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1.
Methods based on nanomaterial labels have been developed for electrochemical immunosensors and immunoassays, but most involved low sensitivity. Herein a novel class of molecular tags, nanogold–polyaniline–nanogold microspheres (GPGs), was first synthesized and functionalized with horseradish peroxidase-conjugated thyroid-stimulating hormone antibody (HRP-Ab2) for sensitive electrochemical immunoassay of thyroid-stimulating hormone (TSH). X-ray diffraction, confocal Raman spectroscopy, scanning electron microscope and transmission electron microscope were employed to characterize the prepared GPGs. Based on a sandwich-type immunoassay format, the assay was performed in pH 5.0 acetate buffer containing 6.0 mmol L−1 H2O2 by using GPG-labeled HRP-Ab2 as molecular tags. Compared with pure polyaniline nanospheres and gold nanoparticles alone, the GPG hybrid nanostructures increased the surface area of the nanomaterials, and enhanced the immobilized amount of HRP-Ab2. Several labeling protocols comprising HRP-Ab2, nanogold particle-labeled HRP-Ab2, and polyaniline nanospheres-labeled HRP-Ab2, were also investigated for determination of TSH and improved analytical features were obtained by using the GPG-labeled HRP-Ab2. With the GPG labeling method, the effects of incubation time and pH of acetate buffer on the current responses of the immunosensors were also studied. The strong attachment of HRP-Ab2 to the GPGs resulted in a good repeatability and intermediate precision down to 7%. The dynamic concentration range spanned from 0.01 to 20 μIU mL−1 with a detection limit (LOD) of 0.005 μIU mL−1 TSH at the 3sB criterion. Significantly, no significant differences at the 0.05 significance level were encountered in the analysis of 15 spiking serum samples between the developed electrochemical immunoassay and the commercially available enzyme-linked immunosorbent assay (ELISA) method for determination of TSH.  相似文献   

2.
This article reports on carbon nanotube/manganese dioxide (CNT–MnO2) composites as electrochemical tags for non-enzymatic signal amplification in immunosensing. The synthesized CNT–MnO2 composites showed good electrochemical activity, electrical conductivity and stability. The electrochemical signal of CNT–MnO2 composites coated glassy carbon electrode (GCE) increased by nearly two orders of magnitude compared to bare GCE in hydrogen peroxide (H2O2) environment. CNT–MnO2 composite was subsequently validated as electrochemical tags for sensitive detection of α-fetoprotein (AFP), a tumor marker for diagnosing hepatocellular carcinoma. The electrochemical immunosensor demonstrated a linear response on a log-scale for AFP concentrations ranging from 0.2 to 100 ng mL−1. The limit of detection (LOD) was estimated to be 40 pg mL−1 (S/N = 3) in PBS buffer. Further measurements using AFP spiked plasma samples revealed the applicability of fabricated CNT–MnO2 composites for clinical and diagnostic applications.  相似文献   

3.
Here we designed a new electrochemical immunoassay protocol for determination of carcinoembryonic antigen (CEA) using nanoplatinum-enclosed gold nanocores (Pt@Au) as catalytically promoted nanolabels on the carbon nanospheres and graphene-modified immunosensor. The Pt@Au nanolabels were synthesized and functionalized with monoclonal anti-CEA antibodies and glucose oxidase (GOx). Using the functional Pt@Au nanolabels as molecular tags, the assay was implemented relative to glucose–hydroquinone system with a sandwich-type immunoassay. Initially, the added glucose was oxidized to gluconolactone and H2O2 by the labeled GOx, and then the generated H2O2 was reduced with the help of platinum nanoparticles, leading to the production of oxygen. The self-produced oxygen could promote the re-oxidation of the glucose, thus resulting in the dual amplification of the electrochemical signal. Several nanolabels, such as multiarmed star-like platinum nanowires, hollow platinum nanospheres and Pt@Au nanostructures, were investigated for CEA detection and improved analytical features were obtained with the Pt@Au nanostructures. Under optimal conditions, the Pt@Au-based immunoassay displayed a wide working range from 0.001 to 120 ng mL−1 with a low detection limit of 0.5 pg mL−1 CEA at 3sB. Intra- and inter-assay coefficients of variation were <10.9%. The system was evaluated with 10 clinical serum samples, receiving good accordance with results from enzyme-linked immunosorbent assay method.  相似文献   

