A novel electrochemical immunosensor based on ordered Au nano-prickle clusters

In this paper, we report a kind of ordered 3D Au nano-prickle clusters by directly electrodeposited on glassy carbon electrode utilizing the spatial obstruction/direction of the polycarbonate membrane. The proposed 3D nanoclusters are applied to fabricate a sandwich-type electrochemical immunosensor with human IgG as a model analyte. The electrodeposited Au nanoclusters build direct electrical contact and immobilization interface for protein molecules, which do not need post-modification and positioning. Scanning electron microscopy, cyclic voltammetry and alternating current impedance spectroscopy were used to investigate the properties of the modified interface. The deposited Au nanoclusters are stable with good biocompatibility, large specific surface area and high electron exchange capability. Under the optimized experimental conditions, a wide linear range from 1.0 to 10000.0 ng/mL was reached with a detection limit of 0.5 ng/mL. The calibration curve fits a second-order polynomial equation very well (R(2)=0.9914). The developed immunosensor based on Au nano-prickle clusters possesses advantages such as simple fabrication, fast response, low detection limit, wide linear range, easy regeneration, excellent reproducibility and long stability. To our knowledge, the Au nanostructure of special ordered 3D nano-prickle clusters is new for electrochemical immunosensor.     

A mediatorless and label-free amperometric immunosensor for detection of h-IgG

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 0.82-90 ng mL(-1) by DPV.     

Electrochemical impedance immunosensor based on gold nanoparticles and aryl diazonium salt functionalized gold electrodes for the detection of antibody

Electrodes modified with passivating organic layers have been shown to, here and previously, to exhibit good Faradaic electrochemistry upon attachment of gold nanoparticles (AuNP). Due to their low background capacitances these constructs have good potential in electrochemical sensing. Herein is reported the application of these electrode constructs for impedance based immunosensing. The immunosensor was constructed by modifying a gold electrode with 4-thiophenol (4-TP) passivating layers by diazonium salt chemistry. Subsequently, the attachment of AuNP and then a biotin derivative as a model epitope to detect anti-biotin IgG were carried out. The interfacial properties of the modified electrodes were evaluated in the presence of Fe(CN)(6)(4-/3-) redox couple as a probe by cyclic voltammetry and electrochemical impedance spectroscopy. The impedance change, due to the specific immuno-interaction at the immunosensor surface was utilized to detect anti-biotin IgG. The increase in charge-transfer resistance (R(ct)) was linearly proportional to the concentration of anti-biotin IgG in the range of 5-500 ng mL(-1), with a detection limit of 5 ng mL(-1).     

Ultrasensitive electrochemical immunosensor based on Au nanoparticles dotted carbon nanotube-graphene composite and functionalized mesoporous materials

A facile and sensitive electrochemical immunosensor for detection of human chorionic gonadotrophin (hCG) was designed by using functionalized mesoporous nanoparticles as bionanolabels. To construct high-performance electrochemical immunosensor, Au nanoparticles (AuNPs) dotted carbon nanotubes (MWCNTs)-graphene composite was immobilized on the working electrode, which can increase the surface area to capture a large amount of primary antibodies (Ab(1)) as well as improve the electronic transmission rate. The as-prepared bionanolabels. composed of mesoporous silica nanoparticles (MCM-41) coated with AuNPs through thionine linking, showed good adsorption of horseradish peroxidase-labeled secondary anti-hCG antibody. Interlayer thionine was not only a bridging agent between MCM-41 and AuNPs but also an excellent electron mediator. The approach provided a good linear response range from 0.005 to 500 mIU mL(-1) with a low detection limit of 0.0026 mIU mL(-1). The immunosensor showed good precision, acceptable stability and reproducibility. Satisfactory results were obtained for determination of hCG in human serum samples. The proposed method provides a new promising platform of clinical immunoassay for other biomolecules.     

