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Abstract: A new sandwich-type electrochemical immunosensor based on nanosilver-doped bovine serum albumin microspheres (Ag@BSA) with a high ratio of horseradish peroxidase (HRP) and detection antibody was developed for quantitative monitoring of biomarkers (carcinoembryonic antigen, CEA, used in this case) by coupling enzymatic biocatalytic precipitation with tyramine signal amplification strategy on capture antibody-modified glassy carbon electrode. Two immunosensing protocols (with and without tyramine signal amplification) were also investigated for the detection of CEA and improved analytical features were acquired with tyramine signal amplification strategy. With the labeling method, the performance and factors influencing the electrochemical immunoassay were studied and evaluated in detail. Under the optimal conditions, the electrochemical immunosensor exhibited a wide dynamic range of 0.005–80ngmL−1 toward CEA standards with a low detection limit of 5.0pgmL−1. Intra- and inter-assay coefficients of variation were below 11%. No significant differences at the 0.05 significance level were encountered in the analysis of 6 clinical serum specimens and 6 spiked new-born cattle serum samples between the electrochemical immunoassay and the commercialized electrochemiluminescent immunoassay method for the detection of CEA. [Copyright &y& Elsevier]

Abstract: A facile and sensitive mediator-free electrochemical immunoassay was developed for determination of alpha-fetoprotein (AFP) in human serum with a sandwich-type mode by using silver nanowire–graphene hybrid nanocomposites (AgNW–GPs) as label. One-pot hydrothermal method was used for the synthesis of the AgNW–GPs with the aid of trisodium citrate. The as-prepared AgNW–GP was not only utilized for the label of horseradish peroxidase-conjugated anti-AFP (HRP-anti-AFP) with highly loading capacity, but also displayed good electrochemical behaviors. Compared with the conventional HRP-anti-AFP-based electrochemical immunoassay, the sensitivity of the proposed immunoassay by using the synthesized nanolabels was greatly improved. Under optimal conditions, the electrochemical immunosensor exhibited a wide linear range of 0.05–400ng/mL with a low detection limit (LOD) of 5pg/mL AFP (at 3s B). Intra- and inter-assay coefficients of variation were below 10% and 8.5%, respectively. The selectivity and stability of the immunosensor were acceptable. In addition, the methodology was also validated by assaying 16 positive serum samples and 7 negative serum samples, receiving a good correlation with the results obtained from the referenced electrochemiluminescence method. [Copyright &y& Elsevier]

Abstract: A new dual-amplification strategy of electrochemical signal based on the catalytic recycling of the product was developed for the antigen–antibody interaction by glucose oxidase (GOD)- conjugated gold–silver hollow microspheres (AuAgHSs) coupled with an artificial catalase, Prussian blue nanoparticles (PB), on a graphene-based immunosensing platform. The first signal amplification introduced in this study was based on the labeled GOD on the AuAgHSs toward the catalytic oxidation of glucose. The generated H2O2 was catalytically reduced by the immobilized PB on the graphene nanosheets with the second amplification. With a sandwich-type immunoassay format, carcinoembryonic antigen (CEA) was monitored as a model analyte by using the synthesized AuAgHSs as labels in pH 6.0 phosphate buffer containing 10mM glucose. Under optimal conditions, the electrochemical immunosensor exhibited a wide dynamic range of 0.005–50ngmL−1 with a low detection limit (LOD) of 1.0pgmL−1 CEA (at 3σ). Both the intra- and inter-assay coefficients of variation (CVs) were lower than 10%. The specificity and stability of the immunosensor were acceptable. In addition, the assay was evaluated for clinical serum specimens, and received a good correlation with those obtained by the referenced electrochemiluminescent (ECL). [Copyright &y& Elsevier]