4.
A novel nonenzymatic sandwich-type electrochemical immunosensor has been developed to detect squamous cell carcinoma antigen (SCCA). Nitrogen-doped graphene sheet (N-GS) was used to increase capacity of capturing primary antibodies (Ab1). Carbon-supported Pd–Au binary nanoparticles (Pd–Au/C) were synthesized and used to label secondary antibodies (Ab2). The specific binding of SCCA and antibodies enabled a quantitative attachment of Pd–Au/C on the electrode surface. Electrocatalytic analysis showed that the prepared Pd–Au/C exhibit excellent electrocatalytic activity towards hydrogen peroxide (H2O2). We use current response of electrocatalytic labels Pd–Au/C to detect the concentration of SCCA. The unique nonenzymatic immunosensor exhibits a relatively wide linear range from 0.005 to 2 ng mL−1 and high sensitivity with a low detection limit of 1.7 pg mL−1. The immunsensor also shows good reproducibility (4.2%) and stability (5.8%), which makes it an enormous application prospect in clinical research.  相似文献   

5.
A new and disposable electrochemical immunosensor was designed for detection of alpha-fetoprotein (AFP), as a model analyte, with sensitivity enhancement based on enzyme-catalyzed silver deposition onto irregular-shaped gold nanoparticles (ISGNPs). The assay was carried out with a sandwich-type immunoassay protocol by using ISGNP-labeled anti-AFP antibodies conjugated with alkaline phosphatase (ALP–Ab2) as detection antibodies. The enzymatically catalytic deposition of silver on the electrode could be measured by stripping analysis in KCl solution due to the Ag/AgCl solid-state voltammetric process. Several labeling protocols including spherical gold nanoparticle-labeled ALP–Ab2 and ISGNP-labeled ALP–Ab2 were investigated for determination of AFP, and improved analytical properties were achieved with the ISGNP labeling. With the ISGNP labeling method, the effects of incubation time and incubation temperature for antigen-antibody reaction, and deposition time of silver on the current responses of the electrochemical immunosensors were also monitored. Under optimal conditions, the electrochemical immunosensor exhibited a wide dynamic range from 0.01 ng mL−1 to 200 ng mL−1 with a detection limit of 5.0 pg mL−1 AFP. The immunosensor displayed a good stability and acceptable reproducibility and accuracy. No significant differences at the 95% confidence level were encountered in the analysis of 10 clinical serum samples between the developed immunoassay and the commercially available electrochemiluminescent method for determination of AFP.  相似文献   

6.
A double electrochemical covalent coupling method based on click chemistry and diazonium chemistry for the fabrication of sensitive amperometric immunosensor was developed. As a proof-of-concept, a designed alkyne functionalized human IgG was used as a capture antibody and a HRP-labeled rabbit anti-goat IgG was used as signal antibody for the determination of the anti-human IgG using the sandwich model. The immunosensor was fabricated by electrochemically grafting a phenylazide on the surface of a glassy carbon electrode, and then, by coupling the alkyne functionalized human IgG with the phenylazide group through an electro-click chemistry in the presence of Cu(II). The amperometric measurement for the determination of the anti-human IgG was performed after the fabricated immunosensor was incubated with the target anti-human IgG and then with the HRP-labeled anti-goat IgG at −0.25 V in 0.10 M PBS (pH 7.0) containing 0.1 mM hydroquinone and 2.0 mM H2O2. The results showed that the increased current was linear with the logarithm of the concentration of the anti-human IgG in the range from 1.0 × 10−10 g mL−1 to 1.0 × 10−8 g mL−1 with a detection limit of 3 × 10−11 g mL−1. Furthermore, the feasibility of the double electrochemical covalent coupling method proposed in this work for fabricating the amperometric immunosensor array was explored. This work demonstrates that the double electrochemical covalent coupling method is a promising approach for the fabrication of the immunosensor and immunosensor array.  相似文献   

