates that can be converted by a labeling enzyme to an electrochemically active product. Here, we report the synthesis of 4-amino-1-naphthylphosphate and it’s application as a new substrate for alkaline phosphatase. The electrochemical and enzymatic properties of this compound were investigated and compared with the properties of other aromatic 1,4-dihydroxy and 1,4-hydroxy-amine derivatives. The product of the enzyme reaction was 4-aminonaphthol, which was rapidly converted in the presences of air to 1,4-iminonaphthoquinone. This compound could then be detected in an amperometric flow injection assay (AFIA) with −200 mV versus Ag/AgCl potential application. The analytical range for mouse IgG, in an alkaline phosphatase amplified sandwich immuoassay with amperometric detection, was 0.01-100 μg ml⁻¹.     

用于高效毛细管电泳的电化学检测器

阐述了高效毛细管电泳电化学检测器（包括电导、电势和安培检测）的研究现状，重点是检测器的研制及接口的制作技术。对各种电化学检测器的应用情况也进行了总结。展望了高效毛细管电泳电化学检测的发展前景。     

Electrochemical enzyme immunoassay using model labels

Enzyme immunoassays (EIAs) based on electrochemical detection offer several potential advantages and have been applied in clinical, medical, biotechnological, food and environmental analysis. Among the enzyme labels employed, horseradish peroxidase (HRP), alkaline phosphatase (ALP) and glucose oxidase (GOx) are the most common. This brief review reflects recent advances, challenges, and trends of electrochemical EIAs focusing on HRP, ALP or GOx as labels over the past five years. We especially emphasize current development of EIAs combined with other developments, including nanotechnology and miniaturization.     

Capillary electrophoresis-based immunoassays: principles and quantitative applications

The use of CE as a tool to conduct immunoassays has been an area of increasing interest over the last decade. This approach combines the efficiency, small sample requirements, and relatively high speed of CE with the selectivity of antibodies as binding agents. This review examines the various assay formats and detection modes that have been reported for these assays, along with some representative applications. Most CE immunoassays in the past have employed homogeneous methods in which the sample and reagents are allowed to react in solution. These homogeneous methods have been conducted as both competitive binding immunoassays and as noncompetitive binding immunoassays. Fluorescent labels are most commonly used for detection in these assays, but enzyme labels have also been utilized for such work. Some additional work has been performed in CE immunoassays with heterogeneous methods in which either antibodies or an analog of the analyte is immobilized to a solid support. These heterogeneous methods can be used for the selective isolation of analytes prior to their separation by CE or to remove a given species from a sample/reagent mixture prior to analysis by CE. These CE immunoassays can be used with a variety of detection modes, such as fluorescence, UV/Vis absorbance, chemiluminescence, electrochemical measurements, MS, and surface plasmon resonance.     

Flow amperometric detection of indigo for enzyme-linked immunosorbent assays with use of screen-printed electrodes

A simple and automated methodology for a sensitive electrochemical detection of enzyme immunoassays that employ alkaline phosphatase (AP) as label has been developed. A flow injection system with programmable pump, valve and cell functions, amperometric detection of indigo and screen-printed electrodes (SPEs) are responsible for the advantages of this methodology. Amperometric detection at a low potential of indigo, the product of the enzymatic hydrolysis of the substrate 3-indoxyl phosphate (IP), is combined with a flow injection system. This incorporates in the flow cell a disposable screen-printed board provided with a graphite working electrode. No electrode pretreatment is necessary to obtain reproducible signals. The system was applied to the determination by an enzyme-linked immunosorbent assays (ELISA) of pneumolysin (PLY), a toxin related to respiratory infections. Linear calibration curves for low and high concentration ranges were obtained. These were also performed in a proteic matrix and linearity was also obtained.     

Artificial electron donors for nitrate and nitrite reductases usable as mediators in amperometric biosensors

Various nitrate and nitrite reductases are capable of accepting electrons from artificial donors. Combining these redox active donors with an amperometric redox electrode which is covered with an immobilized layer of such a nitrate or nitrite reductase, new enzyme sensors can be created for the detection of nitrate or nitrite, respectively. A range of suitable electron donors for nitrate reductases and nitrite reductase from different sources have been selected and characterized by electrochemical methods.     

