Sequential injection/electrochemical immunoassay for quantifying the pesticide metabolite 3,5,6-trichloro-2-pyridinol

An automated and sensitive sequential injection electrochemical immunoassay was developed to monitor a potential insecticide biomarker, 3,5,6-trichloro-2-pyridinol. The current method involved a sequential injection analysis (SIA) system equipped with a thin-layer electrochemical flow cell and permanent magnet, which was used to fix 3,5,6-trichloro-2-pyridinol (TCP) antibody coated magnetic beads (TCP-Ab-MBs) in the reaction zone. After competitive immunoreactions among TCP-Ab-MBs, TCP analyte, and horseradish peroxidase (HRP) labeled TCP, a 3,3′,5,5′-tetramethylbenzidine dihydrochloride and hydrogen peroxide (TMB-H₂O₂) substrate solution was injected to produce an electroactive enzymatic product. The activity of HRP tracers was monitored by a square wave voltammetric scanning electroactive enzymatic product in the thin-layer flow cell. The voltammetric characteristics of the substrate and the enzymatic product were investigated under batch conditions, and the parameters of the immunoassay were optimized in the SIA system. Under the optimal conditions, the system was used to measure as low as 6 ng L⁻¹(ppt) TCP, which is around 50-fold lower than the value indicated by the manufacturer of the TCP RaPID Assay^® kit (0.25 μg/L, colorimetric detection). The performance of the developed immunoassay system was successfully evaluated on tap water and river water samples spiked with TCP. This technique could be readily used for detecting other environmental contaminants by developing specific antibodies against contaminants and is expected to open new opportunities for environmental and biological monitoring.     

Sensitive Electrochemical Detection of Horseradish Peroxidase at Disposable Screen‐Printed Carbon Electrode

A rapid, simple and sensitive electrochemical assay of horseradish peroxidase (HRP) performed on disposable screen‐printed carbon electrode was developed. HRP activities were monitored by square‐wave voltammetric (SWV) measuring the electroactive enzymatic product in the presence of o‐aminophenol and hydrogen peroxide substrate solution. SWV analysis demonstrated a greater sensitivity and shorter analysis time than the widely used amperometric and differential‐pulsed voltammetric methods. The voltammetric characteristics of substrate and enzymatic product as well as the parameters of SWV analysis were optimized. Under optimized conditions, a linear response for HRP from 0.003 to 0.1 U/mL and a detection limit of 0.002 U/mL (1.25×10^?15 mol in 25 μL) were obtained with a good precision (RSD=8%; n=6). This rapid and sensitive HRP assay with microliter‐assay volume could be readily integrated to portable devices and point‐of‐care (POC) diagnosis applications.     

Cyanidin-horseradish peroxidase-hydroperoxide reaction system and its application in enzymelinked immunosensing assays

A cyanidin-based horseradish peroxidase (HRP)-catalyzed reaction system was established in this work. In B-R buffer solutions (pH 6.8), a UV-visible absorbance peak of cyanidin (CAG) at 540 nm (A _p1) appeared. After the oxidation reaction of CAG catalyzed by HRP in the presence of H₂O₂, a significant absorbance peak at 482 nm (A _p2) occurred. The ratio R(A _P2/A _P1) was proportional to the HRP concentration. The application of CAG in the enzyme-linked immunosensing assays was investigated using food and mouth disease virus antigen (FMDVAg) as a model analyte. In sandwich immunoreaction, the analyte FMDVAg and food and mouth disease virus antibody (FMDVAb)-modified magnetic nanoparticles bound the supported conconvalina (Con A) bound with HRP-FMDVAb. After de-absorbing and separating, the HRP-FMDVAb-FMDVAg-FMDVAb-magnetic nanoparticles complexes were subject to enzymatic reaction and UV-visible absorbance measurements. The HRP moiety of the immunoreaction complexes can catalyze the oxidation reaction of CAG by H₂O₂, and the substrate CAG is converted to products. Based on this principle, a sandwich assay model has been employed to determine FMDVAg in rabbit serum samples with the aid of FMDVAb-Fe₃O₄ magnetic nanoparticles. The linear range of the FMDVAg determination is 1.5×10⁻⁸−2.7×10⁻⁶ g/mL with the relatively standard deviation of 3.7% (n = 11). The detection limit is 3.1×10⁻⁹ g/mL. Additional advantages of the typical substrate such as OPD, OAP and TMB are good water-solubility and stability. Supported by the Scientific Research Foundation of Hunan Province (Grant No. 2008SK3052) and the Scientific Research Foundation of Hunan Provincial Education Department (Grant No. 08B004)     