GoldMag nanocomposite-functionalized graphene sensing platform for one-step electrochemical immunoassay of alpha-fetoprotein

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 H(2)O(2). 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 0.01-200 ng mL(-1) with a low detection limit (LOD) of 1.0 pg mL(-1) AFP (at 3s(B)). 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.     

Electrochemiluminescence of peroxydisulfate enhanced by L-cysteine film for sensitive immunoassay

A novel label free electrochemiluminescence (ECL) immunosensor based on the ECL of peroxydisulfate solution for detection of α-1-fetoprotein (AFP) has been developed. For this proposed immunosensor, L-cysteine was firstly electrodeposited on the gold electrode surface, which promoted the electron transfer and largely enhanced the ECL of peroxydisulfate solution. Subsequently, gold nanoparticles (nano-Au) were assembled onto the L-cysteine film modified electrode to improve the absorption capacity of antibody and further amplify the ECL signal. Then, antibody was immobilized onto the electrode through nano-Au. At last bovine serum albumin (BSA) was employed to block the nonspecific binding sites. As a result, a novel ECL immunosensor was firstly obtained by applying the ECL of peroxydisulfate solution without conventional luminescent reagents. The AFP was determined in the range of 0.01-100 ng mL(-1), with a low detection limit of 3.3 pg mL(-1) (S/N=3). The proposed ECL immunosensor provides a rapid, simple, and sensitive immunoassay protocol for protein detection, which might hold a promise for clinical application. Moreover, this work would open up a new field in the application of peroxydisulfate solution ECL for highly sensitive bioassays.     

A novel reagentless amperometric immunosensor based on gold nanoparticles/TMB/Nafion-modified electrode

A novel reagentless immunosensor was fabricated by immobilization of redox mediator 3,3',5,5'-tetramethylbenzidine (TMB) on the Nafion (Nf) film modified glassy carbon electrode. Gold nanoparticles were assembled onto the TMB/Nafion film modified electrode to provide active sites for the immobilization of antibody molecules. The antibody (anti-MIgG), in the present study, was fixed on the electrode for the rapid detection of antigen molecules (MIgG as a model analyte). The results showed that the immunosensor based on the immobilized TMB redox mediator exhibited good electrochemical response. A good linear relationship between peak current and the concentration of the MIgG was obtained in the concentration range from 4 to 120ng/mL. The detection limit was estimated to be 1ng/ml. Under the optimized conditions, the immunosensor exhibits good sensitivity, reproducibility and stability.     

Ag(I)-cysteamine complex based electrochemical stripping immunoassay: ultrasensitive human IgG detection

An ultrasensitive electrochemical immunosensor for a protein using a Ag (I)-cysteamine complex (Ag-Cys) as a label was fabricated. The low detection of a protein was based on the electrochemical stripping of Ag from the adsorbed Ag-Cys complex on the gold nanoparticles (AuNPs) conjugated human immunoglobulin G (anti-IgG) antibody (AuNPs-anti-IgG). The electrochemical immunosensor was fabricated by immobilizing anti-IgG antibody on a poly-5,2':5',2'-terthiophene-3'-carboxylic acid (polyTTCA) film grown on the glassy carbon electrode through the covalent bond formation between amine groups of anti-IgG and carboxylic acid groups of polyTTCA. The target protein, IgG was sandwiched between the anti-IgG antibody that covalently attached onto the polyTTCA layer and AuNPs-anti-IgG. Using square wave voltammetry, well defined Ag stripping voltammograms were obtained for the each target concentration. Various experimental parameters were optimized and interference effects from other proteins were checked out. The immunosensor exhibited a wide dynamic range with the detection limit of 0.4 ± 0.05 fg/mL. To evaluate the analytical reliability, the proposed immunosensor was applied to human IgG spiked serum samples and acceptable results were obtained indicating that the method can be readily extended to other bioaffinity assays of clinical or environmental significance.     

