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.     

Simultaneous immobilization of glucose oxidase on the surface and cavity of hollow gold nanospheres as labels for highly sensitive electrochemical immunoassay of tumor marker

A novel tracer, glucose oxidase (GOD)-functionalized hollow gold nanospheres encapsulating glucose oxidase (Au(shell)@GOD), was designed to label the ferrocenemonocarboxylic-grafted secondary antibodies (Fc@Ab(2)) for highly sensitive detection of tumor marker using carboxyl group functionalized multiwall carbon nanotubes as platform. Initially, Au(shell)@GOD was synthesized specially by reverse micelle approach, and then the labeling of antibody and the preparation of GOD-functionalized Au(shell)@GOD were performed by one-pot assembly of Fc@Ab(2) and GOD on the surface of Au(shell)@GOD. The ferrocene used to label antibodies acted as a mediator of electron transfer between GOD and electrode surface. The high-content glucose oxidase in the tracer (on the surface and in the cavity) could significantly amplify the amperometric signal for sandwich-type immunoassay. Using carcinoembryonic antigen (CEA) as model analyte, the designed tracer showed linear range from 0.02 to 5.0 ng mL(-1) with the detection limit down to 6.7 pg mL(-1). The assay results of serum samples with the proposed method were in an acceptable agreement with the reference values. The new protocol showed acceptable stability and reproducibility, high sensitivity, and good precision, which could provide a promising potential for clinical screening and diagnosis of tumor disease.     

Facile fabrication of magnetic gold electrode for magnetic beads-based electrochemical immunoassay: application to the diagnosis of Japanese encephalitis virus

A novel magnetic beads-based electrochemical immunoassay strategy has been developed for the detection of Japanese encephalitis virus (JEV). The magnetic gold electrode was fabricated to manipulate magnetic beads for the direct sensing applications. Gold-coated magnetic beads were employed as the platforms for the immobilization and immunoreaction process, and horseradish peroxidase was chosen as an enzymatic tracer. The proteins (e.g., antibodies or immunocomplexes) attached on the surface of magnetic beads were found to induce a significant decline in their electric conductivity. Multiwalled carbon nanotubes were introduced to improve sensitivity of the assay. The envelope (E) protein, a major immunogenic protein of JEV, was utilized to optimize the assay parameters. Under the optimal conditions, the linear response range of E protein was 0.84 to 11,200 ng/mL with a detection limit of 0.56 ng/mL. When applied for detection of JEV, the proposed method generated a linear response range between 2×10(3) and 5×10(5) PFU/mL. The detection limit for JEV was 2.0×10(3) PFU/mL, which was 2 orders of magnitude lower than that of immunochromatographic strip and similar to that obtained from RT-PCR. This method was also successfully applied to detect JEV in clinical specimens.     

[AuCl4]− and Fe3+/[Fe(CN)6]3− ions-derivated immunosensing interface for electrochemical immunoassay of carcinoembryonic antigen in human serum

[AuCl₄]⁻ was initially deposited by electrochemical reduction on a glassy carbon electrode (GCE) to form porous nanogold layer, then prussian blue (PB) was electrodeposited onto the as-prepared nanogold layer, and then secondary nanogold particles were fabricated again on the PB surface by electrochemical reduction for the immobilization of anti-CEA antibodies. The presence of double-layer porous gold nanoparticles enhanced the immobilized amount of biomolecules, and improved the sensitivity of the immunoassay. PB, as a good redox probe, was facile to electrochemical analysis and measurement. Under optimal conditions, the developed immunoassay exhibited dynamic range from 3.0 to 80.0 ng/mL with a detection limit of 0.9 ng/mL CEA (S/N = 3). Moreover, the selectivity, reproducibility and stability of the immunosensor were acceptable.     

A novel microfluidic immunoassay system based on electrochemical immunosensors: an application for the detection of NT-proBNP in whole blood

Electrochemical immunosensors have attracted great interest in the search for a selective, simple and reliable system for molecular recognition. Presently, electrochemical immunosensors have been widely studied for biomedical molecular's detection, but the regeneration of these immunosensors has restricted their wide application. To prepare a regeneration-free immunosensor, which may be more suitable for clinical determination, a repeatable immunoassay system was developed based on an electrochemical immunosensor with magnetic nanoparticles, biotin-avidin system (BAS) and Fab antibodies for the heart failure markers aminoterminal pro-brain natriuretic peptides (NT-proBNP). At the same time, a microfluidic system was combined into the proposed system, which enabled continuous determination. Using NT-proBNP as a model system, the proposed immunosensor exhibited rapid and sensitive amperometric response to NT-proBNP with good selectivity, stability, and a wide linear range (0.005-1.67 ng/mL and 1.67-4 ng/mL with a detection limit of 0.003 ng/mL under optimal conditions). Importantly, the proposed immunosensor was also suitable for the detection of other proteins and provided new opportunities for disease diagnosis.     