Nanobioelectroanalysis Based on Carbon/Inorganic Hybrid Nanoarchitectures

The emergence of nanotechnology has opened new horizons for electrochemical biosensors. This review highlights new concepts for electrochemical biosensors based on different carbon/inorganic hybrid nanoarchitectures. Particular attention will be given to hybrid nanostructures involving 1‐ or 2‐dimensional carbon nanotubes or graphene along with inorganic nanoparticles (gold, platinum, quantum dot (QD), metal oxide). Latest advances (from 2007 onwards) in electrochemical biosensors based on such hybrids of carbon/inorganic‐nanomaterial heterostructures are discussed and illustrated in connection to enzyme electrodes for blood glucose or immunoassays of cancer markers. Several strategies for using carbon/inorganic nanohybrids in such bioaffinity and biocatalytic sensing are described, including the use of hybrid nanostructures for tagging or modifying electrode transducers, use of inorganic nanomaterials as surface modifiers along with carbon nanomaterial label carriers, and carbon nanostructure‐based electrode transducers along with inorganic amplification tags. The implications of these nanoscale bioconjugated hybrid materials on the development of modern electrochemical biosensors are discussed along with future prospects and challenges.     

A New Dual-electrode and Multi-channel Electrochemical Detection System for Capillary Electrophoresis

A new type of dual-electrode and multi-channel electrochemical detection technology for capillary electrophoresis is described in this paper.Two detectors(the amperometric detector and the conductometric detector) or two conductometric detectors are connected to the same capillary electrophoresis system.The whole system possesses the advantages of the two electrochemical detectors including sparing time,improving the analytical speed and expanding the sample range.The working electrode and detector cell are handled easily.The system was applied to sample detection with satisfactory results.     

Synthesis of Carbon Nanofibers for Mediatorless Sensitive Detection of NADH

Highly sensitive amperometric detection of dihydronicotinamide adenine dinucleotide (NADH) by using novel synthesized carbon nanofibers (CNFs) without addition of any mediator has been proposed. The CNFs were prepared by combination of electrospinning technique with thermal treatment method and were applied without any oxidation pretreatment to construct the electrochemical sensor. In amperometric detection of NADH, a linear range up to 11.45 μM with a low detection limit of 20 nM was obtained with the CNF‐modified carbon paste electrode (CNF‐CPE). Good selectivity was exhibited for the simultaneous detection of NADH and its common interferent of ascorbic acid (AA) by differential pulse voltammogram. The attractive electrochemical performance and the versatile preparation process of the CNF‐CPE made it a promising candidate for designing effective NADH sensor.     

Determination of horseradish peroxidase and a peroxidase-like iron porphyrin at a Nafion-modified electrode.

A catalytic coupling reaction between 4-amino antipyrine and a N,N-disubstituted aniline derivative has been exploited in the indirect electrochemical detection of horseradish peroxidase (HRP) and of a biomimetic catalyst, the iron(III) sulfonated tetraphenyl porphyrin. In the presence of hydrogen peroxide and one of the two catalysts a cationic electroactive quinone-iminium dye P+ was formed and detected by linear scan voltammetry using a screen-printed electrode coated with a Nafion film. Detection limits of 10(-12) M for HRP and 4 x 10(-10) M for the iron porphyrin have been achieved. In conclusion the iron porphyrin is considered to be a promising alternative to the HRP label in enzyme immunoassays with electrochemical detection.     

Microchip-based amperometric immunoassays using redox tracers

A new chip-based electrochemical immunoassay protocol, based on the use of a ferrocene redox label, is described. Two reaction formats, based on direct (noncompetitive) and competitive modes of operation, were employed for illustrating the use of redox tracers in chip-based electrochemical immunoassays. The direct assay consisted of mixing the ferrocene-tagged antibody and the antigen analyte, a rapid electrophoretic separation of labeled free antibody and the labeled antigen/antibody complex, and a downstream anodic detection of the ferrocene tracer at gold-plated carbon screen-printed electrode detector. The competitive assay integrates precolumn reactions of the labeled antigen and the target antigen with the antibody with electrophoretic separation of the free and bound labeled antigens, along with amperometric detection of the redox tag. An internal standard has been used to normalize the peak area for the construction of calibration plots. Fundamental operating variables are examined and optimized. The use of a redox tracer offers the advantages of simplified protocol, wider linear range, higher stability, and higher separation efficiency compared to an analogous use of enzyme tags. The direct mouse-immunoglobulin G (IgG) assay and the competitive 3,3',5-triiodo-L-thyronine (T(3)) one were accomplished within less than 150 and 130 s (with field strengths of 256 and 192 V/cm), and offer minimum detectable concentrations of 2.5 x 10(-12) and 1 x1 0(-6) g/mL, respectively. Such use of redox labels for chip-based amperometric immunoassay protocols offers considerable promise for decentralized clinical or environmental testing.     