7.
In the present study, a novel and ultrasensitive electrochemiluminescence (ECL) immunosensor based on luminol cathodic ECL was fabricated by using Au nanoparticles and Pt nanoparticles (nano-AuPt) electrodeposited on graphene–carbon nanotubes nanocomposite as platform for the detection of carcinoembryonic antigen (CEA). For this introduced immunosensor, graphene (GR) and single wall carbon nanotubes (CNTs) dispersed in chitosan (Chi-GR-CNTs) were firstly decorated on the bare gold electrode (GE) surface. Then nano-AuPt were electrodeposited (DpAu-Pt) on the Chi-GR-CNTs modified electrode. Subsequently, glucose oxidase (GOD) was employed to block the non-specific sites of electrode surface. When glucose was present in the working buffer solution, GOD immediately catalyzed the oxidation of glucose to in situ generate hydrogen peroxide (H2O2), which could subsequently promote the oxidation of luminol with an amplified cathodic ECL signal. The proposed immunosensor was performed at low potential (−0.1 to 0.4 V) and low concentration of luminol. The CEA was determined in the range of 0.1 pg mL−1 to 40 ng mL−1 with a limit of detection down to 0.03 pg mL−1 (S N−1 = 3). Moreover, with excellent sensitivity, selectivity, stability and simplicity, the as-proposed luminol-based ECL immunosensor provided great potential in clinical applications.  相似文献   

8.
Apurinic/apyrimidinic endonuclease 1 (APE-1), a kind of multifunctional protein widely-distributed in the body, plays an essential role in the DNA base excision repair and serves as multiple possible roles in the response of human cancer to radiotherapy and chemotherapy. In this work, an ultrasensitive solid-state electrochemiluminescence (ECL) immunosensor is designed to determine APE-1 based on the new Ru(bpy)32+/bi-arginine system. The bi-arginine (bi-Arg) is decorated on the Au nanoparticles functionalized magnetic Fe3O4/reduced graphene oxide (bi-Arg/Au@Fe3O4–rGO) according to the self-assembling and covalent cross-linking interaction to obtain the functionalized nanocomposite of bi-Arg/Au@Fe3O4–rGO. Herein, the bi-Arg/Au@Fe3O4–rGO plays not only an amplification label to enhance the ECL signal of Ru(bpy)32+ due to the coreactant of bi-Arg but also an ideal nanocarrier to load numerous secondary antibody. Based on sandwich-type immunoassay format, this proposed method offers a linear range of 1.0 fg mL−1–5.0 pg mL−1 and an estimated detection limit of 0.3 fg mL−1 for the APE-1. Moreover, the reagentless ECL immunosensor also exhibits high sensitivity, excellent selectivity and good stability, which has greatly potential development and application in clinical diagnostics, immunology and biomedical research.  相似文献   

9.
In this paper, a novel sandwich electrochemiluminescence (ECL) immunosensor was constructed by ferrocene for quenching Ag nanoparticles functionalized g-C3N4 (Ag@g-C3N4) emission. The prepared Ag@g-C3N4 had strong and stable ECL signals compared to pure g-C3N4 and primary antibody (Ab1) can be immobilized on Ag@g-C3N4 by adsorption of Ag nanoparticles. Ferrocene carboxylic acid (Fc-COOH) labeled secondary antibody was immobilized on Au doped mesoporous Al2O3 nanorods (Au@Al2O3–Fc-COOH@Ab2) as labels through adsorption ability of Au toward proteins. After a sandwich-type immunoreaction, a remarkable decrease of ECL signal was observed due to the ECL quenching of Ag@g-C3N4 by Au@Al2O3–Fc-COOH@Ab2. As a result, the change of ECL intensity has a direct relationship with the logarithm of CEA concentrations in the range of 1 pg mL−1–100 ng mL−1 with a detection limit of 0.35 pg mL−1 (S/N = 3). Additionally, the proposed immunosensor shows high specificity, good reproducibility, and long-term stability.  相似文献   