Abstract: A facile and sensitive mediator-free electrochemical immunosensor for detection of alpha-fetoprotein (AFP) was designed by using nanosilver-coated silica nanoparticles (Ag-SiO2) as bionanolabels. To construct such an electrochemical immunosensor, silver ions/single-stranded DNA/graphene nanosheets were initially immobilized on a gold electrode in turn, then silver ions were in situ reduced to silver nanoparticles with the aid of NaBH4, and anti-AFP antibodies conjugated to silver nanoparticles were used. In the presence of AFP analyte, the sandwiched immunocomplex was formed on the electrode surface by using horseradish peroxidase-anti-AFP conjugate-labeled Ag-SiO2 (HRP-anti-AFP-Ag-SiO2) as secondary antibodies. Compared with pure silver nanoparticles, Ag-SiO2 nanocomposites could provide a large room for the immobilization of HRP-anti-AFP, and improve the electrochemical responses of the immunosensor. Meanwhile, the presence of highly conductive graphene nanosheets and silver nanoparticles provided a good pathway for electron transfer. Under optimal conditions, the immunosensor exhibited good electrochemical responses toward AFP ranging from 0.3 to 200ng/mL with a detection limit (LOD) of 0.05ng/mL (at 3σ) in pH 6.0 PBS-H2O2 system. Intra- and inter-assay displayed good precisions with coefficient of variation below 9.5%. In addition, the method was evaluated with 23 clinical serum samples, receiving good correlation with results from commercially available electrochemiluminescent analyzer. [Copyright &y& Elsevier]

Abstract: A novel enzyme-free sandwich electrochemical immunoassay with an ultrahigh sensitivity was developed for detection of alpha-fetoprotein (AFP, as a model analyte) using carbon nanotube-enriched gold nanoparticles (CNT-AuNPs) as nanolabels/nanocatalysts on anti-AFP/glutaraldehyde/thionine-modified glassy carbon electrodes (GCEs). The assays were carried out in a pH 8.0 acetic acid-buffered solution containing 6mM p-nitrophenol (NP) and 6mM NaBH4 after the formation of the sandwich-type immunocomplex. Initially, the NP molecules were reduced to p-aminophenol (AP) by the catalysis of the immobilized gold-nanoparticle labels on the CNT-AuNPs with the aid of NaBH4, then the generated AP molecules were electrochemically oxidized to p-quinone imine (QI) by an electron mediator of thionine, and then the oxidized QI molecules were reduced back to APs by NaBH4. The redox cycling of AP and QI continuously increased the signaling, leading to a high sensitivity. Compared with individual gold-nanoparticle labels, the immunosensor using CNT-AuNPs as labels displayed a wider linear range of 8.0×10−7–2.0×102 ng/mL with a lower detection limit (LOD) of 0.8fg/mL AFP at a signal-to-noise ratio of 3, which was lower 6 orders than that of commercially available ELISA. Intra-and inter-assay coefficients of variation were below 10%. In addition, the assay was evaluated with clinical serum samples, and no significant differences at the 5% confidence level were encountered in the analysis of real samples between the proposed immunoassay and commercially available Roche 2010 Electrochemiluminescent Automatic Analyzer for determination of AFP. [Copyright &y& Elsevier]

Abstract: A sensitive electrochemical immunosensor for the fast screening of brevetoxin B (BTX-2) in food samples was developed by means of immobilizing BTX-2–bovine serum albumin conjugate (BTX-2–BSA) on the gold nanoparticles-decorated amine-terminated poly(amidoamine) dendrimers (AuNP–PAADs). The presence of gold nanoparticles greatly improved the conductivity of the PAADs, and three-dimensional PAADs increased the surface coverage of the biomolecules on the electrode. Under optimal conditions, three types of immunosensor, i.e. with AuNPs, PAADs, or AuNP–PAADs, were used for the determination of BTX-2 in a competitive-type immunoassay format using horseradish peroxidase-labeled anti-BTX antibodies (HRP–anti-BTX-2) as trace in the H2O2–o-phenylenediamine (o-PD) system. A low detection limit (LOD) of 0.01ng/mL and a wide dynamic working linear range of 0.03–8ng/mL BTX-2 using AuNP–PAADs as matrices were obtained in comparison with those of only using AuNP or PAADs. Intra-batch assay precision was substantially improved by resorting to the AuNP–PAADs manifold. The proposed method features unbiased identification of negative (blank) and positive samples. No significant differences were encountered in the analysis of the spiking real samples between the electrochemical immunosensor and liquid chromatography for the determination of BTX-2. Importantly, this method provided a biocompatible immobilization and a promising immunosensing platform for analytes with small molecules in the analysis and detection of food safety. [ABSTRACT FROM AUTHOR]

