Renewable amperometric immunosensor for complement 3 assay based on the sol-gel technique

A renewable amperometric immunosensor based on the sol-gel technique has been constructed by dispersing graphite, complement 3 (C3) antiserum, and sol-gel at low temperature. The prepared immunosensor is rigid, porous, and has a renewable external surface. A competitive binding assay has been used to determine C3 in human serum with the aid of C3 labeled with horseradish peroxidase. The enzyme-labeled antigen can readily diffuse toward the encapsulated antibody, which retains its binding properties. The experimental conditions for the assay with the biocomposite, including the loading of C3 antiserum in the biocomposite, the amount of labeled C3 in incubation solution, incubation time, and temperature, have been optimized. Using C3 labeled with horseradish peroxidase, and o-AP as the substrate, amperometric detection at -150 mV (relative to the SCE) results in a linear detection range of 1.17-35.1 microg mL(-1), with a detection limit of 0.56 microg mL(-1). Serum samples have been assayed and the results demonstrate the feasibility of the proposed immunosensor for clinical analysis. The surface of the immunosensor can be renewed simply by polishing to obtain a fresh immunocomposite ready to use in a new competitive assay.     

Renewable amperometric immunosensor for complement 3 assay based on the sol–gel technique

A renewable amperometric immunosensor based on the sol–gel technique has been constructed by dispersing graphite, complement 3 (C3) antiserum, and sol–gel at low temperature. The prepared immunosensor is rigid, porous, and has a renewable external surface. A competitive binding assay has been used to determine C3 in human serum with the aid of C3 labeled with horseradish peroxidase. The enzyme-labeled antigen can readily diffuse toward the encapsulated antibody, which retains its binding properties. The experimental conditions for the assay with the biocomposite, including the loading of C3 antiserum in the biocomposite, the amount of labeled C3 in incubation solution, incubation time, and temperature, have been optimized. Using C3 labeled with horseradish peroxidase, and o-AP as the substrate, amperometric detection at –150 mV (relative to the SCE) results in a linear detection range of 1.17–35.1 μg mL^–1, with a detection limit of 0.56 μg mL^–1. Serum samples have been assayed and the results demonstrate the feasibility of the proposed immunosensor for clinical analysis. The surface of the immunosensor can be renewed simply by polishing to obtain a fresh immunocomposite ready to use in a new competitive assay.     

An amperometric immunosensor based on a conducting immunocomposite electrode for the determination of Schistosoma japonicum antigen.

A renewable amperometric immunosensor based on a graphite-paraffin-Schistosoma japonicum antibody (SjAb) biocomposite electrode has been prepared for the detection of Schistosoma japonicum antigen (SjAg). Competitive ELISA was employed involving HRP-SjAg as a tracer and 3,3',5,5'-tetramethylbenzidine (TMB) as a substrate. The product of an enzyme catalytic reaction was detected at +0.1 V (vs. Ag/AgCl reference electrode) for measuring the amount of HRP-labeled SjAg binding to the electrode surface. The assay conditions were optimized, including the amount of SjAb loading in the electrode and HRP-SjAg in the incubation solution, the pH of the measuring solution and the incubation time. The measuring range was 0.5-30 microg/ml under the optimum conditions. Rabbit serum samples of different infection degree were measured, which demonstrated that the immunosensor meets the demands of clinical analysis. It exhibits some advantages, such as simplicity of fabrication, rapidity of measurement, and satisfactory sensitivity and reproducibility.     

Bacteria-modified amperometric immunosensor for a Brucella melitensis antibody assay.

A novel amperometric immunosensor setup is described which uses horseradish peroxidase (HRP) as a label in conjunction with a current-based Brucella sensor. The Bacteria modified immunosensor was constructed by using a biocomposite formed by dispersing graphite powder into a mixture of Brucella melitensis and silicate polymer gel. The enzyme-labeled antibody can readily diffuse toward the encapsulated antigen (Brucella melitensis), which retains its binding properties, and the association reaction is easily detected at the surface exposed to the solution. The use of an oaminophenol (o-AP) substrate and amperometric detection at -150 mV (vs. SCE) results in a relatively low detection limit of 3.5 ng/ml and a linear detection range of 3.5 ng/ml to 200 ng/ml. Based on an optimized parameter, the prepared sensor was used to detect the Brucella melitensis antibody in serum samples by using a competitive binding assay. The results demonstrate the feasibility of employing the proposed immunosensor for the detection for Brucella melitensis antibody in a clinical analysis.     

