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.     

Nanoporous gold film based immunosensor for label-free detection of cancer biomarker

Nanoporous gold (NPG) film modified electrode for the construction of novel label-free electrochemical immunosensor for ultrasensitive detection of cancer biomarker prostate specific antigen (PSA) is described. Due to its high conductivity, large surface area, and good biocompatibility, NPG film modified electrode was used for the adsorption of anti-PSA antibody (Ab). The sensing signal is based on the monitoring of the electrode's current response towards K(3)Fe(CN)(6), which is extremely sensitive to the formation of immunocomplex within the nanoporous film. Under optimum conditions, the amperometric signal decreases linearly with PSA concentration (0.05-26 ng/mL), resulting in a low limit of detection (3 pg/mL). We demonstrated the application of the novel immunosensor for the detection of PSA in real sample with satisfactory results.     

Electrochemical immunosensor for the milk allergen β-lactoglobulin based on electrografting of organic film on graphene modified screen-printed carbon electrodes

A novel label-free voltammetric immunosensor for sensitive detection of β-lactoglobulin using graphene modified screen printed electrodes has been developed. The derivatization of the graphene electrode surface was achieved by electrochemical reduction of in situ generated 4-nitrophenyl diazonium cations in aqueous acidic solution, followed by electrochemical reduction of the terminal nitro groups to amines. The electrochemical modification protocol was optimized in order to generate monolayer of nitrophenyl groups on the graphene surface without complete passivation of the electrode. Unlike the reported method for graphene functionalization, we demonstrated here the ability of the electrografting of aryl diazonium salt to attach an organic film to the graphene surface in a controlled manner by choosing the suitable grafting protocol. Next, the amine groups on the graphene surface were activated using glutaraldehyde and used for the covalent immobilization of β-lactoglobulin antibodies. Cyclic and differential pulse voltammetry carried out in an aqueous solution containing [Fe(CN)(6)](3-/4-) redox pair have been used for the immunosensor characterization. The results demonstrated that the DPV reduction peak current of [Fe(CN)(6)](3-/4-) decreased linearly with increasing the concentration of β-lactoglobulin due to the formation of antibody-antigen complex on the modified electrode surface. The immunosensor obtained using this novel approach enabled a detection limit of 0.85pgmL(-1) and a dynamic range from 1pgmL(-1) to 100ngmL(-1) of β-lactoglobulin in PBS buffer. In addition, the immunosensor evaluated in different samples including cake, cheese snacks, a sweet biscuit, showing excellent correlation with the results obtained from commercially enzyme-linked immunosorbent assay (ELISA) method.     

A novel label-free amperometric immunosensor for carcinoembryonic antigen based on redox membrane

A label-free immunosensor was developed to detect the presence of an antigen. This immunosensor was based on the modulation of the electrochemistry of the surface bound redox species K(3)Fe(CN)(6) (FC). The model antigen was carcinoembryonic antigen (CEA) and the model epitope was the antibody of CEA (anti-CEA). Glassy carbon (GC) electrode surfaces were first drop-coated with a mixture of FC and chitosan and air-dried. The electrode surface was then covered with nafion membrane, which contained gold nanoparticles. After binding with polyethyleneimine (PEI), glutaraldehyde (GA) was used to cross-link PEI and anti-CEA. Binding of CEA to the surface bound epitope resulted in attenuation of the FC electrochemistry. Under optimal conditions, the response of the label-free immunosensor had a linear range of 0.01-150 ng mL(-1) with a detection limit of 3 pg mL(-1) (S/N = 3). Its response was better than those of radioimmunoassays, enzyme-linked immunosorbent assays, and chemiluminescence assays.     

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).     

Electrochemical immunosensor based on electron transfer mediated by graphene oxide initiated silver enhancement

A new electrochemical immunosensor for the detection of protein biomarker platelet-derived growth factor BB (PDGF-BB) was developed based on graphene oxide (GO) initiated silver enhancement. The immunosensor was fabricated based on the traditional sandwich protocol using secondary anti-PDGF-BB antibody (Ab(2)) modified GO as label. Gold electrode was first modified with self-assembled monolayer (SAM) to block the electron transfer between the electrode and K(3)Fe(CN)(6) solution. After the immobilization of primary anti-PDGF-BB antibody (Ab(1)) onto electrode via aminidation to the carboxylic group of SAM and the formation of the sandwich immuno-structure onto electrode surface, the electrode was immersed into silver enhancement solution for silver deposition. The deposited metal silver onto GO then mediated electron transfer across the SAM, producing redox current. The resulting immunosensor displays a wide range of linear response, low detection limit, good reproducibility and stability. The immunosensor was used to the detection of PDGF-BB contents in serum samples with satisfactory results.     

