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In this paper, an amperometric immunosensor modified with protein A/deposited gold nanocrystals (DpAu) was developed for the ultrasensitive detection of carbofuran residues. First, DpAu were electrodeposited onto the Au electrode surface to absorb protein A (PA) and improve the electrode conductivity. Then PA was dropped onto the surface of DpAu film, used for binding antibody Fc fragments. Next, anti-carbofuran monoclonal antibody was immobilized on the PA modified electrode. Finally, bovine serum albumin (BSA) was employed to block the possible remaining active sites avoiding any nonspecific adsorption. The fabrication procedure of the immunosensor was characterized by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV), respectively. With the excellent electroconductivity of DpAu and the PA's oriented immobilization of antibodies, a highly efficient immuno-reaction and detection sensitivity could be achieved. The influences of the electrodeposition time of DpAu, pH of the detection solution and incubation time on the current response of the fabricated immunosensor were investigated. Under optimized conditions, the current response was proportional to the concentration of carbofuran which ranged from 1 to 100 ng/mL and 100 ng/mL to 100 μg/mL. The detection limit was 0.1924 ng/mL. The proposed carbofuran immnuosensor exhibited high specificity, reproducibility, stability and regeneration performance, which may open a new door for ultrasensitive detection of carbofuran residues in vegetables and fruits.

A new label-free immunosensor for the determination of carbofuran was proposed in this paper. Saturated thiourea (TU) and gold nanoparticles (GNPs) were modified onto the glassy carbon electrode surface to form a GNPs/TU film, which provided an interface containing-NH2 to assemble the second GNPs for the immobilization of anti-carbofuran antibody. Bovine serum albumin was used for blocking the non-specific adsorption sites. Thus, a novel amperometric immunosensor for carbofuran was prepared. The electrochemical properties of the modified processes were characterized by electrochemical impedance spectroscopy and cyclic voltammetry. Under the optimal conditions, the current response was proportional to the concentration of carbofuran ranging from 1.0 ng/mL to 100.0 ng/mL and from 100.0 ng/mL to 200 μg/mL with the detection limit 0.11 ng/mL. The proposed amperometric immunosensor exhibited high sensitivity, low cost and simplified procedures, which provided a new promising tool for the detection of pesticides residues in food and environment. [ABSTRACT FROM PUBLISHER]

Abstract: A novel electrochemical sensing interface, electrodeposition of gold–platinum alloy nanoparticles (Au–PtNPs) on carbon nanotubes, was proposed and used to fabricate a label-free amperometric immunosensor. On the one hand, the multiwalled carbon nanotubes (MWCNTs) could increase active area of the electrode and enhance the electron transfer ability between the electrode and redox probe; on the other hand, the Au–PtNPs not only could be used to assemble biomolecules with bioactivity kept well, but also could further facilitate the shuttle of electrons. In the meanwhile, horseradish peroxidase (HRP) instead of bovine serum albumin (BSA) was employed to block the possible remaining active sites and avoid the nonspecific adsorption. With the synergetic catalysis effect of Au–PtNPs and HRP towards the reduction of hydrogen peroxide (H2O2), the signal could be amplified and the sensitivity could be enhanced. Using alpha-fetoprotein (AFP) as model analyte, the fabricated immunosensor exhibited two wide linear ranges in the concentration ranges of 0.5–20ngmL−1 and 20–200ngmL−1 with a detection limit of 0.17ngmL−1 at a signal-to-noise of 3. Moreover, the immunosensor exhibited good selectivity, stability and reproducibility. The developed protocol could be easily extended to other protein detection and provided a promising potential in clinical diagnosis application. [Copyright &y& Elsevier]

n amperometric immunosensor has been developed for sensitive determination of hepatitis B surface antigen as a model protein. A glassy carbon electrode was modified with an assembly of positively charged poly(allylamine)-branched ferrocene (PAA-Fc) and negatively charged gold nanoparticles (Au NPs). The formation of PAA-Fc effectively avoids the leakage of Fc, retains its electrochemical activity, and enhances the conductivity of the composite. The adsorption of Au NPs onto the PAA-Fc matrix provides sites for the immobilization of the antigen and a favorable micro-environment to maintain its activity. The morphologies and electrochemistry of the sensing film were investigated via scanning electron microscopy, electrochemical impedance spectroscopy, and cyclic voltammetry. Factors influencing the performance of the immunosensor were studied in detail. The concentration of the antigen can be quantitated (by measuring the decrease of the amperometric response resulting from the specific binding between antigen and antibody) in the range between 0.1 and 150 ng mL, with a detection limit of 40 pg mL (S/N = 3). The method is economical, efficient, and potentially attractive for clinical immunoassays. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