Schistosoma japonicum antibody assay by immunosensing with fluorescence detection using 3,3',5,5'-tetramethylbenzidine as substrate

An immunosensing system for Schistosoma Japonicum antibody (SjAb) assay has been developed which is useful for the diagnosis of schistosomaisis. To circumvent the difficulty of regeneration of the immunosensing device, the sol-gel technique is used which results in a considerable retention of the activity of the encapsulated antigen (SjAb) and easily diffusing into the pores of the polymeric silica matrix. The surface of the immunosensing device prepared can easily be renewed by simply polishing. The regenerated surface serves as a platform for the competitive immuno-reaction of HRP-SjAb and SjAb with SjAg bound at the surface. By using 3, 3', 5, 5'-tetramethylbenzidine (TMB) as the substrate, the amount of HRP-SjAb bound is quantitated fluorimetrically, which is in turn related with the SjAb content. An amplification effect is obtained by using the enzymatic reaction, and an improved detection limit of 4.5 ng ml(-1) is thus realized. The optimum analytical conditions such as pH, amount of the labeled antibody and flow rates of substrate carrier solution were established. The immunosensing procedure shows a pseudo linear response range from 4.5 to 55 ng ml(-1). The proposed procedure has been employed to determine SjAb in serum samples.     

Electrochemical Detection of Schistosoma Japonicum Antibody Using Biocatalytic Deposition

Abstract

An ultrasensitive electrochemical immunoassay based on biocatalytic deposition has been proposed for the detection of Schistosoma japonicum antibody (SjAb) in infected rabbit serum. Schistosoma japonicum antigen (SjAg) was immobilized on the gold electrode surface via glutaraldehyde crosslink and then incubated with infected rabbit serum containing SjAb; finally, the goat anti-rabbit IgG labeled with alkaline phosphatase was sandwiched to form the immunocomplex on the gold electrode surface. The alkaline phosphatase converted nonelectroactive substrate into the reducing agent and the latter, in turn, reduced metal ions to form electroactive metallic product on the electrode surface. Linear sweep voltammetry (LSV) was used to quantify the amount of the deposited silver and give the analytical signal for SjAb. Assay conditions such as the antigen concentration and enzymatic silver deposition time were optimized. The electrochemical immunosensor was able to realize a reliable determination of SjAb in the dilution range from 1:5000 to 1:100 with a detection limit of 1:6457 of dilution ratio. The feasibility of the proposed immunosensor for possible clinical applications was also investigated by analyzing real serum samples.     

Organic-inorganic matrix for electrochemical immunoassay: Detection of human IgG based on ZnO/chitosan composite

A new strategy to construct amperometric immunosensor for human IgG assay based on ZnO/chitosan composite as sensing platform has been described. This material, which combined the advantages of inorganic species, ZnO and organic polymer, chitosan, can maintain biological activity well. A sequential sandwich immunoassay format was performed on the ZnO/chitosan composite supported by glass carbon electrode (GCE) using goat-anti-human IgG antibody (IgG Ab) and human IgG as a model system. Amperometry was used to determine the amount of horse-radish peroxidase (HRP) fixed on the sensor surface, which was related to the content of the desired human IgG. Assay conditions that were optimized included the amount of labeled antibody, the incubation time and temperature, the pH of the substrate solution, etc. Using hydroquinone as a mediator, amperometric detection at −150 mV (versus SCE) resulted in a detection range 2.5-500 ng mL⁻¹, with a detection limit of 1.2 ng mL⁻¹. The simple manipulations of the construction of ZnO/chitosan composite, as well as low-cost and broad linear range, are the main features of the proposed immunosensing method.     

Amperometric immunosensor for the detection of Vibrio cholerae O1 using disposable screen-printed electrodes.

