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We report on a protocol for a simultaneous competitive immunoassay for tetracycline (TC) and chloramphenicol (CAP) on the same sensing interface. Conjugates of TC and of CAP with bovine serum albumin were first co-immobilized on a glassy carbon electrode modified with gold nanoparticles. In parallel, monoclonal anti-TC and anti-CAP antibodies were conjugated onto CdS and PbS nanoclusters, respectively. In a typical assay, the immobilized haptens and the added target analytes competed for binding to the corresponding antibodies on the nanoclusters. Subsequently, Cd(II) and Pb(II) ions are released from the surface of the corresponding nanoclusters by treatment with acid and then were detected by square wave anodic stripping voltammetry. The currents at the peak potentials for Cd(II) and Pb(II) were used as the sensor signal for TC and CAP, respectively. This multiplex immunoassay enables the simultaneous determination of TC and CAP in a single run with dynamic ranges from 0.01 to 50 ng mL for both analytes. The detection limits for TC and for CAP are 7.5 pg mL and 5.4 pg mL, respectively. No obvious nonspecific adsorption and cross-reactivity was observed in a series of analyses. Intra-assay and inter-assay coefficients of variation were less than 10 %. The method was evaluated by analyzing TC and CAP in spiked samples of milk and honey. The recoveries range from 88 % to 107 % for TC, and from 91 % to 119 % for CAP. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Abstract: A novel class of molecular tags, cadmium ion-doped magnetic poly(styrene-acrylic acid) nanospheres (Cd-MPSA), was first synthesized and functionalized with polyclonal rabbit anti-human luteinizing hormone antibodies (PAb2) for highly efficient electrochemical immunoassay of luteinizing hormone (LH). Transmission electron microscope (TEM) and Fourier transform infrared spectroscope (FTIR) were employed to characterize the prepared Cd-MPSA. By using Cd-MPSA-labeled PAb2 as molecular tags, a novel sandwich-type immunoassay protocol was built for determination of LH on monoclonal mouse anti-human luteinizing hormone antibody (MAb1)-functionalized gold electrode. The assay was carried out in pH 5.3 HAc-NaAc buffer solution by square wave voltammetry (SWV). The signal was obtained by the reduction of the doped cadmium ions in the Cd-MPSA. Under optimal conditions, the currents increased with the increasing LH level in the sample, and exhibited a linear range from 0.25 to 240mIUmL−1 with a detection limit of 0.08mIUmL−1 LH at 3s B. The precision, reproducibility, and specificity were acceptable. No obvious difference was encountered in the analysis of spiking LH samples into newborn calf serum with the referenced values. [Copyright &y& Elsevier]

Abstract: A new electrochemical immunosensor for sensitive determination of thyroid-stimulating hormone (TSH) was designed by using redox-active nanogold-functionalized magnetic beads (GoldMag) as signal tags on the nanogold–graphene interface. To construct such GoldMag nanostructures, polyethyleneimine-functionalized magnetic beads (PEI-MBs) were initially prepared by using a wet chemical method, and the electroactive thionine molecules and gold nanoparticles were then alternately immobilized on the surface of PEI-MBs by using an opposite-charged adsorption technique and an in situ synthesis method, respectively. The synthesized GoldMag nanostructures were utilized as signal tags for the label of horseradish peroxidase-anti-TSH conjugates (HRP-anti-TSH). With a sandwich-type immunoassay format, the conjugated signal tags on the transducer were increased with the increasing TSH concentration in the sample, thus enhancing the signal of the electrochemical immunosensor due to the labeled HRP toward the catalytic reduction of H2O2. Under optimal conditions, the current was proportional to the logarithm of TSH concentration ranging from 0.01 to 20μIUmL−1 in pH 6.0 HAc–NaAc containing 6mM H2O2. The detection limit (LOD) was 0.005μIUmL−1 TSH at 3s B. The immunosensor displayed an acceptable reproducibility, stability and selectivity. In addition, the methodology was evaluated with human serum specimens, receiving good correlation with results from commercially available electrochemiluminescent analyzer. [Copyright &y& Elsevier]

Abstract: A new flow-through electrochemical immunosensor was designed for sensitive detection of alpha-fetoprotein (AFP) in human serum by using nanogold-functionalized magnetic graphene nanosheets as immunosensing probes. Initially, amino functionalized magnetic beads were covalently immobilized on the surface of graphene oxide nanosheets (MGPs), then nanogold particles were adsorbed on the amino groups of the MGPs to construct GoldMag nanocomposites functionalized graphene nanosheets (GMGPs), and then horseradish peroxidase-anti-AFP conjugates (HRP-anti-AFP) were assembled onto the surface of nanogold particles (bio-GMGP). With the aid of an external magnet, the formed bio-GMGPs were attached onto the base electrode in the flow system. With a non-competitive immunoassay format, the injected sample containing AFP antigens was produced transparent immunoaffinity reaction with the immobilized HRP-anti-AFP on the bio-GMGPs. The formed immunocomplex inhibited partly the active center of HRP, and decreased the labeled HRP toward the reduction of H2O2. The performance and factors influencing the performance of the immunosensor were investigated in detail. Under optimal conditions, the electrochemical immunosensor displayed a wide working range of 0.01–200ngmL−1 with a low detection limit (LOD) of 1.0pgmL−1 AFP (at 3sB). Intra- and inter-assay coefficients of variation (CV) were below 10%. In addition, the methodology was validated with real serum samples, receiving a good correlation with the results obtained from commercially available electrochemiluminescence automated analyzer. [Copyright &y& Elsevier]

Abstract: A novel redox-active magnetic nanostructure was synthesized by using a wet chemical method for high-efficiency electrochemical immunoassay of carcinoembryonic antigen (CEA, as a model analyte). The nanostructures based on the combination of a magnetic nanocore, a layer of electroactive poly(o-phenylenediamine) (PPD), and a silver metallic shell displayed good adsorption properties for the attachment of anti-CEA antibody selective to CEA. The magnetic nanostructure presented good redox behaviors to facilitate and modulate the way it was integrated into a magnetic carbon paste electrode. The assay was based on a sandwich-type immunoassay protocol by using nanogold-patterned graphene oxide nanoscales (AuNP-GO), conjugated with horseradish peroxidase-labeled anti-CEA, as secondary antibodies and biofunctionalized magnetic nanostructures as immunosensing probes. Under optimal conditions, the nanoparticle-based immunocomposites exhibited good electrochemical responses for the determination of CEA, and allowed the detection of CEA at a concentration as low as 1.0pgmL−1 at a signal-to-noise ratio of 3. In addition, the magnetic immunosensing had good reproducibility, and acceptable accuracy, and could be successfully applied for the detection of CEA in the clinical serum specimens. Significantly, by controlling the target biomolecules, this assay can be easily extended for use with other immunosensings, and thus represents a versatile design routine. [Copyright &y& Elsevier]