Electrochemical immune-biosensor for immunoglobulin G based bioelectrocatalytic reaction on micro-comb electrodes

A new amplification strategy of electrochemical signaling from antigen-antibody interactions was proposed via back-filling immobilization of horseradish peroxidase (HRP), immunoglobulin G antibodies (anti-IgG) and gold nanoparticles onto a three-dimensional sol-gel (3DSG)-functionalized biorecognition interface. The 3DSG sol-gel network was employed not only as a building block for the surface modification but also as a matrix for ligand functionalization. The signal-amplification was based on the bioelectrocatalytic reaction of the back-filling immobilization of HRP to H(2)O(2). With the non-competitive format, the formation of the antigen-antibody complex by a simple one-step immunoreaction between the immobilized anti-IgG and IgG in sample solution inhibited partly the active center of HRP, and decreased the immobilized HRP towards H(2)O(2) reduction. Under optimal conditions, the proposed immunosensor exhibited a good electrochemical behavior to IgG in a dynamic range of 1.12-162 ng/mL with a detection limit of 0.56 ng/mL (at 3delta). Moreover, the precision, reproducibility and stability of the as-prepared immunosensor were acceptable. Importantly, the proposed methodology would be valuable for diagnosis and monitoring of biomarkers and its metastasis.     

Amperometric immunosensor for the determination of IgA deficiency in human serum samples

An electrochemical immunosensor for the detection of human IgA deficiency in real human blood serum has been developed. The performance of the immunosensor presents a large but sensitive dynamic range that allows the determination of non-deficient IgA levels (>70 μg/mL) as well as of severe IgA deficiencies (0.5-5.0 μg/mL). The assay architecture involves the immobilisation of a coating antibody on an electrode surface using carboxylic-ended bipodal alkane-thiol self-assembled monolayers (SAMs). The long chain bipodal SAM presents intercalated poly(ethylenglycol) groups that confer the immunosensor the ability to retain its optimum performance in very complex matrices and serum with negligible non-specific adsorption phenomena. Amperometric optimisation of the assay resulted in limits of detection of 142 ng/mL in just 30 min total assay time. Real patients' serum samples were analysed using the developed electrochemical immunosensor demonstrating an excellent correlation in terms of sensitivity and reproducibility compared with standard enzyme linked immunosorbent assays (ELISA).     

A novel amperometric immunosensor based on acetone-extracted propolis for the detection of the HIV-1 p24 antigen

A novel amperometric immunosensor for the detection of the p24 antigen (p24Ag) from HIV-1 was constructed using gold nanoparticles (GNP), multi-walled carbon nanotubes (MWCNTs), and an acetone-extracted propolis film (AEP). First, amino-functionalized MWCNTs (MWCNTNH?) were prepared and dispersed in an HAuCl? solution to synthesize GNPs in situ. Next, the GNP/CNT/AEP nanocomposite was prepared by mixing an AEP solution and the GNP/CNT powder. The nanocomposite was dripped onto a gold electrode (GE), and then p24 antibody (anti-p24 Ab) was immobilized on the resulting modified gold electrode to construct the immunosensor. The assembly process was characterized using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The factors that were likely to influence the performance of the proposed immunosensor were studied in detail. Under optimal conditions, the proposed immunosensor exhibited good electrochemical sensitivity to the presence of p24 in a concentration range of 0.01 to 60.00 ng/mL, with a relatively low detection limit of 0.0064 ng/mL (S/N = 3). Moreover, the proposed immunosensor showed a rapid (≤ 18 s) and highly sensitive amperometric response (0.018 and 1.940 μA/ng/mL) to p24 with acceptable stability and reproducibility.     