Abstract: A new electrochemical immunoassay protocol for the determination of carcinoembryonic antigen (CEA) in human serum was developed by means of immobilizing horseradish peroxidase-labeled anti-CEA antibodies (HRP-anti-CEA) on the nanogold/nano-Fe3O4 particles-functionalized polyion complex (PIC) membrane. With a one-step immunoassay format, the formed immunocomplex inhibited partly the bioactive center of the immobilized HRP, and decreased the HRP toward the reduction of H2O2. Nanogold-actuated nano-Fe3O4 mimetic peroxidase was used for the sensitivity improvement of the electrochemical immunosensor with Prussian blue (PB) as mediators. Under optimal conditions, the electrochemical immunosensor displayed a wide dynamic range of 0.1–220ngmL−1 with a relative low detection limit (LOD) of 10pgmL−1 CEA (at 3σ). Intra- and inter-assay show good precisions with a coefficient of variation (CV) lower than 7.8% and 7.3%, respectively. No significant difference at the 95% confidence level was encountered in the analysis of 17 human serum specimens between the electrochemical immunosensor and automatic electrochemiluminescent (ECL) analyzer for the determination of CEA. [ABSTRACT FROM AUTHOR]

Abstract: A new electrochemical immunoassay protocol for sensitive detection of α-fetoprotein (AFP, as a model) is designed using carbon nanoparticles (CNPs)-functionalized biomimetic interface as immunosensing probe and irregular-shaped gold nanoparticles (ISNGs)-labeled horseradish peroxidase-anti-AFP conjugates (HRP-anti-AFP-ISNG) as trace label. The low-toxic and high-conductive CNPs provided a high capacity nanoparticulate immobilization surface and a facile pathway for electron transfer. In comparison with conventional label methods, i.e. spherical gold nanoparticles-labeled HRP-anti-AFP and HRP-labeled anti-AFP, the electrochemical immunosensor using HRP-anti-AFP-ISNGs as trace labels exhibited high bioelectrocatalytic response toward enzyme substrate and a wide dynamic range from 0.02 to 4.0ng/mL with a low detection limit of 10pg/mL toward AFP (at 3σ). The developed immunoassay method showed good selectivity and acceptable reproducibility. Clinical serum samples with various AFP concentrations were evaluated by using the electrochemical immunosensor and the referenced enzyme-linked immunosorbent assay (ELISA), respectively, and received in good accordance with results obtained from these two methods. [Copyright &y& Elsevier]

Abstract: An integrated automatic electrochemical immunosensor array has been designed for the simultaneous detection of 5-type hepatitis virus antigens (i.e. hepatitis A, hepatitis B, hepatitis C, hepatitis D, and hepatitis E). Initially, 5-type hepatitis virus antibodies were immobilized onto a self-made electrochemical sensor array using nanogold particles and protein A as matrices, and the immunosensor array was then used to capture their corresponding antigens from sample solution with a 1-step capture format. The detection is based on the potential change before and after the antigen-antibody reaction by using a 2-electrode system. The developed immunosensor array with allowed simultaneous determination of 5-type hepatitis virus antigens in 5min. The detection limit of the sensor array was ≤1.0ng/mL for most analytes. The intra- and inter-assay imprecision (CVs) of the sensor array were ≤7.3% and 8.1%, respectively. No nonspecific adsorption was evaluated, and the crossing talk between neighboring sites was ≤7.5% (CV). The developed immunoassay system offers promise for label-free, simple, rapid, cost-effective analysis of multi-analyte. Importantly, the chip-based immunosensor array could open new opportunities for high-throughput multi-analyte immunoassays in point-of-care testing of miniaturized lab-on-a-chip devices. [Copyright &y& Elsevier]