Developments in microscale and nanoscale sensors for biomedical sensing

The widespread use of point of care testing in biomedical and clinical applications is a major aim of the electrochemical field. A large number of groups are working on lab-on-a-chip systems or sensor arrays which are underpinned by electrochemical detection methodologies. Miniaturized transducers have the potential to be adopted in such systems for diagnosis of a range of diseases in both clinical and nonclinical settings. In this review, we will present the current trends and state of the art for a selection of miniaturized sensing elements (microelectrodes, nanoelectrodes, and field-effect transistors) and provide an impression of current technologies, their associated performance characteristics, and also considering the major barriers to adoption and how they might be surmounted in future so these technologies can fulfil their early promise.     

Nanogold-penetrated poly(amidoamine) dendrimer for enzyme-free electrochemical immunoassay of cardiac biomarker using cathodic stripping voltammetric method

Methods based on immunoassays have been developed for cardiac biomarkers, but most involve the low sensitivity and are unsuitable for early disease diagnosis. Herein we design an electrochemical immunoassay for sensitive detection of myoglobin (a cardiac biomarker for acute myocardial infarction) by using nanogold-penetrated poly(amidoamine) dendrimer (AuNP-PAMAM) for signal amplification without the need of natural enzymes. The assay was carried out on the monoclonal mouse anti-myoglobin (capture) antibody-anchored glassy carbon electrode using polyclonal rabbit anti-myoglobin (detection) antibody-labeled AuNP-PAMAM as the signal tag. In the presence of target myoglobin, the sandwiched immunocomplex could be formed between capture antibody and detection antibody. Accompanying AuNP-PAMAM, the carried gold nanoparticles could be directly determined via stripping voltammetric method under acidic conditions. Under optimal conditions, the detectable electrochemical signal increased with the increasing target myoglobin in the sample within a dynamic working range from 0.01 to 500 ng mL⁻¹ with a detection limit of 3.8 pg mL⁻¹. The electrochemical immunoassay also exhibited high specificity and good precision toward target myoglobin. Importantly, our strategy could be applied for quantitative monitoring of myoglobin in human serum specimens, giving well matched results with those obtained from commercialized enzyme-linked immunosorbent assay (ELISA) method.     

Carbon fiber nanoelectrodes applied to microchip electrophoresis amperometric detection of neurotransmitter dopamine in rat pheochromocytoma (PC12) cells

Microelectrodes have been adopted in electrochemical detection for CE or microchip CE in recent years. In this paper, the use of nanoelectrodes (with tip diameter of 100-300 nm) as the electrochemical detector in microchip CE is firstly reported. The experimental results indicated that both the sensitivity and resolution of microchip CE with the carbon fiber nanoelectrode (CFNE) amperometric detection have been improved markedly comparing with the traditional microelectrodes. The detection limit of dopamine (S/N = 3) is 5.9x10(-8) M, which is one or two orders of magnitude lower than that reported so far, and the resolution of dopamine (DA) and isoprenaline (IP) has also improved from 0.6 (using 7 mum carbon fiber microelectrodes, CFME) to 1.0. We assembled a novel and easily operated microchip CE system with end-column amperometric detection, which allows the convenient and fast replacement of the passivated electrodes. Under the optimized condition, the RSDs of peak height and migration time are 1.47 and 0.31%, respectively (n = 40), indicating that the system displays excellent reproducibility. The nanoelectrode-based microchip CE system has been successfully applied to the determination of DA in cultured rat pheochromocytoma (PC12) cells, and the average content of DA in an individual PC12 cell is 0.54 +/- 0.07 fmol, which is in good agreement with that reported in the literature.     

Recent developments in amperometric detection for microchip capillary electrophoresis

The interest in microfluidic devices has increased considerably over the past decade due to the numerous advantages of working within a miniature, microfabricated format. This review focuses on recent advances in coupling amperometric detection with microchip capillary electrophoresis (CE). Advances in electrochemical cell design, isolation of the detector from the separation field, and integration of both pre- and postseparation reaction chambers are discussed. The use of microchip CE with amperometric detection for enzyme/immunoassays, clinical and environmental assays, and the determination of neurotransmitters is described.     