4-羟基苯乙烯基吡啶为HRP底物的酶荧光免疫传感体系测定布氏杆菌抗体

本文合成了一种新型辣根过氧化物酶（HRP）荧光底物—4-羟基苯乙基吡啶（pHSP）,并首次将它运用于酶联荧光免疫传感体系。对pHSP化学性质的研究证实,pHSP在空气中较稳定,对HRP、H2O2的荧光响应性能优于传统HRP荧光底物如对羟苯乙酸、Amplex Red和佳味醇等。pHSP本身只有极弱的荧光,在HRP催化下可被 H2O2氧化成二聚体产物,该二聚体在300 nm的激发光下能发射波长为437 nm的强荧光,并且反应体系的荧光增加与HRP量在一定浓度范围内成线形相关。根据此原理,建立了兔布氏杆菌抗体的酶联荧光传感分析新方法。运用制备的传感装置测定兔布氏杆菌抗体的线形范围为110-5 1.6 10-3 g/L,抗体检出限为110-5 g/L,相对标准偏差为4.1%（n=11）。 pHSP的二聚体产物水溶性很低,利用设计的装置较好地解决了传统测定溶液体系方法灵敏度打折的问题。     

Magnetic beads-based bioelectrochemical immunoassay of polycyclic aromatic hydrocarbons

A simple, rapid, and sensitive bioelectrochemical immunoassay method based on magnetic beads (MBs) has been developed to detect polycyclic aromatic hydrocarbons (PAHs). The principle of this bioassay is based on a direct competitive enzyme-linked immunosorbent assay using PAH-antibody-coated MBs and horseradish peroxidase (HRP)-labeled PAH (HRP-PAH). A magnetic process platform was used to mix and shake the samples during the immunoreactions and to separate free and unbound reagents after the liquid-phase competitive immunoreaction among PAH-antibody-coated MBs, PAH analyte, and HRP-PAH. After a complete immunoassay, the HRP tracers attached to MBs were transferred to a substrate solution containing 3,3′,5,5′-tetramethylbenzidine (TMB) and hydrogen peroxide (H₂O₂) for electrochemical detection. The voltammetric characteristics of the substrate were investigated, and the reduction peak current of TMB was used to quantify the concentration of PAH. The different parameters, including the amount of HRP-PAH conjugates, the enzyme catalytic reaction time, and the pH of the supporting electrolyte that governs the analytical performance of the immunoassay have been studied in detail and optimized. The detection limit of 50 pg mL⁻¹ was obtained under optimum experimental conditions. The performance of this bioelectrochemical magnetic immunoassay was successfully evaluated with tap water spiked with PAHs, indicating that this convenient and sensitive technique offers great promise for decentralized environmental applications.     

Bioelectrochemical immunoassay of polychlorinated biphenyl

A simple, rapid, and highly sensitive bioelectrochemical immunoassay method based on magnetic beads (MBs) and disposable screen-printed electrodes (SPE) has been developed to detect polychlorinated biphenyls (PCBs). The principle of this bioassay is based on a direct competitive enzyme-linked immunosorbent assay using PCB-antibody-coated MBs and horseradish peroxidase (HRP)-labeled PCB (HRP-PCB). A magnetic process platform was used to mix and shake the samples during the immunoreactions and to separate free and unbound reagents after the liquid-phase competitive immunoreactions among PCB-antibody-coated MBs, PCB analyte, and HRP-PCB. After a complete immunoassay, the HRP tracers attached to MBs were transferred to a substrate solution containing o-aminophenol and hydrogen peroxide for electrochemical detection. The different parameters, including the amount of HRP-PCB conjugates, immunoreaction time, and the concentration of substrate that governs the analytical performance of the immunoassay have been studied in detail and optimized. The detection limit of 10 pg mL⁻¹ was obtained under optimum experimental conditions. The performance of this bioelectrochemical immunoassay was successfully evaluated with untreated river water spiked with PCBs, and the results were validated by commercial PCB enzyme-linked immunosorbent assay kit, indicating that this convenient and sensitive technique offers great promise for decentralized environmental application and trace PCBs monitoring.     

