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Su B.,Fuzhou University | Tang J.,Fuzhou University | Chen H.,Fuzhou University | Huang J.,Clinical Laboratory and Medical Diagnostics Laboratory | And 2 more authors.
Analytical Methods | Year: 2010

A simple and sensitive electrochemical immunoassay method was developed for the detection of alpha-fetoprotein (AFP) in human serum. The immunoassay is based on the use of an AFP-functionalized thionine/nanogold multilayer film ({Thi/AuNP}n) on a single-walled carbon nanotubes (CNT)-coated gold electrode and double-codified gold nanoparticles with horseradish peroxidase-labelled anti-AFP antibodies (HRP-anti-AFP-AuNPs). With a competitive immunoassay format, the assay was performed using HRP-anti-AFP-AuNP as trace and H2O2 as enzyme substrate. The electrochemical behavior of the multilayer {Thi/AuNP}n films was studied. The maximal signal was obtained at n = 3 (i.e. {Thi/AuNP}3). Under optimal conditions, the obtained electrochemical responses were proportional to the AFP levels in the sample. The dynamic range for AFP quantification was 0.25-45 ng mL -1. Both the intra- and inter-assay coefficients of variation were less than 9.5%. The proposed assay had a detection limit of 0.05 ng mL -1, which was 20-fold vs. 10-fold lower than that obtained using monolayer Thi/AuNP film vs. conventional HRP-labelled anti-AFP antibodies. Importantly, no significant differences at the 0.05 confidence level were encountered in the analysis of clinical serum samples between the proposed immunoassay and the commercially available Roche 2010 Electrochemiluminescent (ECL) Automatic Analyzer for AFP determination. © 2010 The Royal Society of Chemistry. Source


Tang J.,Fuzhou University | Huang J.,Clinical Laboratory and Medical Diagnostics Laboratory | Su B.,Fuzhou University | Chen H.,Fuzhou University | Tang D.,Fuzhou University
Biochemical Engineering Journal | Year: 2011

A simple and sensitive conductometric immunosensor for detection of alpha-fetoprotein (AFP) was designed using carbon nanoparticles as labels. The immunosensing probe was fabricated by means of the immobilization of monoclonal anti-AFP primary antibodies on an interdigitated conductometric transducer, while the detection antibodies were prepared using nanocarbon-conjugated horseradish peroxidase-labeled anti-AFP (CNP-HRP-anti-AFP). With a sandwich-type immunoassay format, the conjugated CNP-HRP-anti-AFP on the transducer was increased with the increase of AFP in the sample, and the conductivity of the immunosensor was decreased in the H2O2-KI system. Under optimal conditions, the immunosensor exhibited a wide dynamic range of 0.1-500ng/mL with a detection limit of 50pg/mL AFP at 3σ. The reproducibility and recovery were <10% and 83.9-112.3%, respectively. Interestingly, 45 clinical serum specimens were assayed using the conductometric immunosensor, and the results were in accordance with those obtained from our Clinical Laboratory using Roche 2010 Electrochemiluminescent Automatic Analyzer. © 2010 Elsevier B.V. Source


Tang J.,Fuzhou University | Tang D.,Fuzhou University | Su B.,Fuzhou University | Huang J.,Clinical Laboratory and Medical Diagnostics Laboratory | And 2 more authors.
Biosensors and Bioelectronics | Year: 2011

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 8.0×10-7-2.0×102ng/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. © 2010 Elsevier B.V. Source


Chen H.,Fuzhou University | Tang J.,Fuzhou University | Su B.,Fuzhou University | Chen G.,Fuzhou University | And 2 more authors.
Analytica Chimica Acta | Year: 2010

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-Fe 3O 4 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 H 2O 2. Nanogold-actuated nano-Fe 3O 4 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. © 2010 Elsevier B.V. Source


Su B.,Fuzhou University | Tang J.,Fuzhou University | Yang H.,Fuzhou University | Chen G.,Fuzhou University | And 2 more authors.
Electroanalysis | Year: 2011

A new graphene immunosensing platform based on a new electrochemical immunosensor was designed for sensitive screening of carcinoembryoninc antigen (CEA) as a model biomarker in clinical immunoassay. Gold nanoflowers and single-stranded DNA (ssDNA) molecules were initially assembled onto the surface of graphene for the fabrication of the electrochemical immunosensor using layer-by-layer strategy, and then a sandwich-type immunoassay format was developed for CEA detection based on gold nanoflower-labeled signal antibodies. Graphene nanosheets with high conductivity and biocompatibility could greatly enhance the bound force with ssDNA, and improve the analytical properties of the immunosensor. Gold nanoflowers with electrochemical activity provide a large surface area for the label of biomolecules. The graphene platform displayed a good electrochemical behaviour toward the detection of CEA. Under optimal conditions, the electrochemical immunosensor exhibited a wide dynamic range of 0.05 to 45ng mL-1 with a relatively low detection limit of 0.01ng mL-1 CEA at signal-to-noise ratio of 3. Intra- and interassay coefficients of variation were below 12%. The feasibility of the electrochemical immunosensor was evaluated for real-life clinical specimens, and no significant differences at the 95% confidence level were encountered between the immunosensor and commercially available Roche Elecsys 2010 Electrochemiluminescent Automatic Analyzer. In addition, the analytical properties of the immunosensor were comparatively favourable with those of other electrochemical immunosensors. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Source

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