Methyl parathion hydrolase based nanocomposite biosensors for highly sensitive and selective determination of methyl parathion

This article reports the fabrication of a nanocomposite biosensor for the sensitive and specific detection of methyl parathion. The nanocomposite sensing film was prepared via the formation of gold nanoparticles on silica particles, mixing with multiwall carbon nanotubes and subsequent covalent immobilization of methyl parathion hydrolase. The composite of the individual materials was finely tuned to offer the sensing film with high specific surface area and high conductivity. A significant synergistic effect of nanocomposites on the biosensor performance was observed in biosensing methyl parathion. The square wave voltammetric responses displayed well defined peaks, linearly proportional to the concentrations of methyl parathion in the range from 0.001 μg mL?1 to 5.0 μg mL?1 with a detection limit of 0.3 ng mL?1. The application of this biosensor in the analysis of spiked garlic samples was also evaluated. The proposed protocol can be used as a platform for the simple and fast construction of biosensors with good performance for the determination of enzyme-specific electroactive species.     

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.     

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.     

A chemiluminescent metalloimmunoassay based on copper‐enhanced gold nanoparticles for quantification of human growth hormone

A chemiluminescence (CL) immunoassay was developed to determine human growth hormone (hGH) based on copper‐enhanced gold nanoparticles. In this method, gold nanoparticles were deposited on polystyrene wells for adsorption of human growth antibodies as well as catalyst for reducing of copper ions from the copper enhancer solution. The reduction of copper ions was prevented where the gold nanoparticles were covered by the antibody–antigen immunocomplex. The deposited copper on Au nanoparticles was then dissolved in HNO₃ solution and quantified using the CL method. The CL intensity response was logarithmically dependent on the hGH concentrations over the range 0.2–50 ng/mL, with a detection limit (3σ) of 0.036 ng/mL. Copyright © 2012 John Wiley & Sons, Ltd.     

Single Walled Carbon Nanotube-Based Junction Biosensor for Detection of Escherichia coli

Foodborne pathogen detection using biomolecules and nanomaterials may lead to platforms for rapid and simple electronic biosensing. Integration of single walled carbon nanotubes (SWCNTs) and immobilized antibodies into a disposable bio-nano combinatorial junction sensor was fabricated for detection of Escherichia coli K-12. Gold tungsten wires (50 µm diameter) coated with polyethylenimine (PEI) and SWCNTs were aligned to form a crossbar junction, which was functionalized with streptavidin and biotinylated antibodies to allow for enhanced specificity towards targeted microbes. In this study, changes in electrical current (ΔI) after bioaffinity reactions between bacterial cells (E. coli K-12) and antibodies on the SWCNT surface were monitored to evaluate the sensor''s performance. The averaged ΔI increased from 33.13 nA to 290.9 nA with the presence of SWCNTs in a 10⁸ CFU/mL concentration of E. coli, thus showing an improvement in sensing magnitude. Electrical current measurements demonstrated a linear relationship (R² = 0.973) between the changes in current and concentrations of bacterial suspension in range of 10²–10⁵ CFU/mL. Current decreased as cell concentrations increased, due to increased bacterial resistance on the bio-nano modified surface. The detection limit of the developed sensor was 10² CFU/mL with a detection time of less than 5 min with nanotubes. Therefore, the fabricated disposable junction biosensor with a functionalized SWCNT platform shows potential for high-performance biosensing and application as a detection device for foodborne pathogens.     