10.
Some nanostructures are reported to possess enzyme-mimetic activities similar to those of natural enzymes. Herein, highly-dispersed Pt nanodots on Au nanorods (HD- PtNDs@AuNRs) with mimetic peroxidase activity were designed as an active electrode modifier for fabrication of a hydrogen peroxide (H2O2) electrochemical sensor. The HD-PtNDs@AuNRs were synthesized by a seed-mediated growth approach and confirmed by scanning electron microscopy, transmission electron microscopy, energy dispersive X-ray spectroscopy, and UV–vis spectroscopy. The electrochemical and catalytical performances of HD-PtNDs@AuNRs towards H2O2 reduction were investigated in detail by cyclic voltammetry and amperometry. The HD-PtNDs@AuNRs modified electrode displayed a high catalytic activity to H2O2 at −0.10 V (versus SCE), a rapid response within 5 s, a wide linear range of 2.0–3800.0 μM, a detection limit of 1.2 μM (S/N = 3), and a high sensitivity of 181 μA mM−1 cm−2. These results suggested a promising potential of fabricating H2O2 electrochemical sensor using HD- PtNDs@AuNRs.  相似文献   

11.
In this work, poly(diallyldimethylammonium chloride) (PDDA) protected Prussian blue/gold nanoparticles/ionic liquid functionalized reduced graphene oxide (IL-rGO-Au-PDDA-PB) nanocomposite was fabricated. The resulting nanocomposite exhibited high biocompatibility, conductivity and catalytic activity. To assess the performance of the nanocomposite, a sensitive sandwich-type immunosensor was constructed for detecting alpha-fetoprotein (AFP). Greatly enhanced sensitivity for this immunosensor was based on triple signal amplification strategies. Firstly, IL-rGO modified electrode was used as biosensor platform to capture a large amount of antibody due to its increased surface area, thus amplifying the detection response. Secondly, a large number of Au-PDDA-PB was conjugated on the surface of IL-rGO, which meant the enrichment of the signal and the more immobilization of label antibody. Finally, the catalytic reaction between H2O2 and the IL-rGO-Au-PDDA-PB nanocomposite further enhanced the signal response. The signals increased linearly with AFP concentrations in the range of 0.01–100 ng mL−1. The detection limit for AFP was 4.6 pg mL−1. The immunosensor showed high sensitivity, excellent selectivity and good stability. Moreover, the immunosensor was applied to the analysis of AFP in serum sample with satisfactory result.  相似文献   

12.
In this work, we reported a sandwiched luminol electrochemiluminescence (ECL) immunosensor using ZnO nanoparticles (ZnONPs) and glucose oxidase (GOD) decorated graphene as labels and in situ generated hydrogen peroxide as coreactant. In order to construct the base of the immunosensor, a hybrid architecture of Au nanoparticles and graphene by reduction of HAuCl4 and graphene oxide (GO) with ascorbic acid was prepared. The resulted hybrid architecture modified electrode provided an excellent platform for immobilization of antibody with good bioactivity and stability. Then, ZnONPs and GOD functionalized graphene labeled secondary antibody was designed for fabricating a novel sandwiched ECL immunosensor. Enhanced sensitivity was obtained by in situ generating hydrogen peroxide with glucose oxidase and the catalysis of ZnONPs to the ECL reaction of luminol–H2O2 system. The as-prepared ECL immunosensor exhibited excellent analytical property for the detection of carcinoembryonic antigen (CEA) in the range from 10 pg mL−1 to 80 ng mL−1 and with a detection limit of 3.3 pg mL−1 (S N−1 = 3). The amplification strategy performed good promise for clinical application of screening of cancer biomarkers.  相似文献   

13.
An ultrasensitive multiplexed immunoassay method was developed at a disposable immunosensor array using mesoporous platinum nanoparticles (M-Pt NPs) as nonenzymatic labels. M-Pt NPs were prepared by ultrasonic method and employed to label the secondary antibody (Ab2) for signal amplification. The immunosensor array was constructed by covalently immobilizing capture antibody (Ab1) on graphene modified screen printed carbon electrodes (SPECs). After the sandwich-type immunoreactions, the M-Pt-Ab2 was bound to immunosensor surface to catalyze the electro-reduction of H2O2 reaction, which produced detectable signals for readout of analytes. Using breast cancer related panel of tumor markers (CA125, CA153 and CEA) as model analytes, this method showed wide linear ranges of over 4 orders of magnitude with the detection limits of 0.002 U mL−1, 0.001 U mL−1 and 7.0 pg mL−1 for CA125, CA153 and CEA, respectively. The disposable immunosensor array possessed excellent clinical value in cancer screening as well as convenient point of care diagnostics.  相似文献   

