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A novel electrochemical multiplexed immunoassay was designed for simultaneous determination of alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA) using recombinant apoferritin-encoded metallic nanoparticles (rApo-M) as labels and dual-template magnetic molecularly imprinted polymers (MMIPs) as capture probes. The labels were prepared by loading recombinant apoferritin (r-Apo) and separately immobilize primary antibodies (anti-AFP and anti-CEA) via Au nanoparticles of in site growth on graphene (G). The capture probes were synthesized by self-polymerization of dopamine (DA) on the Fe3O4 nanoparticles (Fe3O4 NPs) and using AFP and CEA as the template proteins, which were used to enrich the targets simultaneously. After a sandwich-type immunoreaction, the labels were captured to the surface of MMIPs. The subsequent electrochemical stripping analysis of the metal components from the immunocomplex provide a means for quantification of targets based on the peak currents of Cd and Pb. Experimental results showed the immunoassay enabled the simultaneous determination of AFP and CEA in a single run with wide dynamic ranges of 0.001-5ngmL(-1). And the detection limits of AFP and CEA were 0.3 and 0.35pgmL(-1) (S/N=3), respectively. These results suggested that the proposed multiplexed immunoassay would be applied for clinical screening of other biomarkers., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The CdTe quantum dots (QDs), graphene nanocomposite (CdTe-G) and dextran-Fe3O4 magnetic nanoparticles have been synthesized for developing an ultrasensitive electrochemiluminescence (ECL) immunoassay for Carcinoembryonic antigen 19-9 (CA 19-9) in serums. Firstly, the capture probes (CA 19-9 Ab1/Fe3O4) for enriching CA 19-9 were synthesized by immobilizing the CA 19-9's first antibody (CA 19-9 Ab1) on magnetic nanoparticles (dextran-Fe3O4). Secondly, the signal probes (CA 19-9 Ab2/CdTe-G), which can emit an ECL signal, were formed by attaching the secondary CA 19-9 antibody (CA 19-9 Ab2) to the surface of the CdTe-G. Thirdly, the above two probes were used for conjugating with a serial of CA 19-9 concentrations. Graphene can immobilize dozens of CdTe QDs on their surface, which can emit stronger ECL intensity than CdTe QDs. Based on the amplified signal, ultrasensitive antigen detection can be realized. Under the optimal conditions, the ECL signal depended linearly on the logarithm of CA 19-9 concentration from 0.005 to 100 pg/mL, and the detection limit was 0.002 pg/mL. Finally, five samples of human serum were tested, and the results were compared with a time-resolved fluorescence assay (TRFA). The novel immunoassay provides a stable, specific and highly sensitive immunoassay protocol for tumor marker detection at very low levels, which can be applied in early diagnosis of tumor.

We report on an on-site ultrasensitive sandwich immunoassay for ractopamine (RAC) that is based on the use of a personal glucose meter. RAC is preconcentrated with the help of magnetic nanoparticles coated with β-cyclodextrin (FeO@SiO-CD), and a novel polymer tag labeled with invertase is employed to catalyze the hydrolysis of sucrose to form glucose which is detected with the glucose meter. Invertase-modified gold nanoparticles were prepared and immobilized, along with antibodies against RAC, on an Envision reagent (a polymer that contains many anti-IgG antibodies). Because of its high loading with the enzyme, the resulting copolymers strongly amplify the signal. The concentration of RAC is calculated on the basis of the linear relationship between the concentration of RAC and the amount of glucose formed. The method exhibits a detection limit of 0.34 μg kg as determined for a sample of pork. This on-site testing method may also be applied to the detection of other low-molecular toxins by simply changing the antibody. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

We report on a novel enzyme-enhanced label for the electrochemical determination of diethylstilbestrol (DES). The label was obtained by orientation-controlled immobilization of a multiplex horseradisch peroxidase (HRP) conjugated polymer on gold nanoparticles (AuNPs) using the Envision reagent (EV) which is an enzyme-polymer complex that contains HRP and anti-IgG antibody in a polydextrin amine skeleton. The AuNPs were modified with Concanavalin A (Con A) and served as a carrier for immobilization of the EV−DES antibody composite. This resulted in a bioconjugate of the type AuNP−Con A−EV−DES Ab which was employed as the label. On exposure to samples containing DES, a sandwich immunocomplex is formed between antibody against DES (which was immobilized on a glassy carbon electrode and is acting as a capture probe), DES (the analyte), and the above label as the signal tracer. Hemin was used as an electronic mediator in the reaction of HRP. The HRP on the label catalyzes the oxidative formation of hydrogen peroxide at pH 7.0, and this induces an increased reductive current in the presence of hemin as an electron mediator. Under optimal conditions, the current increases linearly with increasing concentrations of DES in the range from 5 to 500 pg · mL, with a detection limit as low as 2 pg · mL (at an S/N of 3). The method exhibits high selectivity and good stability. It works without incubation so that the time for an assay is shortened to 5 min. The assays was successfully applied to the determination of DES in milk samples. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