Hierarchical Hybrid Peroxidase Catalysts for Remediation of Phenol Wastewater

We report a new family of hierarchical hybrid catalysts comprised of horseradish peroxidase (HRP)–magnetic nanoparticles for advanced oxidation processes and demonstrate their utility in the removal of phenol from water. The immobilized HRP catalyzes the oxidation of phenols in the presence of H₂O₂, producing free radicals. The phenoxy radicals react with each other in a non‐enzymatic process to form polymers, which can be removed by precipitation with salts or condensation. The hybrid peroxidase catalysts exhibit three times higher activity than free HRP and are able to remove three times more phenol from water compared to free HRP under similar conditions. In addition, the hybrid catalysts reduce substrate inhibition and limit inactivation from reaction products, which are common problems with free or conventionally immobilized enzymes. Reusability is improved when the HRP–magnetic nanoparticle hybrids are supported on micron‐scale magnetic particles, and can be retained with a specially designed magnetically driven reactor. The performance of the hybrid catalysts makes them attractive for several industrial and environmental applications and their development might pave the way for practical applications by eliminating most of the limitations that have prevented the use of free or conventionally immobilized enzymes.     

3‐Indoxyl Phosphate as an Electrochemical Substrate for Horseradish Peroxidase

In this work 3‐indoxyl phosphate (3‐IP), an alkaline phosphatase substrate, is demonstrated to be a suitable substrate for horseradish peroxidase (HRP). HRP catalyzes the oxidation of 3‐IP in presence of hydrogen peroxide (H₂O₂) generating the product indigo blue, which is an aromatic heterocycle compound insoluble in aqueous solutions. This product was easily converted into its soluble parent compound indigo carmine (IC) (by addition of fuming sulfuric acid to the reaction media) which has a reversible voltammetric peak at the formal potential of ?0.15 V (vs. Ag pseudo‐reference electrode) when a screen‐printed carbon electrode (SPCE) is used. Parameters that influence the enzymatic reaction, such as pH, temperature, substrate concentration and reaction time have been optimized. Moreover, the enzyme apparent kinetic constants (V_max, K_M) for both substrates (3‐IP and H₂O₂) have been calculated. Indirect measurements of HRP activity in solution were carried out not only by cyclic voltammetry but also using amperometric detection in a flow system. The detection limits were 6.86×10^?12 and 5.68×10^?12 M, respectively. Thus, 3‐IP is the first substrate that could be used for alkaline phosphatase (AP) and HRP, the most common enzymatic labels in affinity assays.     

Amplified Electrochemical Immunosensor for Calmodulin Detection Based on Gold‐Silver‐Graphene Hybrid Nanomaterials and Enhanced Gold Nanorods Labels

Calmodulin (CaM) is an important intracellular calcium‐binding protein. It plays a critical role in a variety of biological and biochemical processes. In this paper, a new electrochemical immunosensing protocol for sensitive detection of CaM was developed by using gold‐silver‐graphene (AuAgGP) hybrid nanomaterials as protein immobilization matrices and gold nanorods (GNRs) as enhanced electrochemical labels. Electrode was first modified with thionine‐chitosan film to provide an immobilization support for gold‐silver‐graphene hybrid nanomaterials. The hybrid materials formed an effective matrix for binding of CaM with high density and improved the electrochemical responses as well. Gold nanorods were prepared for the fabrication of enhanced labels (HRP‐Ab₂‐GNRs), which provided a large capacity for HRP‐Ab₂ immobilization and a facile pathway for electron transfer. With two‐step immunoassay format, the HRP‐Ab₂‐GNRs labels were introduced onto the electrode surface, and produced electrochemical responses by catalytic reaction of HRP toward enzyme substrate of hydrogen peroxide (H₂O₂) in the presence of thionine. The proposed immunosensor showed an excellent analytical performance for the detection of CaM ranging from 50 pg mL^?1 to 200 ng mL^?1 with a detection limit of 18 pg mL^?1. The immunosensor has also been successfully applied to the CaM analysis in two cancer cells (HepG2 and MCF‐7) with high sensitivity, which has shown great potency for improving clinic diagnosis and treatment for cancer study.     