14.
An ultrasensitive electrochemiluminescence (ECL) immunosensor based on CdSe quantum dots (QDs) has been designed for the detection of clenbuterol. The immunosensor was fabricated by layer by layer and characterized with atomic force microscopic images (AFM) and electrochemical impedance spectra (EIS). In oxygen-saturated pH = 9.0 Tris-HCl buffer, a strong ECL emission of QDs could be observed during the cathodic process due to the H2O2 product from electrochemical reduction of dissolved oxygen. Upon the formation of immunocomplex, the second antibody labeled with horseradish peroxidase was simply immobilized on the electrode surface. The ECL emission decreased since steric hindrance of the immunocomplex slowed down the electron-transfer speed of dissolved oxygen, and also could be greatly amplified by an enzymatic cycle to consume the self-produced coreactant. Using clenbuterol as model analyte, the ECL intensity was determined by the concentration of competitive immunoassay of clenbuterol with a wide calibration in the range of 0.05 ng mL−1 to 1000 ng mL−1, and a low detection limit was 0.02 ng mL−1. The immunosensor shows good stability and fabrication reproducibility. It was applied to detecting practical samples with the satisfactory results. This immunosensing strategy opens a new avenue for detection of residue and application of QDs in ECL biosensing.  相似文献   

15.
In this contribution, mesoporous carbon nanospheres (MCN) were used to fabricate a label-free electrochemical immunosensor for breast cancer susceptibility gene (BRCAl). The detection platform was constructed by conjugation of anti-BRCA1 on glassy carbon electrodes which were modified by mesoporous carbon nanospheres–toluidine blue nanocomposite (MCN–TB)/room temperature ionic-liquid (RTIL) composited film. TB was adsorbed onto MCN and acted as a redox probe. The electroactivity of TB was greatly enhanced in the presence of MCN. The good conductivity of MCN and BMIM·BF4 could promote the electron transfer and thus enhance the detection sensitivity. Moreover, the large surface area of MCN and the protein-binding properties of BMIM·BF4 could greatly increase the antibody loading. The specific antibody–antigen immunoreaction on the electrode surface resulted in a decrease of amperometric signal of the electrode. Under optimized conditions, the amperometric signal decreased linearly with BRCAl concentration in the range of 0.01–15 ng mL−1 with a low detection limit of 3.97 pg mL−1. The immunosensor exhibits high sensitivity, good selectivity and stability.  相似文献   

16.
A new kind of signal amplification strategy based on ferrocene (Fc) incorporated polystyrene spheres (PS-Fc) was proposed. The synthesized PS-Fc displayed narrow size distribution and good stability. PS-Fc was applied as label to develop immunosensors for prostate specific antigen (PSA) after the typical sandwich immunoreaction by linking anti-PSA antibody (Ab2) onto PS-Fc. After the fabrication of the immunosensor, tetrahydrofuran (THF) was dropped to dissolve PS and release the contained Fc for the following stripping voltammetric detection. PS-Fc as a new electrochemical label prevented the leakage of Fc and greatly amplified the immunosensor signal. In addition, the good biocompatibility of PS could maintain the bioactivity of the antibodies. The response current was linear to the logarithm of PSA concentration in the range from 0.01 ng mL−1 to 20 ng mL−1 with a detection limit of 1 pg mL−1. The immunosensor results were validated through the detection of PSA in serum samples with satisfactory results.  相似文献   

17.
For the first time, a simple and highly sensitive label-free electrochemical carcinoembryonic antigen (CEA) immunosensor based on a cryogel electrode has been developed and tested. The as-prepared nanocomposite combined the advantages of the graphene, AuNPs and chitosan (AuNPs–GP–CS) together with the ease of preparing a cryogel coupled to a silver deposition, to act as a redox mediator, on a Au electrode. Under the optimal conditions, the decrease of the cyclic voltammetry (CV) silver peak current was proportional to the CEA concentration over a range of from 1.0 × 10−6 to 1.0 ng mL−1 with a detection limit of 2.0 × 10−7 ng mL−1. This AuNPs–GP–CS cryogel electrode gave a 1.7 times higher sensitivity and 25 times lower detection limit than the non-cryogel electrode. Moreover, the proposed electrochemical immunosensor exhibited good selectivity, reproducibility and stability. When applied to analyse clinical serum samples, the data determined by the developed immunosensor were in agreement with those obtained by the current hospital analysis system (enzyme linked fluorescent assay) (P > 0.05), to indicate that the immunosensor would be potentially useful for clinical diagnostics.  相似文献   