A disposable amperometric immunosensor for alpha-1-fetoprotein based on enzyme-labeled antibody/chitosan-membrane-modified screen-printed carbon electrode

A screen-printed three-electrode system is fabricated to prepare a novel disposable screen-printed immunosensor for rapid determination of alpha-1-fetoprotein (AFP) in human serum. The immunosensor is prepared by entrapping horseradish peroxidase (HRP)-labeled AFP antibody in chitosan membrane to modify the screen-printed carbon electrode. The membrane is characterized with scanning electron microscope and electrochemical methods. After the immunosensor is incubated with AFP at 30 degrees C for 35 min, the access of the active center of HRP catalyzing the oxidation reaction of thionine by H(2)O(2) is partly inhibited. In presence of 1.2 mM thionine and 6 mM H(2)O(2), the electrocatalytic current decreases linearly in two concentration ranges of AFP from 0 to 20 and from 20 to 150 ng/mL with a detection limit of 0.74 ng/mL. The immunosensor shows an acceptable accuracy compared with those obtained from immunoradiometric assays. The interassay coefficients of variation are 6.6 and 4.2% at 10 and 100 ng/mL, respectively. The storage stability is acceptable in pH 7.0 phosphate buffer solution at 4 degrees C for more than 10 days. The proposed method can detect the AFP through one-step immunoassay and would be valuable for clinical immunoassay.     

Electrochemical immunosensor for casein based on gold nanoparticles and poly(L-Arginine)/multi-walled carbon nanotubes composite film functionalized interface

In this paper, a novel electrochemical immunosensor for the determination of casein based on gold nanoparticles and poly(L-Arginine)/multi-walled carbon nanotubes (P-L-Arg/MWCNTs) composite film was proposed. The P-L-Arg/MWCNTs composite film was used to modify glassy carbon electrode (GCE) to fabricate P-L-Arg/MWCNTs/GCE through electropolymerization of L-Arginine on MWCNTs/GCE. Gold nanoparticles were adsorbed on the modified electrode to immobilize the casein antibody and to construct the immunosensor. The stepwise assembly process of the immunosensor was characterized by cyclic voltammetry and differential pulse voltammetry. Results demonstrated that the peak currents of [Fe(CN)(6)](3-/4-) redox pair decreased due to the formation of antibody-antigen complex on the modified electrode. The optimization of the adsorption time of gold nanoparticles, the pH of supporting electrolyte and the incubation time were investigated in details. Under optimal conditions, the peak currents obtained by DPV decreased linearly with the increasing casein concentrations in the range from 1 × 10(-7) to 1 × 10(-5) g mL(-1) with a linear coefficiency of 0.993. This electrochemical immunoassay has a low detection limit of 5 × 10(-8) g mL(-1) and was successfully applied to the determination of casein in cheese samples.     

Novel electrochemical catalysis as signal amplified strategy for label-free detection of neuron-specific enolase

A label-free electrochemical immunoassay for neuron-specific enolase (NSE), a kind of lung cancer marker, was developed in this work via novel electrochemical catalysis for signal amplification. The new amplified strategy was based on the electrochemical catalysis of nickel hexacyanoferrates nanoparticles (NiHCFNPs) in the presence of dopamine (DA). NiHCFNPs, which were assembled on the porous gold nanocrystals (AuNCs) modified glassy carbon electrode (GCE), could exhibit a distinct pair of redox peaks corresponding to anodic and cathodic reactions of hexacyanoferrate (II/III). Subsequently, gold nanoparticles functionalized graphene nanosheets (Au-Gra) were coated on the surface of NiHCFNPs/AuNCs film. Then an enhanced amount of neuron-specific enolase antibody (anti-NSE) could be loaded to obtain a sensitive immunosensor of anti-NSE/Au-Gra/NiHCFNPs/AuNCs/GCE due to the strong adsorption capacity and large specific surface area of Au-Gra. More importantly, the oxidation peak current can be enormously enhanced towards the electrocatalytic oxidation of DA based on NiHCFNPs, resulting in the further improvement of the immunosensor sensitivity. Under optimal conditions, the electrochemical immunosensor exhibited a linear range of 0.001-100 ng/mL with a detection limit of 0.3 pg/mL (S/N=3). Thus, the proposed immunosensor provides a rapid, simple, and sensitive immunoassay protocol for NSE detection, which may hold a promise for clinical diagnosis.     