The hepatotoxic microcystins, especially microcystin-LR (MC-LR), are causing serious problems to public health and fisheries. We describe here a label-free amperometric immunosensor for rapid determination of MC-LR in water sample. The sensor was prepared by immobilizing antibody on a gold electrode coated with L-cysteine-modified gold nanoparticles. The stepwise self-assembly of the immunosensor was monitored and characterized by means of electrochemical impedance spectroscopy and differential pulse voltammetry. A 0.60 mmol L solution of hydroquinone was used as the electron mediator. The immunosensor was incubated with MC-LR at 25 °C for 20 min, upon which the differential pulse voltammetric current changed linearly over the concentration range from 0.05 to 15.00 μg L, with a detection limit of 20 ng L. The developed biosensor was used to determine MC-LR in spiked crude algae samples. The recovery was in the range from 95.6 to 105%. This method is simple, economical and efficient, this making it potentially suitable for field analysis of MC-LR in crude algae and water samples. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Abstract: The aim of this study is to elaborate a simple and sensitive electrochemical immunoassay using ferrocenecarboxylic (Fc-COOH)-doped silica nanoparticles (SNPs) as an immobilized affinity support for cancer antigen 15-3 (CA 15-3) detection. The Fc-COOH-doped SNPs with redox-active were prepared by using a water-in-oil microemulsion method. The use of colloidal silica could prevent the leakage of Fc-COOH and were easily modified with trialkoxysilane reagents for covalent conjugation of CA 15-3 antibodies (anti-CA 15-3). The Fc-COOH-doped SNPs were characterized by X-ray photoelectron spectroscopy (XPS) and transmission electron microscopy (TEM). The fabrication process of the electrochemical immunosensor was demonstrated by using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) techniques. Under optimal conditions, the developed immunosensor showed good linearity at the studied concentration range of 2.0–240UmL−1 with a coefficient 0.9986 and a detection limit of 0.64UmL−1 at S/N=3. [Copyright &y& Elsevier]

Abstract: A novel experimental methodology for studying a mediatorless and label-free immunosensor is proposed by immobilizing antibody on gold nanoparticle/l-cysteine coated electrode (nano-Au/l-cysteine electrode). Differential pulse voltammograms (DPV) resulting from the assembled immunosensor indicate that the immunosensor shows excellent electrochemical response to dopamine so that the electrochemical response is utilized for the signal generation step of the immunosensor. Therefore, by means of unenzymatic-labeling procedure combined with the amperometric detection using dopamine as substrate, the immunological reaction can be detected. After the immunosensor is incubated with h-IgG solution, the access of electrocatalytic behavior center of the immunosensor to dopamine is partly inhibited, which leads to a linear decrease in amperometric response of the immunosensor with h-IgG concentration over a range 0.82–90ngmL−1 by DPV. [Copyright &y& Elsevier]

Abstract: A sensitive amperometric immunosensor for carcinoembryonic antigen (CEA) was prepared. Firstly, a porous nano-structure gold (NG) film was formed on glassy carbon electrode (GCE) by electrochemical reduction of HAuCl4 solution, then nano-Au/Chit composite was immobilized onto the electrode because of its excellent membrane-forming ability, and finally the anti-CEA was adsorbed onto the surface of the bilayer gold nanoparticles to construct an anti-CEA/nano-Au/Chit/NG/GCE immunosensor. The characteristics of the modified electrode at different stages of modification were studied by cyclic voltammetry (CV). The gold colloid, chitosan and nano-Au/Chit were characterized by transmission electron microscopy and UV–vis spectroscopy. In addition, the performances of the immunosensor were studied in detail. The resulting immunosensor offers a high-sensitivity (1310 nA/ng/ml) for the detection of CEA and has good correlation for detection of CEA in the range of 0.2 to 120.0 ng/ml with a detection limit of 0.06 ng/ml estimated at a signal-to-noise ratio of 3. The proposed method can detect the CEA through one-step immunoassay and would be valuable for clinical immunoassay. [Copyright &y& Elsevier]