Fluorogenic assays for activated protein C using aptamer modified magnetic beads

We describe a fluorogenic assay for activated protein C (APC) by using magnetic beads modified with DNA aptamers, taking advantage of strong binding affinity of aptamer, facile magnetic separation, and signal amplification via an enzymatic reaction. APC is specifically captured from a sample by the DNA aptamers on magnetic beads, and the concentrated APC then catalyzes the conversion of a fluorogenic substrate of APC to a fluorescent product. Detection of APC is achieved by measuring the generated product. This method is simple, sensitive, and specific. APC can be detected at 0.4 pM concentration level in a sample volume of 250 μL, corresponding to 0.1 femtomole of APC, when 2-h enzymatic reaction is employed. The proteins thrombin, trypsin, proteinase K, chymotrypsin, and elastase do not interfere.

Fluorometric enzyme immunosensing system based on natural product resveratrol for horseradish peroxidase and Ag/SiO<Subscript>2</Subscript> nanoparticles

The properties of resveratrol (3′, 4′, 5-trihydroxystlbene, RST) were for the first time evaluated as a potential substrate for horseradish peroxidase (HRP)-catalyzed fluorogenic reaction. The properties of RST for use as fluorogenic substrates for HRP and its application in immunoassays were compared with commercially available substrates such as p-hydroxyphenylpropionic acid (pHPPA), chavicol and Amplex red by a fluoroimmunosensing method in the use of Schistosomia japonicum antibody (SjAb) as a model analyte. The fluoroimmunosensing device was constructed by dispersing Schistosomia japonicum antigen (SjAg), nano-Ag/SiO₂ particles and sol-gel at low temperature. In pH 5.8 Britton-Robinson buffer (B-R), HRP-SjAb conjugates can catalyze the polymerization reaction of RST by H₂O₂ forming fluorescent dimmers. The increase of the fluorescence intensity of the dimmers product at emission of 462 nm (excitation: 315 nm) is proportional to the concentration of HRP-SjAb binding to the SjAg entrapped in the nano-Ag/SiO₂ particles-sol-gel matrix. A competitive binding assay has been used to determine SjAb in rabbit serum with the aid of SjAb labeled with HRP. Substrate RST showed comparable ability for HRP detection and its enzyme-linked immunosensing reaction system, in a linear detection ranging of 1.5×10⁻⁶–7.3×10⁻⁴ g/L and with a detection limit of 1.5×10⁻⁶ g/L. The immobilized biocomposites surface could be regenerated by simply polishing with an alumina paper, with an excellent reproducibility (RSD = 4.7%). The proposed method has been successfully used for analysis of the rabbit serum sample with satisfactory results. Supported by the Projects of Scientific Research Fund of Hunan Provincial Education Department of China (Grant Nos. 05B020 and 06C098)     

Graphene and Nanogold‐Functionalized Immunosensing Interface with Enhanced Sensitivity for One‐Step Electrochemical Immunoassay of Alpha‐Fetoprotein in Human Serum