Detection of estradiol at an electrochemical immunosensor with a Cu UPD|DTBP-Protein G scaffold

A copper monolayer was formed on a gold electrode surface via underpotential deposition (UPD) method to construct a Cu UPD|DTBP-Protein G immunosensor for the sensitive detection of 17β-estradiol. Copper UPD monolayer can minimize the non-specific adsorption of biological molecules on the immunosensor surface and enhance the binding efficiency between immunosensor surface and thiolated Protein G. The crosslinker DTBP (Dimethyl 3,3'-dithiobispropionimidate · 2HCl) has strong ability to immobilize Protein G molecules on the electrode surface and the immobilized Protein G provides an orientation-controlled binding of antibodies. A monolayer of propanethiol was firstly self-assembled on the gold electrode surface, and a copper monolayer was deposited via UPD on the propanethiol modified electrode. Propanethiol monolayer helps to stabilize the copper monolayer by pushing the formation and stripping potentials of the copper UPD monolayer outside the potential range in which copper monolayer can be damaged easily by oxygen in air. A droplet DTBP-Protein G was then applied on the modified electrode surface followed by the immobilization of estradiol antibody. Finally, a competitive immunoassay was conducted between estradiol-BSA (bovine serum albumin) conjugate and free estradiol for the limited binding sites of estradiol antibody. Square wave voltammetry (SWV) was employed to monitor the electrochemical reduction current of ferrocenemethanol and the SWV current decreased with the increase of estradiol-BSA conjugate concentration at the immunosensor surface. Calibration of immunosensors in waste water samples spiked with 17β-estradiol yielded a linear response up to ≈ 2200 pg mL(-1), a sensitivity of 3.20 μA/pg mL(-1) and a detection limit of 12 pg mL(-1). The favorable characteristics of the immunosensors such as high selectivity, sensitivity and low detection limit can be attributed to the Cu UPD|DTBP-Protein G scaffold.     

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.     

A simple assay to amplify the electrochemical signal by the aptamer based biosensor modified with CdS hollow nanospheres

A simple method to amplify the electrochemical signal by an aptamer with 22 bases modified with CdS hollow nanospheres (CdSHNs) was described. Using the thrombin as a model, the interaction between the aptamer and CdSHNs was characterized by cyclic voltammetry, electrochemical impedance spectroscopy and circular dichroism spectroscopy. CdSHNs promoted the electron transfer between the gold electrode and K(3)[Fe(CN)(6)] and facilitated the conformation conversion of the aptamer from hairpin to G-quadruplex after the aptamer interacted with thrombin. Under optimal conditions, the modified electrode could be used for the determination of thrombin from 0 to 33 μg mL(-1) and the sensitivity was 1.34 μA mL μg(-1)cm(-2), while the linear range of the modified electrode without the immobilization of CdSHNs was from 2.75 to 27.5 μg mL(-1) and the sensitivity was 0.062 μA mL μg(-1)cm(-2). This constructed biosensor also had a good stability, specificity, reproducibility and accuracy which could provide a promising platform for fabrication of aptamer based biosensors.     

New amperometric and potentiometric immunosensors based on gold nanoparticles/tris(2,2'-bipyridyl)cobalt(III) multilayer films for hepatitis B surface antigen determinations

Two generic, fast, sensitive and novel electrochemical immunosensors have been developed. Initially, a layer of plasma-polymerized Nafion film (PPF) was deposited on the platinum electrode surface, then positively charged tris(2,2'-bipyridyl)cobalt(III) (Co(bpy)(3)(3+)) and negatively charged gold nanoparticles were assembled on the PPF-modified Pt electrode by layer-by-layer technique. Finally, hepatitis B surface antibody (HBsAb) was electrostatically adsorbed on the gold nanoparticles surface. Electrochemical behavior of the {Au/Co(bpy)(3)(3+)}(n) multilayer film-modified electrodes was studied. Cyclic voltammetry, electrochemical impedance spectroscopy (EIS) were adopted to monitor the regular growth of the multilayer films. The performance and factors influencing the performance of the resulting immunosensors were studied in detail. The multilayer film-modified immunosensor was used for hepatitis B surface antigen (HBsAg) determination via the amperometric and potentiometric immunosensor systems, and both systems provided the same linear ranges from 0.05 to 4.5 microg/mL with different detection limits for the amperometric system 0.005 microg/mL and for the potentiometric system 0.015 microg/mL. The immunosensors were used to analyse HBsAg in human serum samples. Analytical results of clinical samples show that the developed immunoassay is comparable with the enzyme-linked immunosorbent assays (ELISAs) method, implying a promising alternative approach for detecting HBsAg in the clinical diagnosis. In addition, the multilayer films also showed better stability for 1 month at least.     

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.     

Comparison of electrochemical immunosensors based on gold nano materials and immunoblot techniques for detection of histidine-tagged proteins in culture medium

In this work, the direct electrochemical determination of poly-histidine tagged proteins using immunosensor based on anti-His (C-term) antibody immobilized on gold electrodes modified with 1,6-hexanedithiol, gold colloid particles or gold nanorods is described. The recombinant histidine-tagged silk proteinase inhibitor protein (rSPI2-His(6)) expressed in Pichia system selected as antigen for this immonosensor. An electrochemical impedance spectroscopy was used as label free detection technique for immune conjugation. The gold nanorods modified electrode layer showed better analytical response than gold nano particles. The linear calibration range was observed between 10pg/ml and 1ng/ml with limit of detection 5pg/ml (S/N=3). Up to four successive assay cycles with retentive sensitivity were achieved for the immunosensors regenerated with 0.2M glycine-HCl buffer, pH 2.8. The performance of this immnosensor were compared with immuoblotting techniques.     