Abstract: This work describes a two-step conjugate synthesis of a porous organometallic nanostructured materials composed of ferrocenemonocarboxylic (Fc–COOH) and 3,4,9,10-perylenetetracarboxylic dianhydride (PTCDA) and then a reagentless amperometric immunosensor prepared with positively charged gold nanoparticles (PGN) immobilized in this nanostructure conductive film is developed. This nanostructured material containing ferrocenyl (PTC–Fc) could easily form stable film on the electrode surface with efficient redox-activity and excellent conductivity. Furthermore, with the negatively charged surface, this film can be used as an interface to adsorbing the PGN, which was prepared in organic solvents at relatively high concentrations with improved monodispersity compared to those prepared in aqueous solution. The presence of PGN provided a congenial microenvironment for adsorbed biomolecules and decreased the electron-transfer impedance. Thus, with carcinoembryonic antibody (anti-CEA) as a model antibody, the proposed immunosensor showed rapid and highly sensitive amperometric response to carcinoembryonic antigen (CEA) with acceptable preparation reproducibility and stability. [Copyright &y& Elsevier]

Abstract: A novel amperometric immunosensor with amplified sensitivity for the determination of carcinoembryonic antigen (CEA) has been developed based on the assembly of {Thi+/nano-u} n layer-by-layer films by alternate adsorption of negatively charged gold nanoparticles (nano-Au) and positively charged thionine (Thi+) on Titania nanoparticles/gold nanoparticles composite film formed previously on the electrode via self-assembly and deposition method, which provided an interface to assemble carcinoembryonic antibody (anti-CEA). Subsequently, HRP was backing-filled into the CEA-modified electrode surface to amplify the response of the antigen–antibody interactions. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were used to monitor and confirm the films growth. Assay conditions were optimized including the incubation temperature and time, the pH of working buffer, the concentration of the H2O2 and the electrodeposition time of nano-Au, etc. The reduction current of the immunosensor decreases linearly in the range of CEA from 0.2 to 80.0ng/mL with a detection limit of 0.07ng/mL in presence of 0.55mM H2O2 in working solution. Moreover, the immunosensor showed acceptable reproducibility, high sensitivity and long-term stability. Clinical serum samples were assayed with this method and the results were in acceptable agreement with those obtained from the enzyme-linked immunosorbent assays (ELISAs). Therefore, the platform that combines the advantages of nanostructured materials with those of the layer-by-layer self-assembling technique opens the doors to the new and exciting possibilities for the development of immunosensor using different transduction modes. [Copyright &y& Elsevier]

Abstract: A highly hydrophilic, non-toxic and conductive TiO2 nanoparticles/gold nanoparticles bilayer films as immobilization matrix via self-assembly (SA) and deposition method was prepared on a gold electrode. Subsequently, positively charged horseradish peroxidase (HRP) was assembled onto the bilayer films, which provided an interface to assemble gold nanoparticles for immobilization of carcinoembryonic antibody (anti-CEA). Finally, HRP was used to block sites against non-specific binding. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were applied to characterize the electrochemical properties of the SA process. The CVs reduction current of the immunosensor decreases linearly in two concentrations ranges of CEA from 0.3 to 10ng/ml and from 10 to 80ng/ml with a detection limit of 0.2ng/ml in presence of 0.7mM H2O2 in analyte solution. Effects of deposition time, pH of working buffer, temperature and incubation time were also explored for optimum analytical performance by using the amperometric method. Moreover, the proposed immunosensor exhibited good accuracy, high sensitivity, long-term stability and made it to determine CEA in serum samples with satisfactory results. [Copyright &y& Elsevier]

A novel amperometric immunosensor was developed by immobilizing ferritin antibody (FeAb) on the surface of Fe3O4 magnetic nanoparticles/chitosan composite film modified glassy carbon electrode (GCE). This material combined the advantages of inorganic Fe3O4 nanoparticles with the organic polymer chitosan. The stepwise assembly procedure of the immunosensor was characterized by means of differential pulse voltammetry (DPV) and ac impedance. The K3Fe(CN)6/K4Fe(CN)6 was used as a marker to probe the interface and to determinate ferritin. The factors that could influence the performance of the resulting immunosensor were studied in detail. After the immunosensor was incubated with ferritin for 32 min at 35 °C, the DPV current decreased linearly with the logarithm of ferritin concentration in the range from 20 to 500 ng mL−1 with a correlation coefficient of 0.995 and a detection limit of 7.0 ng mL−1. This immunosensor was used to analyze ferritin in human serum samples. The analytical results showed that the developed immunoassay was comparable with the radioimmunoassay (RIA), and the studied immunosensor exhibited good accuracy, high sensitivity, and long-term stability for 3 weeks, which implies a promising alternative approach for detecting ferritin in clinical diagnosis. [ABSTRACT FROM AUTHOR]