Label-free electrochemical immunosensor for the determination of fetoprotein based on core-shell-shell nanocomposite particles

A new approach toward the development of advanced immunosensors based on chemically functionalized core-shell-shell magnetic nanocomposite particles, and the preparation, characteristics, and measurement of relevant properties of the immunosensor useful for the detection of alpha-1-fetoprotein (AFP) in clinical immunoassays. The core-shell NiFe2O4/3-aminopropyltriethoxysilance (APTES) (NiFe2O4@APTES) was initially prepared by covalent conjugation, then gold nanoparticles were adsorbed onto the surface of NiFe2O4@APTES, and then anti-AFP molecules were conjugated on the gold nanoparticles. The core-shell-shell nanocomposite particles not only had the properties of magnetic nanoparticles, but also provided a good biocompatibility for the immobilization of biomolecules. The core-shell-shell nanostructure present good magnetic properties to facilitate and modulate the way it was integrated into a carbon paste. The analytical performance of the immunosensor was investigated by using an electrochemical method. Under optimal conditions, the resulting composite presents good electrochemical response for the detection of AFP, and exhibits wide linear range from 0.9 to 110 ng/mL AFP with a detection limit of 0.5 ng/mL. Moreover, the proposed immunosensors were used to analyze AFP in human serum specimens. Analytical results, obtained for the clinical serum specimen by the developed immunosensor, were in accordance with those assayed by the standard ELISA. Importantly, the proposed immunoassay system could be further developed for the immobilization of other antigens or biocompounds.     

The direct electron transfer of glucose oxidase and glucose biosensor based on carbon nanotubes/chitosan matrix

The glucose oxidase (GOD) is entrapped in the composite of carbon nanotubes/chitosan and direct electron transfer reaction between GOD and electrode takes place. The electron transfer rate of GOD is greatly enhanced to 7.73 s(-1) in the system, which is more than one-fold higher than that of flavin adenine dinucleotide adsorbed on the carbon nanotubes (3.1 s(-1)). This maybe results from the conformational change of GOD in the microenvironment enabling the accessibility of active site for GOD to the electrode. Additionally, the bioactivity of GOD modified in the composite on electrode surface is kept. So as-prepared electrode can be used as a glucose biosensor exhibiting higher sensitivity (0.5 microA mM(-1)) and better stability. The facile procedure of immobilizing GOD will promote the developments of electrochemical research for protein, biosensors and other bioelectrochemical devices.     

Sensitive label-free electrochemical immunoassay based on a redox matrix of gold nanoparticles/Azure І/multi-wall carbon nanotubes composite

A sensitive label-free electrochemical immunosensing platform was designed by a redox matrix of gold nanoparticles (GNPs), Azure І and multi-wall carbon nanotubes (MWCNT) self-assemblying nanocomposite. To construct the immunosensor, MWCNT was first dispersed in Nafion (Nf) to obtain a homogeneous solution and then it was dropped on the surface of the gold electrode (Au). Then the positively-charged redox molecule, Azure І, was entrapped into MWCNT–Nf film to form a redox nanostructural membrane. Next, the negatively charged gold nanoparticles (GNPs) were assembled to the interface through the electrostatic force. Finally, carcinoembryonic antibody molecules could be absorbed into the GNPs/Azure І/MWCNT–Nf immobilization matrix. Using carcinoembryonic antigen (CEA) as a model protein, the electrochemical immunosensor exhibited good stability and reproducibility, as well as good selectivity and storage stability. This strategy presented a promising platform for sensitive and facile monitoring of CEA.     

Bimetallic AuPt nanochains: Synthesis and their application in electrochemical immunosensor for the detection of carcinoembryonic antigen

The recent development in the nanotechnology has paved the way for a large number of alloyed nanomaterials and devices of desirable properties which have useful functions for electrochemical sensor and biosensor applications. In this paper, bimetallic AuPt nanochains were synthesized through a mild chemical method, with which anti-horseradish peroxidase-conjugated anti-carcinoembryonic antigen (HRP-anti-CEA-NCAuPt) was developed for electrochemical detection of carcinoembryonic antigen (CEA) in a sandwich-type immunoassay format. The alloyed nanocrystals exhibit not only sound signal amplification effect of Au nanoparticles, but also further new combination of interfacial, electrical and structural properties arising from the disparate AuPt components. As a result, the electrochemical signal was significantly amplified by using the HRP-anti-CEA-NCAuPt as tracer and hydrogen peroxide as enzyme substrate. The linear range of the developed immunosensor is 0.01-200ng/mL and the detection limit is 0.11pg/mL of CEA, which makes the biometallic nanochains promising candidates for the next-generation sandwich-type electrochemical immunoassays.     