A new electrochemical immunosensing protocol for sensitive detection of alpha‐fetoprotein (AFP, as a model) in human serum was developed by means of immobilization of horseradish peroxidase‐anti‐AFP conjugates (HRP‐anti‐AFP) onto graphene and nanogold‐functionalized biomimetic interfaces. The low‐toxic and high‐conductive graphene complex provided a large capacity for nanoparticulate immobilization and a facile pathway for electron transfer. With a one‐step immunoassay format, the antigen‐antibody complex was formed between the immobilized HRP‐anti‐AFP on the electrode and AFP in the sample. The formed immunocomplex was coated on the electrode surface, inhibited partly the active center of HRP, and decreased the catalytic reduction of HRP toward the enzyme substrate of H₂O₂. Under optimal conditions, the decrease of reduction currents was proportional to AFP concentration, and the dynamic range was 1.0–10 ng/mL with a relative‐low detection limit (LOD) of 0.7 ng/mL AFP. Intra‐ and inter‐assay coefficients of variation (CVs) were less than 10 %. The assay was evaluated for clinical human serum samples, including 8 (possible) patients with hepatocarcinoma and 3 normal human sera. Correct identification of negative/positive samples and perfect accordance with results from Elecsys 2010 Electrochemiluminescent Automatic Analyzer as a reference was obtained. Importantly, the graphene and nanogold‐based sensor provided a promising platform for the detection of other biocompounds, and could be further applied for development of other potential electrochemical bio/chemosensors.     

Electrochemical Immunosensing Chip Using Selective Surface Modification,Capillary‐Driven Microfluidic Control,and Signal Amplification by Redox Cycling

A sensitive electrochemical immunosensing chip is presented by employing (i) selective modification of protein‐resistant surfaces; (ii) fabrication of a stable Ag/AgCl reference electrode; (iii) capillary‐driven microfluidic control; (iv) signal amplification by redox cycling along with enzymatic reaction. Purely capillary‐driven microfluidic control is combined with electrochemical sandwich‐type immunosensing procedure. Selective modification of the surfaces is achieved by chemical reactivity‐controlled patterning and electrochemical deposition. Fluidic control of the immunosensing chip is achieved by spontaneous capillary‐driven flows and passive washing. The detection limit for mouse IgG in the immunosensing chip is 10 pg/mL.     

Improvement of analytical performances of a disposable electrochemical immunosensor by using magnetic beads

A comparison of two electrochemical immunosensing strategies for PCBs detection, based on the use of two different solid phases, is here discussed. In both cases, carbon-based screen-printed electrodes (SPEs) are used as transducers in a direct competitive immunoassay scheme, where PCBs in solution compete with the tracer PCB28-alkaline phosphatase (AP) labeled for antibodies immobilized onto the solid-phase.In the standard format (called EI strategy), SPEs are both the solid-phase for immunoassay and electrochemical transducers: in this case the immunochemical reaction occurs onto the working electrode. Finally, the enzymatic substrate is added and an electroactive product is generated and detected by electrochemical measurement. In order to improve the performances of the system, a new approach (called EMI strategy) is developed by using functionalized magnetic beads as solid phase for the competitive assay; only after the immunosensing step they are captured by a magnet onto the working surface of the SPE for the electrochemical detection.Experimental results evidenced that the configuration based on the use of separate surfaces for immunoassay and for electrochemical detection gave the best results in terms of sensitivity and speed of the analysis. The improvement of analytical performances of the immunosensor based on EMI strategy was also demonstrated by the analysis of some spiked samples.     

Magnetic beads-based immunoassay as a sensitive alternative for atrazine analysis

A new immunoassay strategy for sensitive atrazine determination based on magnetic beads is reported. The immuno-method is a competitive solid-phase immunoassay where the anti-atrazine antibody is immobilized on the magnetic beads surface and fixed at the reaction cell bottom using a simple magnet, which generates a magnetic field. Analyte and HRP (horseradish peroxidase) tracer compete for active sites of antibody. After the immunointeractions antibody-analyte and antibody-tracer, atrazine quantification from the sample is performed by injection of the chemiluminescence substrate (luminol, hydrogen peroxide and p-iodophenol). Different antibodies (polyIgG anti-atrazine Ab I and affinity purified polyIgG anti-atrazine AbI) were tested in this configuration. Also, optimum concentration of antibody-covered magnetic beads was set up (8 mg/l Ab II). Finally, the performance of magnetic beads-based immunoassay for atrazine determination was evaluated demonstrating that the magnetic beads-based immunoassay is one of the most sensitive method for atrazine determination (LoD = 3 pg/l, IC₅₀ = 37 pg/l, DR = 10-1000 pg/l).     