A novel and sensitive immunosensor has been developed by electro-depositing gold nanoparticles on to a Prussian Blue-modified glassy carbon electrode for determination of hepatitis B surface antigen (HBsAg). After the developed immunosensor was incubated with different concentrations of HBsAg samples at 37°C for 15 min, the current response decreased with an increasing HBsAg concentration in the sample solution. The decreased percentage of the current was proportional to HBsAg concentration ranging from 2 to 300 ng HbsAg ml−1 with a detection limit of 0.42 ng HbsAg ml−1 ( S/ N = 3). Analytical results of 50 specimens using the developed immunosensor showed satisfactory agreement with those using ELISA, indicating the method to be a promising alternative for detecting HBsAg in clinical diagnosis. [ABSTRACT FROM AUTHOR]

Abstract: A novel amperometric immunosensor for the detection of α-fetoprotein (AFP) based on the integration of microelectronic technology, mixed self-assembled monolayers (mixed SAMs), gold nanoparticles (nanogold) and enzyme amplification has been developed. Using microelectronic technology, an immunosensor was fabricated which has an “Au, Pt, Pt” three-microelectrode system and two microwells constructed by SU-8 photoresist on silicon wafer. Using mixed SAMs and nanogold, a mixed monolayer comprising cysteamine and 1,6-hexanedithiol was formed on the working electrode surface to assemble nanogold and further to immobilize AFP antibody for detecting AFP in human serum samples. The stepwise mixed SAMs and nanogold based immobilization procedure was characterized by means of cyclic voltammetry. The factors influencing the performance of the resulting immunosensor were studied in detail. After the addition of H2O2 and KI to the immunosensor incubated with AFP and further with horseradish peroxidase-labeled AFP antibody, the cathodic current varied linearly in concentration range of AFP from 15 to 350ng/ml with a detection limit of 5ng/ml. Moreover, the studied immunosensor has attractive advantages, such as miniaturization, compatibility with the complementary metal oxide semiconductor (CMOS) techniques, high specificity, good reproducibility and long-term stability, which make it potentially attractive for clinical immunoassays. [Copyright &y& Elsevier]

Abstract: A conventional three-electrode system is fabricated to prepare a novel modified amperometric immunosensor for rapid determination of α-1-fetoprotein (AFP) in human serum. The immunosensor was prepared by entrapping thionine (Thi) into Nafion (Nf) to form a composite Thi/Nf membrane, which yield an interface containing amine groups to assemble gold nanoparticles (nano-Au) layer for immobilization of α-1-fetoprotein antibody (anti-AFP). The stepwise self-assemble procedure of the immunosensor was further characterized by means of electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The factors influencing the performance of the resulting immunosensor were studied in detail. After the immunosensor was incubated with AFP at 37°C for 10min, the CVs current decreased linearly in concentration ranges of AFP from 5.0 to 200.0ng/mL with a detection limit of 2.4ng/mL. Moreover, the studied immunosensor exhibited good accuracy, high sensitivity and long-term stability (>3months). The proposed method is economical and efficient, making it potentially attractive for clinical immunoassays. [Copyright &y& Elsevier]

A new strategy to construct amperometric immunosensor for Schistosoma japonicum antigen (SjAg) assay based on nano-size particulate gold (nano-Au) monolayer as sensing platform has been described. The nano-Au monolayer was obtained through chitosan-entrapped carbon paste electrode (CCPE). The high affinity of chitosan entrapped in CCPE for nano-Au associated with its amino groups facilitated the formation of nano-Au monolayer on the surface of CCPE. A sequential competitive immunoassay format was performed on the nano-Au monolayer supported by CCPE using S. japonicum antibody (SjAb) and SjAg as a model system. The assay comprised first loading of SjAb on nano-Au monolayer, then blocking in BSA solution, followed by a competitive incubation in the buffer containing the SjAg (analyte) and SjAg labeled with horseradish peroxidase (HRP) and finally the amperometric detection with hydroquinone as an enzymatic substrate. The dynamic concentration range for SjAg assay is 0.11–22.4 μg ml−1 with a detection limit of 0.06 μg ml−1. The feasibility of regenerating nano-Au monolayer for consecutive assays was demonstrated by a simple chemical treatment after each determination. The simple manipulations of construction of nano-Au monolayer, low-cost and improved sensitivity are main features of proposed immunosensing method. [Copyright &y& Elsevier]