A novel sandwich-type electrochemiluminescence (ECL) immunosensor was developed to enable the sensitive detection of HIV-1 antibodies. This system incorporated mesoporous silica (mSiO2) complexed with quantum dots (QDs) and nano-gold particles, which were assembled to enhance signal detection. Magnetic beads were used by immobilizing the secondary anti-IgG antibody. This was first employed to capture HIV-1 antibody (Ab) to form a Fe3O4/anti-IgG/Ab complex. A high loading and signal-enhanced nanocomposite (hereafter referred to as Au-mSiO2-CdTe) was used as a HIV-1 antigen label. The Au-mSiO2-CdTe nanocomposite was conjugated with the Fe3O4/anti-IgG/Ab complex to form an immunocomplex (hereafter referred to as Fe3O4/anti-IgG/Ab/HIV-1/CdTe-mSiO2-Au). This complex could be further separated by an external magnetic field to produce ECL signals. Due to the large specific surface area and pore volume of mSiO2, the loading of the CdTe QDs was markedly increased. Thus, the loaded QDs released a powerful chemiluminescent signal with a concordantly increased sensitivity of the immunosensor. The immunosensor was highly sensitive, and displayed a linear range of responses for HIV-1 antibody across a dilution range of 1 : 1500 through 1 : 50 with the detection limit of 1 : 4500. The immunoassay can be a promising candidate in early diagnosis of HIV infection. [ABSTRACT FROM AUTHOR]

An ultrasensitive and amplified piezoelectric immunosensor (PEI) with highly sensitivity has been proposed for the detection of ultra-trace of Myoglobin (Mb) in human serum based on precipitation of an insoluble product on the functionalized gold electrode. The core-shell Fe3O4@Au (with 85 nm as particle size)-multi-walled carbon nanotubes nanostructures labeling with Myoglobin first antibody (Mb Ab1) was firstly synthesized as capture probe(Fe3O4@Au-Mb Ab1-MWCNTs). It was modified on the surface of quartz crystal gold electrode. After a sandwich-type immunoreactions, the immunocomplex formed between Fe3O4@Au-Mb Ab1-MWCNTs/Mb and the HRP linked secondary antibody (HRP-Mb Ab2), can lead to the decrease of frequency (Δf) of the piezoelectric crystal electrode. Moreover, HRP enzyme labeled on the immunocomplex can catalyze 3- Amino-9-ethylcarbazole (AEC) in the presence of H2O2, resulting in an insoluble product on the electrode surface. The above two matters, immunocomplex and AEC's oxidation product, can add more weight on the detector and achieve a great amplification of Δf. Based on the change of Δf, ultrasensitive Mb detection was achieved. Under optimal conditions, Δfdepended linearly on the Mb concentration with a range from 0.001 to 5 ng/mL, with a detection limit of 0.3 pg/mL. Comparing with the conventional ELISA, the immunoassay provides a more stable, specific protocol for Mb, which can greatly lower the sensitivity for detection of ultra-trace level of Mb in serum. It can be employed to quantify other biomarker if changed the detection antibody. [ABSTRACT FROM AUTHOR]

A novel immunosensing strategy was designed to simultaneous analysis of alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA) by using electrochemiluminescence (ECL) coupled with cyclic voltammetry (CV) methods. In the assay, two kinds of graphene-polymer based nanotags were fabricated for tumor makers (TMs) distinction and signal amplification. The CV nanotags (rGO-H/Pt-PV) were prepared by immobilizing PowerVision doped platium nanoparticles complex (Pt-PV) on the surface of hemin modified reduced graphene oxide sheets (rGO-H) composites. Pt-PV is a kind of compact enzyme-linker linear polymer with a great number of horseradish peroxidase and was then modified with Pt NPs, which have the double catalyst abilities of the reduction of H 2 O 2 at cathode potential in the presence of hemin. The ECL signal nanotags (rGO-PLL/Ru-Si@Au) were prepared by immobilizing Ru(bpy) 3 2+ -silica doped gold nanoparticles composites (Ru-Si@Au) on reduced graphene oxide sheets (rGO) using poly-L-lysine (PLL) as linker and co-reactant. Moreover, the biosensor platform was prepared by co-immobilizing CEA and AFP's first antibody on gold nanoparticles modified glassy carbon electrode. Based on a sandwich-type immunoreaction, CEA and AFP, CV and ECL nanotags were conjugated on the electrode sequentially. In one-cycle voltammetric test, the CV nanotags produce a cathode current signal at −0.3 V and the ECL nanotags emit an anode luminescent signal at 1.25 V, which were respectively used for CEA and AFP detections. Under the optimum conditions, CEA and AFP could be assayed in the linear ranges of 5 pg mL −1 - 40 ng mL −1 and 3 pg mL −1 - 50 ng mL −1 , with the detection limits of 1 pg mL −1 and 0.5 pg mL −1 (at 3s B ), respectively. More importantly, this designed method was used in real serum samples analysis and has significant potential for TMs detection in a clinical laboratory setting. [ABSTRACT FROM AUTHOR]

Highlights: [•] A facile multiplex electrochemical immunoassay of biomarkers was proposed using bidirectional (anodic and cathodic) stripping voltammetry. [•] The multiplex labels were made by polymer enriched metal sulfide and gold nanoparticles to amplify the signal. [•] A self-made electrochemical reaction microcell was used for detection. [•] Trifunctionalized magnetic beads were used as capture probes. [ABSTRACT FROM AUTHOR]