Horseradish peroxidase-functionalized gold nanoparticle label for amplified immunoanalysis based on gold nanoparticles/carbon nanotubes hybrids modified biosensor

This paper describes the combination of electrochemical immunosensor using gold nanoparticles (GNPs)/carbon nanotubes (CNTs) hybrids platform with horseradish peroxidase (HRP)-functionalized gold nanoparticle label for the sensitive detection of human IgG (HIgG) as a model protein. The GNPs/CNTs nanohybrids covered on the glass carbon electrode (GCE) constructed an effective antibody immobilization matrix and made the immobilized biomolecules hold high stability and bioactivity. Enhanced sensitivity was obtained by using bioconjugates featuring HRP labels and secondary antibodies (Ab₂) linked to GNPs at high HRP/Ab₂ molar ratio. The approach provided a linear response range between 0.125 and 80 ng/mL with a detection limit of 40 pg/mL. The immunosensor showed good precision, acceptable stability and reproducibility and could be used for the detection of HIgG in real samples, which provided a potential alternative tool for the detection of protein in clinical laboratory.     

Electrochemical immunoassay for carcinoembryonic antigen based on signal amplification strategy of nanotubular mesoporous PdCu alloy

Interests in using nanoporous metals for biosensing applications have been increasing. Herein, nanotubular mesoporous PdCu (NM-PdCu) alloy is used to fabricate a novel label-free electrochemical immunosensor for cancer biomarker carcinoembryonic antigen (CEA). It operates through physisorption of anti-CEA on NM-PdCu and the mixture of sulfonated graphene sheets (HSO(3)-GS) and thionine (TH) functionalized glassy carbon electrode interface as the detection platform. In this study, chitosan (CS)-PdCu is bound very strongly to carcinoembryonic antibody (anti-CEA), because of the good electron conductivity, high surface area, and good biocompatibility. CS-PdCu is immobilized on electrodes by electrostatic interactions between the negatively charged sulfo group of HSO(3)-GS and the abundant positively charged amino groups of chitosan. TH acts as the redox probe. Under the optimized conditions, the electrochemical immunosensor exhibits a wide working range from 0.01 to 12 ng/mL with a low detection limit of 4.86 pg/mL. The accuracy, reproducibility, and stability of the immunosensor are acceptable. The assay is evaluated for real serum samples, receiving satisfactory results. The nanoporous metal materials-based immunoassay provides a promising approach in clinical application and thus represents a versatile detection method.     

Sensitive amperometric immunosensor for alpha-fetoprotein based on carbon nanotube/gold nanoparticle doped chitosan film

A novel strategy for the fabrication of sensitive immunosensor to detect alpha-fetoprotein (AFP) in human serum has been proposed. The immunosensor was prepared by immobilizing AFP antigen onto the glassy carbon electrode (GC) modified by gold nanoparticles and carbon nanotubes doped chitosan (GNP/CNT/Ch) film. GNP/CNT hybrids were produced by one-step synthesis based on the direct redox reaction. The electrochemical properties of GNP/CNT/Ch films were characterized by impedance spectroscopy and cyclic voltammetry. It was indicated that GNP/CNT nanohybrid acted as an electron promoter and accelerated the electron transfer. Sample AFP, immobilized AFP, and alkaline phosphatase (ALP)-labeled antibody were incubated together for the determination based on a competitive immunoassay format. After the immunoassay reaction, the bound ALP label on the modified GC led to an amperometric response of 1-naphthyl phosphate (1-NP), which was changed with the different antigen concentrations in solution. Under the optimized experimental conditions, the resulting immunosensor could detect AFP in a linear range from 1 to 55 ng ml(-1) with a detection limit of 0.6 ng ml(-1). The proposed immunosensor, by using GNP/CNT/Ch as the immobilization matrix of AFP, offers an excellent amperometric response of ALP-anti-AFP to 1-NP. The immunosensor provided a new alternative to the application of other antigens or other bioactive molecules.     