A disposable amperometric immunosensor was studied for the rapid detection of Vibrio cholerae (V. cholerae), the causative agent of cholera, employing an indirect sandwich enzyme linked immunosorbent assay (ELISA) principle. Screen-printed electrodes (SPEs) were fabricated (by using commercial and homemade carbon inks), electrochemically characterized and the assay conditions were optimized for capturing antibodies and antigen. Whole cell lysate (WCL) of V. cholerae was used to raise antibodies in rabbits and mice. The antibodies raised against WCL of V. cholerae were found to be specific, and no cross reactivity was observed with other enteric bacteria. 1-Naphthyl phosphate was used as a substrate with the amperometric detection of its enzymatic hydrolysis product 1-naphthol at a potential of +400 mV vs. Ag/AgCl reference electrode. A comparison between the amperometric detection technique and the standard ELISA was made in terms of the total assay time, the amount of biological materials used and the sensitivity of detection. The minimum detection limit of the amperometric immunosensor for V. cholerae was found to be 10(5) cells/ml in 55 min, while ELISA detected 10(6) cells/ml in 4 h.     

Renewable amperometric immunosensor based on paraffin-graphite-transferrin antiserum biocomposite for transferrin assay

A renewable electrochemical immunosensor was developed for the determination of transferrin in human serum. It is based on a paraffin-graphite-transferrin antiserum biocomposite, which needs no additional curing. A competitive binding assay was used to determine transferrin in human serum with the aid of transferrin labeled with horseradish peroxidase. The assay conditions were optimized, including the loading of transferrin antiserum in the biocomposite, the amount of labeled transferrin in the incubation solution, incubation time and temperature. Serum samples were analyzed and the results demonstrate that the concentration range of determination with this system meets the demands of clinical analysis. The surface of the immunosensor can be regenerated by simply polishing to obtain a fresh immunocomposite ready to be used in a new competitive assay.     

A label-free amperometric immunosenor based on multi-layer assembly of polymerized o-phenylenediamine and gold nanoparticles for determination of Japanese B encephalitis vaccine

A label-free amperometric immunosensor for fast and sensitive assay of Japanese B encephalitis vaccine is presented. Antiserum of Japanese B encephalitis were immobilized on bilayer nano-Au/o-phenylenediamine polymer film with deposited Prussian blue as an electronic mediator on the Pt electrode. The electrochemical behavior of the biosensor was studied with Fe^2+/3+ as probe on the Pt surface using cyclic voltammetry technique. The variation of amperometric response to the concentration of Japanese B encephalitis vaccine, the target antigen, was evaluated by cyclic voltammetry in PBS. The immunosensor showed a specific response to Japanese B encephalitis vaccine in the range 1.1 × 10⁻⁸ to 1.9 × 10⁻⁶ lg pfu/ml (pfu/ml is plaque forming unit and lg is common logarithm) with a detection limit of 6 × 10⁻⁹ lg pfu/ml. The correlation coefficient is 0.9955. The incubation time, incubation temperature, pH, reproducibility and stability of the immunosensor were also studied. The present work supplied a promising test method for biological products.     

基于等离子体聚合膜的日本血吸虫压电免疫传感器的研究

提出了一种测定日本血吸虫抗体的可逆压电免疫传感器。先在石英晶振上沉积正丁胺等离子体聚合膜,再自组装聚电解质,用以静电吸附固定日本血吸虫抗原。然后采用BSA和NRS作封闭剂,以封闭晶振上非特异必吸附位点,实现对日本血吸虫感染兔血清的测定。探讨了聚电解质(PSS和AASS)自组装、抗原包被和免疫反应等实难条件的影响;考察了该传感器的响应特性与再生性能,并与采用戊二醛共价键合固定法进行比较。发现该传感器具有灵敏度高、重现性好、非物异性吸附低、再生简便等优点。将它用于测定一系列不同感染程度的兔血清样本,结果表明,该传感系统是临床定性和定量诊断日本血吸虫病的一种有效工具。     

Increasing the sensitivity of Listeria monocytogenes assays: evaluation using ELISA and amperometric detection

An immunosensor for the detection of Listeria monocytogenes was developed. ELISA and amperometric studies were run in parallel to develop a more sensitive and rapid assay for the bacterium. Conditions for the immunosensor were primarily characterised using ELISA. A direct sandwich assay was employed and the affinities of two polyclonal (goat and rabbit) and one monoclonal (mouse) anti-L. monocytogenes antibodies were compared using this format. Owing to low sensitivity being obtained with all antibodies, biotin-avidin amplification and an indirect sandwich assay were employed. The system was then transferred to screen-printed electrodes (SPEs), the primary antibody being immobilised by cross-linking with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, and the mode of detection being amperometric. Various parameters (limit of detection, working range, incubation time, cross-reactivity) of the systems were characterised. The effect of direct incubation in milk is also discussed. The final immunosensor had a working range of 1 x 10(6)-1 x 10(3) cells ml-1 and a detection limit of 9 x 10(2) cells ml-1. The assay took about 3.5 h to complete.     