18.
A sandwich-type electrochemical immunosensor for the detection of carbohydrate antigen 19-9 (CA 19-9) antigen based on the immobilization of primary antibody (Ab1) on three dimensional ordered macroporous magnetic (3DOMM) electrode, and the direct electrochemistry of horseradish peroxidase (HRP) that was used as both the label of secondary antibody (Ab2) and the blocking reagent. The 3DOMM electrode was fabricated by introducing core–shell Au–SiO2@Fe3O4 nanospheres onto the surface of three dimensional ordered macroporous (3DOM) Au electrode via the application of an external magnet. Au nanoparticles functionalized SBA-15 (Au@SBA-15) was conjugated to the HRP labeled secondary antibody (HRP-Ab2) through the Au–SH or Au–NH3+ interaction, and HRP was also used as the block reagent. The formation of antigen–antibody complex made the combination of Au@SBA-15 and 3DOMM exhibit remarkable synergistic effects for accelerating direct electron transfer (DET) between HRP and the electrode. Under the optimal conditions, the DET current signal increased proportionally to CA 19-9 concentration in the range of 0.05 to 15.65 U mL−1 with a detection limit of 0.01 U mL−1. Moreover, the immunosensor showed high selectivity, good stability, satisfactory reproducibility and regeneration. Importantly, the developed method was used to assay clinical serum specimens, achieving a good relation with those obtained from the commercialized electrochemiluminescent method.  相似文献   

19.
Three-dimensional macroporous gold nanoparticles/graphene composites (3D-AuNPs/GN) were synthesized through a simple two-step process, and were used to modify working electrode sensing platform, based on which a facile electrochemical immunoassay for sensitive detection of carcinoembryonic antigen (CEA) in human serum was developed. In the proposed 3D-AuNPs/GN, AuNPs were distributed not just on the surface, but also on the inside of graphene. And this distribution property increased the area of sensing surface, resulting in capturing more primary antibodies as well as improving the electronic transmission rate. In the presence of CEA, a sandwich-type immune composite was formed on the sensing platform, and the horseradish peroxidase-labeled anti-CEA antibody (HRP-Ab2)/thionine/nanoporous silver (HRP-Ab2/TH/NPS) signal label was captured. Under optimal conditions, the electrochemical immunosensor exhibited excellent analytical performance: the detection range of CEA is from 0.001 to 10 ng mL−1 with low detection limit of 0.35 pg mL−1 and low limit of quantitation (LOQ) of 0.85 pg mL−1. The electrochemical immunosensor showed good precision, acceptable stability and reproducibility, and could be used for the detection of CEA in real samples. The proposed method provides a promising platform of clinical immunoassay for other biomolecules  相似文献   

20.
A highly sensitive impedimetric immunosensor based on a gold nanoparticles/multiwall carbon nanotube-ionic liquid electrode (AuNPs/MW-CILE) was developed for the determination of human epidermal growth factor receptor 2 (HER2). Gold nanoparticles were used to enhance the extent of immobilization and to retain the immunoactivity of the antibody Herceptin on the electrode. Cyclic voltammetry and electrochemical impedance spectroscopy were employed for characterization of various layers coated onto the AuNPs/MW-CILE. The impedance measurements at different steps were based on the charge transfer kinetics of the [Fe(CN)6]3−/4− redox pair. The immobilization of antibody and the corresponding antigen–antibody interaction at the electrode surface altered the interfacial electron transfer. The interactions of antibody with various concentrations of antigen were also monitored via the change of impedance response. The results showed that the charge transfer resistance increases linearly with increasing concentrations of HER2 antigen. The linear range and limit of detection were found as 10–110 ng mL−1 and 7.4 ng mL−1, respectively. The sensitivity and specificity of the immunosensor were validated. The results showed that the prepared immunosensor is a useful tool for screening of trace amounts of HER2 in serum samples of breast cancer patients.  相似文献   

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