Amperometric Magnetoimmunosensors for Direct Determination of D‐Dimer in Human Serum

Two different D‐dimer disposable amperometric immunosensing designs based on indirect competitive or sandwich formats and the use of carboxylic acid‐modified magnetic beads (COOH‐MBs) and screen‐printed carbon electrodes (SPCEs) have been developed and compared. In both approaches, the resulting modified MBs were magnetically captured on the surface of a SPCE which was used as the transducer for the electrochemical detection at ?0.20 V upon addition of H₂O₂, and hydroquinone (HQ). Both configurations exhibited linear ranges of clinical usefulness and detection limits quite below the clinical threshold (0.5 µg mL^?1 D‐dimer). The sandwich configuration has been successfully tested with serum samples.     

An improved ELISA for the determination of southern bean mosaic virus with linear sweep voltammetry detection based on new system of PAP-H_2O_2-HRP

An improved enzyme-linked immunosorbent assay (ELISA) for the determination of southern bean mosaic virus (SBMV) with linear sweep voltammetry based on a new system of p-aminophenol (PAP)-H2O2-horseradish peroxidase (HRP) has firstly been developed. The enzymatic product 3-[(4-hydrox-yphenyl) amino]-4-(2-amino-5-hydroxyphenyl)-6-[ (4-hydrox-yphenyl)imino]-2,4-cyclohexadiene-l-one, produced from the oxidation of PAP with H2O2 catalyzed by HRP, yielded a sensitive linear sweep voltammetric response at - 0.45 V ( vs. SCE) in Britton-Robinson (BR) buffer solution. Based on the voltammetric peak, HRP can be measured with a detection limit of 0.4 mU/L and a linear range of 1.0-1.0 × 102 mU/ L. The detection limit for the SBMV is 8.0 ng/mL and the highest dilution ratio for the detection of infected leaf sap is 1: 1.5×105.     

Electrochemical immunoassay using magnetic beads for the determination of zearalenone in baby food: An anticipated analytical tool for food safety

In this work, electrochemical immunoassay involving magnetic beads to determine zearalenone in selected food samples has been developed. The immunoassay scheme has been based on a direct competitive immunoassay method in which antibody-coated magnetic beads were employed as the immobilisation support and horseradish peroxidase (HRP) was used as enzymatic label. Amperometric detection has been achieved through the addition of hydrogen peroxide substrate and hydroquinone as mediator.Analytical performance of the electrochemical immunoassay has been evaluated by analysis of maize certified reference material (CRM) and selected baby food samples. A detection limit (LOD) of 0.011 μg L⁻¹ and EC₅₀ 0.079 μg L⁻¹ were obtained allowing the assessment of the detection of zearalenone mycotoxin. In addition, an excellent accuracy with a high recovery yield ranging between 95 and 108% has been obtained. The analytical features have shown the proposed electrochemical immunoassay to be a very powerful and timely screening tool for the food safety scene.     

Ultrasensitive analysis of glucose in serum by capillary electrophoresis with LIF detection in combination with signal amplification strategies and on‐column enzymatic assay

A highly specific and sensitive method for glucose quantification in human serum samples based on on‐column enzymatic assay is described. In this method, the head of the capillary was used as a nanoliter‐microreactor, the diluted samples spiked with a novel fluorogenic reagent named 2‐[6‐(4′‐amino) phenoxy‐3H‐xanthen‐3‐on‐9‐yl] benzoic acid (APF), and the mixed enzyme solutions of glucose oxidase (GOx) and horseradish peroxidase (HRP), were individually injected into the capillary. Hydrogen peroxide (H₂O₂) generated in situ by catalytic reaction between GOx and glucose, activates APF in the presence of HRP to form a highly fluorescent product, which was electrophoretically separated from the unreacted APF and detected by the LIF detector. The proposed method allowed the determination of glucose down to 10 nM in real samples, with RSD values lower than 3.5%, which also has the potential for measurements of multicomponents in many other systems including measurement of α‐glucosidase activity and screening for its inhibitors.