Specific Detection of Salmonella typhi Using Renewable Amperometric Immunosensor

A renewable amperometric immunosensor was developed for the specific detection of Salmonella typhi (S. typhi) using flagellin specific antibodies. An immunocomposite comprising paraffin, graphite, and capturing antibodies against S. typhi was used to construct the electrode. The detection technique involved a sandwich ELISA system. The assay conditions were optimized for loading of capturing antibody, incubation time for S. typhi cells, rotation speed and minimum amount of substrate needed. 1‐naphthyl phosphate was used as the substrate with an amperometric detection of its enzymatic hydrolysis product 1‐naphthol at a potential of +400 mV vs Ag/AgCl reference electrode. The minimum detection limit for S. typhi was found to be 10⁵ cells/mL in 90 min, while ELISA detects 10⁶ cells/mL in five hours.     

Amperometric Immunosensor for the Determination of 2′,3′-dideoxyinosine

Abstract

An amperometric immunosensor, based on carbon paste impregnated with solubilized antidideoxyinosine (from rabbit lyophilized powder, whole antiserum) has been constructed for the assay of anti-HIV agent dideoxyinosine (didanosine, DDI). The amperometric immunosensor was reliably used for dideoxyinosine assay in the 900 pmol/L to 9 nmol/L concentration range, with a detection limit of 180 pmol/L, at E = +1.04 V vs. Ag/AgCl. The construction of the immunosensor is reproducible. Its surface can be easily renewed by simple polishing on an aluminium paper. The new amperometric immunosensor is reliable for the assay of 2′,3′-dideoxyinosine in raw material as well as in its pharmaceutical formulation (Videx tablets).     

Immunosensor for the determination of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> using a tyrosinase–mercaptopropionic acid modified electrode as an amperometric transducer

An amperometric immunosensor for the quantification of Staphylococcus aureus based on the coimmobilization of rabbit immunoglobulin G (RbIgG) and tyrosinase on a mercaptopropionic acid self-assembled monolayer modified gold electrode is reported. A competitive mode in which protein-A-bearing S. aureus cells and antiRbIgG labeled with alkaline phosphatase (AP) compete for the binding sites of immobilized RbIgG was used. Monitoring of the affinity reaction was carried out by the amperometric detection at -0.15 V of phenol generated in the enzyme reaction with AP, at the tyrosinase-modified electrode through the electrochemical reduction of the o-quinone formed. Optimization of the working variables, such as the immunosensor composition and incubation times, the applied potential, the working pH and the concentration of phenyl phosphate used as the AP substrate, was carried out. Under the optimized conditions, both the repeatability of the measurements and the reproducibility of the responses obtained with different immunosensors yielded relative standard deviation values for the steady-state current lower than 10%. The immunosensor showed a dynamic range from 4.4x10(5) to 1.8x10(7) S. aureus cells mL(-1), with a detection limit of 1.7x10(5) cells mL(-1). The limit of detection was remarkably improved by subjecting S. aureus cells to wall lysis by heat treatment. The value obtained was 2.3x10(3) cells mL(-1), which is adequate for the monitoring of S. aureus contamination levels in some foodstuffs. As an application, milk samples spiked with bacteria at the 4.8x10(3) cells mL(-1) level were analyzed.     

A piezoelectric immunoassay based on self-assembled monolayers of cystamine and polystyrene sulfonate for determination of Schistosoma japonicum antibodies