Electrochemical immunosensors for cancer biomarker with signal amplification based on ferrocene functionalized iron oxide nanoparticles

Ultrasensitive sandwich type electrochemical immunosensors for the detection of cancer biomarker prostate specific antigen (PSA) is described which uses graphene sheet (GS) sensor platform and ferrocene functionalized iron oxide (Fe(3)O(4)) as label. To fabricate the labels, dopamine (DA) was first anchored onto Fe(3)O(4) surface followed by conjugating ferrocene monocarboxylic acid (FC) and secondary-antibody (Ab(2)) onto Fe(3)O(4) through the amino groups of DA (DA-Fe(3)O(4)-FC-Ab(2)). The great amount of DA molecules anchored onto Fe(3)O(4) surface increased the immobilization of FC and Ab(2) onto the Fe(3)O(4) nanoparticle, which in turn increased the sensitivity of the immunosensor. GS used as biosensor platform increased the surface area to capture a great amount of primary antibodies (Ab(1)) and the good conductivity of GS enhanced the detection sensitivity to FC. Using the redox current of FC as signal, the immunosensor displays high sensitivity, wide linear range (0.01-40 ng/mL), low detection limit (2 pg/mL), good reproducibility and stability. In addition, this method could be extended to the immobilization of other interesting materials (fluorescence dyes) onto Fe(3)O(4) for preparing various kinds of labels to meet the different requirements in immunoassays.     

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.     

Electrocatalytic oxidation of NADH with Meldola's blue functionalized carbon nanotubes electrodes

Meldola's blue (MB) functionalized carbon nanotubes (CNT) nanocomposite film (MB/CNT) electrode was prepared by non-covalent adsorbing MB on the surface of a carbon nanotubes modified glassy carbon electrode (CNT/GCE). Electrochemical behaviors of the resulting electrode were investigated thoroughly with cyclic voltammetry in the potential range of -0.6 to 0.2V, and two well-defined redox couples were clearly visualized. We also studied the electron transfer kinetics of MB loaded on CNT (MB/CNT) in comparison with that of MB on conventional graphite powder (MB/GP). The heterogeneous electron transfer rate constant (k(s)) of MB/CNT was calculated to be about three times larger than that of MB/GP. The accelerated electron transfer kinetics was attributed to the unique electrical and nanostructural properties of CNT supports as well as the interaction between MB and CNT. In connection with the oxidation of nicotinamide adenine dinucleotide (NADH), excellent electrocatalytic activities were observed at MB/CNT/GCE compared with MB/GP modified glassy carbon electrode (MB/GP/GCE). Based on the results, a new NADH sensor was successfully established using the MB/CNT/GCE. Under a lower operation potential of -0.1V, NADH could be detected linearly up to a concentration of 500 microM with an extremely lower detection limit of 0.048+/-0.02 microM estimated at a signal-to-noise ratio of 3. Sensitivity, selectivity, reproducibility and stability of the NADH sensor were also investigated and the main analytical data were also compared with those obtained with the MB/GP/GCE.     

A sensitive amperometric immunosensor for carcinoembryonic antigen detection with porous nanogold film and nano-Au/chitosan composite as immobilization matrix

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.     

Ultrasensitive luminol electrochemiluminescence for protein detection based on in situ generated hydrogen peroxide as coreactant with glucose oxidase anchored AuNPs@MWCNTs labeling

In this study, an ultrasensitive luminol electrochemiluminescence (ECL) immunosensor was constructed using carboxyl group functionalized multi-walled carbon nanotubes (MWCNTs) as platform and glucose oxidase (GOD) supported on Au nanoparticles (AuNPs) decorated MWCNTs (AuNPs@MWCNTs-GOD) as labels. Firstly, using poly(ethylenimine) (PEI) as linkage reagents, AuNPs@MWCNTs were prepared and introduced for binding of the secondary antibody (Ab(2)) and glucose oxidase (GOD) with high loading amount and good biological activity due to the improved surface area of AuNPs@MWCNTs and excellent biocompatibility of AuNPs. Then the GOD and Ab(2) labeled AuNPs@MWCNTs were linked to the electrode surface via sandwich immunoreactions. These localized GOD and AuNPs amplified luminol ECL signals dramatically, which was achieved by efficient catalysis of the GOD and AuNPs towards the oxidation of glucose to in situ generate improved amount of hydrogen peroxide (H(2)O(2)) as coreactant and the enhancement of AuNPs to the ECL reaction of luminol-H(2)O(2). The experimental results demonstrated that the proposed immunosensor exhibited sensitive and stable response for the detection of α-1-fetoprotein (AFP), ranging from 0.0001 to 80 ng mL(-1) with a limit of detection down to 0.03 pg mL(-1) (S/N=3). With excellent stability, sensitivity, selectivity and simplicity, the proposed luminol ECL immunosensor showed great potential in clinical applications.