We report on a new kind of electrochemical immunosensors for simultaneous determination of the biomarkers carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP). Thionine and ferrocene were applied as distinguishable electrochemical tags (and mediators) which were covalently conjugated on anti-AFP and anti-CEA antibodies, respectively, via carboxy groups. The resulting conjugates were co-immobilized on a glassy carbon electrode functionalized with gold nanoparticles. Finally, horseradish peroxidase (HRP) was immobilized onto the modified electrode. Labeled thionine and ferrocene, respectively, act as distinguishable tags for simultaneous determination of AFP and CEA due to the difference in the location of their voltammetric peaks. With a one-step immunoassay format, the analytes in the sample produced transparent immunoaffinity reaction with the corresponding antibodies on the electrode. Once the immunocomplex is formed, it partially inhibits the active center of the immobilized HRP, and this decreased the activity of HRP in terms of reduction of hydrogen peroxide. This immunosensor enables the simultaneous determination of AFP and CEA in a single run and within the same dynamic range (0.01-50 ng mL) and the same lower detection limit (0.01 ng mL). The reproducibility and stability of the immunosensors are acceptable. The dual immunosensor was applied to evaluate several specimens, and the assay results are in acceptable agreement with clinical data. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Abstract: A new electrochemical immunoassay strategy for direct detection of carcinoembryoninc antigen (CEA) in serum was developed by using multiarmed star-like platinum nanowires (PtNWs) with biomolecular assembly as signal tags on an anti-CEA-functionalized graphene sensing platform. Initially, the PtNWs were synthesized via a wet chemical method, and then the synthesized PtNWs were used for the co-immobilization of CEA and horseradish peroxidase (HRP). Compared with platinum nanoparticles, the prepared PtNWs could provide a large room for the conjugation of HRP and CEA. With a competitive-type immunoassay format, the assay was performed in two types of supporting electrolytes including new born cattle serum (NBCS) and acetate buffer solution (ABS, pH 5.5), respectively. Similar detection limit (LOD) of 5.0pgmL−1 vs. 1.0pgmL−1 but narrower dynamic working linear range of 0.01–60ngmL−1 vs. 0.002–80ngmL−1 was obtained toward CEA standards in the NBCS compared to the ABS. The intra-assay coefficients of variation (CVs) were 4.3%, 8.6%, and 6.2% at 0.05, 10, and 40ngmL−1 CEA, respectively, while the inter-assay CVs were 7.6%, 10.5%, and 8.9% at the above-mentioned levels, respectively. In addition, the selectivity and stability of the electrochemical immunosensor were acceptable. Importantly, the developed method was used to assay clinical serum specimens, receiving a good relation with those obtained from the referenced method. [Copyright &y& Elsevier]

Abstract: A feasible and practicable amperometric immunoassay strategy for sensitive screening of carcinoembryonic antigen (CEA) in human serum was developed using carbon nanotube (CNT)-based symbiotic coaxial nanocables as labels. To construct such a nanocable, a thin layer of silica nanoparticles was coated on the CNT surface by sonication and sol–gel methods, and then colloidal gold nanoparticles were assembled on the amino-functionalized SiO2/CNTs, which were used for the label of horseradish peroxidase-anti-CEA conjugates (HRP-anti-CEA-Au/SiO2/CNT). In the presence of analyte CEA, the sandwich-type immunocomplex was formed on an anti-CEA/Au/thionine/Nafion-modified glassy carbon electrode by using HRP-anti-CEA-Au/SiO2/CNTs as detection antibodies. To embody the advantages of the protocol, the analytical properties of variously modified electrodes were compared in detail on the basis of different nanolabels. Under optimal conditions, the cathodic peak currents of the electrochemical immunosensor were proportional to the logarithm of CEA concentration over the range from 0.01 to 12ngmL−1 in pH 5.5 HAc-NaAc containing 5mM H2O2. At a signal-to-noise ratio of 3, the detection limit (LOD) is 5pgmL−1 CEA. Intra- and inter-assay coefficients of variation were below 9.5%. Meanwhile, the selectivity and stability of the immunosensor were acceptable. In addition, the technique was evaluated by spiking CEA standards in pH 7.4 PBS and with 35 clinical serum specimens, receiving excellent accordance with results from commercially available electrochemiluminescent enzyme-linked immunoassay. [Copyright &y& Elsevier]

Abstract: A new sandwich-type electrochemical immunosensor with enhanced sensitivity was developed for detection of alpha-fetoprotein (AFP, as a model analyte) in biological fluids by using nanogold-enclosed titania nanoparticle (AuTi)-labeled secondary antibody on a gold–silver–graphene hybrid nanosheet (AuAgGP)-functionalized glassy carbon electrode (GCE). The presence of the AuAgGP nanosheets not only enhanced the immobilized amount of biomolecules, but also improved the electrochemical properties of the immunosensor. With the aid of AuTi nanolabels, the electrochemical signal was greatly amplified in comparison with pure nanogold or titania-based labels. Under optimal conditions, the sensitivity and dynamic range of the immunosensor were evaluated by using the labeled horseradish peroxidase on the AuTi as trace and H2O2 as enzyme substrate, and exhibited a wide dynamic range of 0.001–200ngmL−1 with a low detection limit (LOD) of 0.5pgmL−1 AFP (at 3σ). Both the intra- and inter-assay coefficients of variation were less than 10%. The current of the immunosensor at 13th day was as much as 90% of the initial current. In addition, the methodology was evaluated for 8 positive serum specimens obtained from hepatocarcinoma patients and 19 negative sera, and validated with the commercially available Roche 2010 Electrochemiluminescent (ECL) Automatic Analyzer. No significant differences at the 95% confidence level were encountered between two methods. [Copyright &y& Elsevier]