Bi-enzyme synergetic catalysis to in situ generate coreactant of peroxydisulfate solution for ultrasensitive electrochemiluminescence immunoassay

A novel electrochemiluminescence (ECL) immunosensor for ultrasensitive detection of α-1-fetoprotein (AFP) was designed based on the in situ bi-enzymatic reaction to generate coreactant of peroxydisulfate for signal amplification. In this work, AuNPs were electrodeposited on the glassy carbon electrode (GCE) surface, which promoted the electron transfer. Then, L-cysteine and another layer of AuNPs were, respectively assembled onto the modified electrode surface, which formed the multilayer films for amplifying the ECL signal of peroxydisulfate and immobilizing antibody. At last, glucose oxidase (GOD) and horseradish peroxidase (HRP) were employed to block the nonspecific binding sites. When proper amounts of glucose were added in the detection solution, GOD catalyzed the oxidation of glucose to generate H(2)O(2), which could be further catalyzed by HRP to generate O(2) for the signal amplification. The linear range for AFP detection was 0.001-100 ng mL(-1), with a low detection limit of 3.3 × 10(-4) ng mL(-1). The novel strategy has the advantages of simplicity, sensitivity, good selectivity and reproducibility which might hold a new promise for highly sensitive bioassays applied in clinical detection.     

Label-free electrochemical immunosensor based on graphene/methylene blue nanocomposite

For the specificity of prostate cancer markers, prostate specific antigen (PSA) has been widely used in prostate cancer screening, diagnosis, and treatment after monitoring. In normal male serum, PSA can only be detected in traces of 0-4 ng mL(-1). In this paper, we constructed an electrochemical immunosensor for PSA detection using a nanocomposite film of graphene sheets-methylene blue-chitosan (GS-MB-CS) as electrode material. The nanocomposite film showed high binding affinity to the electrode and was used to immobilize the antibody of PSA. The modification procedure was monitored by cyclic voltammetry (CV). An amperometric biosensor was easily developed based on the response of peak current to the capture of PSA induced by specific antigen-antibody reactions. Under optimum conditions, the amperometric signal decreased linearly with PSA concentration (0.05-5.00 ng mL(-1)). A low limit of detection (13 pg mL(-1)) and a high selectivity are obtained. Moreover, the prepared immunosensor was applied for the analysis of PSA in serum samples with satisfactory results. The proposed method may have a promising future in biochemical assays for high selectivity, good reproducibility, and stability.     

Sensitive immunoassay of human chorionic gonadotrophin based on multi-walled carbon nanotube–chitosan matrix

A novel amperometric immunosensor for human chorionic gonadotropin (HCG) assay has been fabricated through incorporating toluidine blue (TB) and hemoglobin (Hb) on the multiwall carbon nanotube (MWNT)-chitosan (CS) modified glassy carbon electrode, followed by electrostatic adsorption of a conducting gold nanoparticles (nanogold) film as sensing interface. The MWNT-CS matrix provided a congenial microenvironment for the immobilization of biomolecules and promoted the electron transfer to enhance the sensitivity of the immunosensor. Due to the strong electrocatalytic properties of Hb and MWNT toward H(2)O(2), the Hb and MWNT significantly amplified the current signal of the antigen-antibody reaction. The immobilized toluidine blue as an electron transfer mediator exhibited excellent electrochemical redox property. After the immunosensor was incubated with HCG solution, the access of activity center of the Hb to toluidine blue was partly inhibited, which leaded to a linear decrease in the catalytic efficiency of the Hb to the oxidation of immobilized toluidine blue by H(2)O(2) over HCG concentration ranges from 0.8 to 500 mIU/mL. Under optimal condition, the detection limit for the HCG immunoassay was 0.3 mIU/mL estimated at a signal-to-noise ratio of 3. Moreover, the proposed immunosensor displayed a satisfactory stability and reproducibility.