Nonaqueous capillary electrophoresis with indirect electrochemical detection

Nonaqueous capillary electrophoresis (NACE) which makes use of organic solvents in place of conventional aqueous electrophoresis buffers is gaining increasing importance among modern separation techniques. Recently, it has been shown that amperometric detection in conjunction with acetonitrile-based NACE offers an extended accessible potential range and an enhanced long-term stability of the amperometric responses generated at solid electrodes. The present contribution takes advantage of the latter aspect to develop reliable systems for NACE with indirect electrochemical detection (IED). In this context, several compounds such as (ferrocenylmethyl)trimethylammonium perchlorate, tris(1,10-phenanthroline)cobalt(III) perchlorate and bis(1,4,7-triazacyclononane)nickel(II) perchlorate were studied regarding their suitability to act as electroactive buffer additives for IED in NACE. The performance characteristics for the respective buffer systems were evaluated. Tetraalkylammonium perchlorates served as model compounds for the optimization of the NACE-IED system. Target analytes choline and acetylcholine could easily be separated and determined by means of NACE-IED. In the case of a buffer system containing 10(-4) M tris(1,10-phenanthroline)cobalt(III) perchlorate the limits of detection were 2.5 x 10(-7) M and 4.6 x 10(-7) M for choline and acetylcholine, respectively. With the elaborated analytical procedure choline could be determined in pharmaceutical preparations.     

Effect of extraction pH on metal speciation in plant root extracts

The species patterns of nutrient and trace metals (K, Ca, Mg, Mn, Fe, Zn) obtained by extraction of plant roots have been determined as a function of extraction pH in the range 4-9. The extractable metal concentrations were subdivided into low-molecular-weight (<10 kDa) and high-molecular-weight (>10 kDa) metal species by TXRF analysis. Except for pH 9, the low-molecular-weight fraction is predominant. This fraction was further separated by HPLC with AAS detection. It was demonstrated that the nature of the metal species changes dramatically when the extraction pH is changed, especially in the range 9-7. Information about the chemical nature of chromatographically separated metal species is obtained by use of different electrochemical detectors (amperometric detection at a glassy carbon or copper electrode and pulsed amperometric detection) and diode-array UV detection.     

Biocompatible and label-free amperometric immunosensor for hepatitis B surface antigen using a sensing film composed of poly(allylamine)-branched ferrocene and gold nanoparticles

An amperometric immunosensor has been developed for sensitive determination of hepatitis B surface antigen as a model protein. A glassy carbon electrode was modified with an assembly of positively charged poly(allylamine)-branched ferrocene (PAA-Fc) and negatively charged gold nanoparticles (Au NPs). The formation of PAA-Fc effectively avoids the leakage of Fc, retains its electrochemical activity, and enhances the conductivity of the composite. The adsorption of Au NPs onto the PAA-Fc matrix provides sites for the immobilization of the antigen and a favorable micro-environment to maintain its activity. The morphologies and electrochemistry of the sensing film were investigated via scanning electron microscopy, electrochemical impedance spectroscopy, and cyclic voltammetry. Factors influencing the performance of the immunosensor were studied in detail. The concentration of the antigen can be quantitated (by measuring the decrease of the amperometric response resulting from the specific binding between antigen and antibody) in the range between 0.1 and 150?ng?mL^?C1, with a detection limit of 40?pg?mL^?C1 (S/N = 3). The method is economical, efficient, and potentially attractive for clinical immunoassays.

Determination of taurine in human tear fluid by capillary electrophoresis with indirect amperometric detection based on electrogenerated bromine

A new method for the determination of taurine was developed based on indirect amperometric detection after capillary electrophoresis. A serial dual‐electrode detector comprising an on column Pt film electrode (upstream electrode) and an end column Pt microdisk electrode (downstream electrode) was utilized to conduct the indirect amperometric detection. Bromide is oxidized to bromine at upstream electrode and reduced back to bromide at downstream electrode. Since taurine can react with bromine quantitatively and rapidly, its concentration can therefore be determined by the decrease of the current for bromine reduction at the downstream electrode. Principal experimental parameters governing the analytical performance were investigated and optimized. Under the optimal conditions, taurine can be baseline separated from interfering amino acids and the detection limit of 0.18 μM was obtained with a linear correlation coefficient of 0.999 over the concentration range of 0.5–60 μM. The developed method has been successfully applied in the determination of taurine in human tear fluid. The taurine level obtained was in good agreement with previous reports and recoveries for taurine spiked ranged from 92–95% with relative standard deviations within 4.6%, demonstrating the reliability of the developed method in the determination of taurine in human tear fluid.