Application of electrochemical impedance spectroscopy for monitoring allergen-antibody reactions using gold nanoparticle-based biomolecular immobilization method

Gold nanoparticles were used to enhance the immobilization amount and retain the immunoactivity of recombinant dust mite allergen Der f2 immobilized on a glassy carbon electrode (GCE). The interaction between allergen and antibody was studied by electrochemical impedance spectroscopy (EIS). Self-assembled Au colloid layer (?=16nm) deposited on (3-mercaptopropyl)trimethoxysilane (MPTS)-modified GCE offered a basis to control the immobilization of allergen Der f2. The impedance measurements were based on the charge transfer kinetics of the [Fe(CN)(6)](3-/4-) redox pair, compared with bare GCE, the immobilization of allergen Der f2 and the allergen-antibody interaction that occurred on the electrode surface altered the interfacial electron transfer resistance and thereby slowed down the charge transfer kinetics by reducing the active area of the electrode or by preventing the redox species in electrolyte solution from approaching the electrode. The interactions of allergen with various concentrations of monoclonal antibody were also monitored through the change of impedance response. The results showed that the electron transfer resistance increased with increasing concentrations of monoclonal antibody.     

A label-free electrochemical immunoassay for carcinoembryonic antigen (CEA) based on gold nanoparticles (AuNPs) and nonconductive polymer film

A simple and sensitive label-free electrochemical immunoassay electrode for detection of carcinoembryonic antigen (CEA) has been developed. CEA antibody (CEAAb) was covalently attached on glutathione (GSH) monolayer-modified gold nanoparticle (AuNP) and the resulting CEAAb-AuNP bioconjugates were immobilized on Au electrode by electro-copolymerization with o-aminophenol (OAP). Electrochemical impedance spectroscopy and cyclic voltammetry studies demonstrate that the formation of CEA antibody-antigen complexes increases the electron transfer resistance of [Fe(CN)(6)](3-/4-) redox pair at the poly-OAP/CEAAb-AuNP/Au electrode. The use of CEA antibody-AuNP bioconjugates and poly-OAP film could enhance the sensitivity and anti-nonspecific binding of the resulting immunoassay electrode. The preliminary application of poly-OAP/CEAAb-AuNP/Au electrode for detection of CEA was also evaluated.     

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.     

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.     

Sensitive electrochemical immunosensor for cancer biomarker with signal enhancement based on nitrodopamine-functionalized iron oxide nanoparticles

A novel electrochemical immunosensor for sensitive detection of cancer biomarker prostate specific antigen (PSA) based on nitrodopamine (NDA) functionalized iron oxide nanoparticles (NDA-Fe(3)O(4)) is described. NDA-Fe(3)O(4) was used both for the immobilization of primary anti-PSA antibody (Ab(1)) and as secondary anti-PSA antibody (Ab(2)) label. For the preparation of the label, mediator thionine (TH) was first conjugated onto NDA-Fe(3)O(4) based on the amino groups of NDA, and then the amino group of TH was used to immobilize horseradish peroxidase (HRP) and Ab(2). Due to the high amount of NDA anchored onto Fe(3)O(4) surface, the loading of antibodies as well as mediator and enzyme onto NDA-Fe(3)O(4) was substantially increased, which increased the sensitivity of the immunosensor. The resulting immunosensor displayed a wide range of linear response (0.005-50 ng/mL), low detection limit (4 pg/mL), good reproducibility and stability. The immunosensor was used to detect the PSA contents in serum samples with satisfactory results.     

Amperometric immunosensor based on toluidine blue/nano-Au through electrostatic interaction for determination of carcinoembryonic antigen

A new current amplified immunosensor for the determination of carcinoembryonic antigen (CEA) was demonstrated in this work. The electrode architecture was fabricated by positively charged toluidine blue (TB) coated on negatively charged poly-sulfanilic acid (PSAA) modified glassy carbon electrode (GCE) surface through electrostatic interactions to form a TB/PSAA film, which provided an interface containing amine groups to assemble gold nanoparticles (nano-Au) for immobilization of carcinoembryonic antibody (anti-CEA) and horseradish peroxidase (HRP) instead of bovine serum albumin (BSA) to block sites against non-specific binding. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were employed to characterize the electrochemical properties of the modified processes. The CVs reduction current of the immunosensor charged linearly in two concentration ranges of CEA from 0.5 to 5.0 and 5.0 to 120.0 ng/ml in presence of 0.3mM H2O2 in analyte solution, and the detection limit was 0.2 ng/ml at three times background noise. The proposed method is economical, efficient and potentially attractive for clinical immunoassays.     

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.     

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.