A piezoelectric immunosensor based on an improved immobilization strategy combining self-assembled monolayers (SAM) of cystamine (Cys) and polystyrene sulfonate (PSS) has been developed for the determination of Schistosoma japonicum antibodies (SjAb) in rabbit serum. Cys SAM were first applied to the gold electrode surface of the crystal, serving as a positively-charged base. Schistosoma japonicum antigen (SjAg) was then electrostatically immobilized on the crystal by means of a negatively-charged PSS layer. When sealed by use of an appropriately selected blocking reagent for BSA and normal rabbit serum (NRS), non-specific adsorption could be substantially reduced.The immunosensor was used to determine SjAb in optimized buffer medium with addition of poly(ethylene glycol) (PEG), which served as an immunoreaction enhancer. It was shown experimentally that SjAg immobilized by the Cys-PSS adsorption procedure had higher immunological activity or binding efficiency than those immobilized by the glutaraldehyde (GLU) binding or direct attachment procedures. The immunosensor developed had satisfactory sensitivity and detection limit, and regeneration of the piezoelectric quartz-crystal was easy. Analytical results obtained with infected rabbit serum samples indicated that the proposed immunosensor is a promising alternative for qualitative and quantitative determination of SjAb in clinical diagnosis of infection with Schistosoma japonicum.     

Probe functionalization with a Rhop-3 antibody: toward a Rhop-3 antigen immunosensor for detection of malaria

The antibody specific for the malaria protein, Rhop-3, and FL-Rhop-3, were immobilized on the surface of a gold electrode modified with cysteamine. Colloidal gold was used to enhance the detection signal for Rhop-3 antigens. The Rhop-3 antibody was also immobilized on gold electrodes preactivated with dithiobis(succinimidyl proprionate) (DSP). Immobilization was performed at room temperature and at 37 °C. Cyclic voltammetry (CV) was used to monitor the interaction between the immobilized antibody and its cognate antigen in solution, using ferricyanide, K₃Fe(CN)₆, as reporting electroactive probe. Tests indicate recognition of Rhop-3 protein by the immobilized antibody. Antigen recognition was enhanced by incubation at 37 °C compared with room-temperature incubation. Our results suggest that an immunosensor can be developed and optimized to aid detection of Rhop-3 antigens in samples from malaria patients. As far as we are aware, this is the first amperometric immunosensor targeting Rhop-3 antigen as a malaria biomarker.     

Immunosensor for the determination of azidothymidine: its utilization as detector in a sequential injection analysis system

An amperometric immunosensor based on graphite paste (graphite powder and paraffin oil) has been constructed for the assay of azidothymidine (AZT). The graphite paste is impregnated with anti-AZT. The immunosensor can be reliably used for the assay of AZT in its pharmaceutical formulation. The potential used for AZT assay was 435 mV vs Ag/AgCl electrode. The surface of the immunosensor can be regenerated by simply polishing, obtaining fresh immunocomposite ready to be used in a new assay. Due to its reliability, the immunosensor was successfully used as a detector in a sequential injection analysis system, and gave reliable results for on-line assay of AZT purity in raw material and AZT contents in pharmaceutical formulations.     

基于纳米银与有机多孔材料/纳米金修饰的甲胎蛋白免疫传感器研究

在金电极表面电沉积银为氧化还原探针,利用有机多孔材料(PTC-NH2)、纳米金(nano-Au)固载甲胎蛋白抗体(anti-AFP),制备出用于检测甲胎蛋白(AFP)的安培型免疫传感器。通过交流阻抗技术、循环伏安法研究了电极的电化学特性,考察了孵育时间、测试液pH值等实验条件对传感器性能的影响,并利用扫描电子显微镜(SEM)对电极的修饰过程进行了表征。该传感器对AFP有良好的电流响应,线性范围分别为1.0～20.0ng/mL和20.0～60.0 ng/mL,检测限为0.6 ng/mL。     

Colorimetric assay for lysozyme using Micrococcus luteus labeled with a blue dye, Remazol brilliant blue R, as a substrate.

Micrococcus luteus (M. lysodeikticus) labeled with Remazol brilliant blue R (blue ML) was prepared as a novel substrate for the colorimetric assay of lysozyme. The treatment of the labeled substrate with lysozyme resulted in the release of soluble blue products which can be easily measured spectrophotometrically at 600 nm. The blue color was most efficiently released at pH 7 and ionic strength of 0.2 on incubation with hen lysozyme at 40 degrees C. A new colorimetric method for the assay of lysozyme using this substrate was developed. The assay system gave a linear dose-response curve, and as little as 0.1 microgram of human lysozyme (1 microgram/ml, 100 microliters) can be detected. The present method is more convenient and reproducible than the conventional lysozyme assay with bacterial cells. Application of the system to the determination of lysozyme in human serum is described.