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1.
《Electroanalysis》2017,29(12):2818-2831
Immobilization of biomolecules with a proper orientation is considered as a basis for diverse biotechnological applications. Herein, we report a host‐guest inclusion complexation between β‐cyclodextrin (β‐CD) and biotin as a versatile approach for the immobilization of biomolecules. As a practical application, a sandwich‐type electrochemical immunosensor was designed for the determination of prostate specific antigen (PSA). The immunosensor was fabricated by in situ electropolymerization of poly(N‐acetylaniline) onto a rGO‐modified Pt electrode. Then, β‐CD was covalently grafted onto the over‐oxidized polymer backbone. For improving the efficiency of the assay, AuNPs were casted on the polymeric film, on the surface of which thionine (TH) as an electron mediator was covalently immobilized. Using a host‐guest inclusion complexation between β‐CD and biotin, a β‐CD/biotin‐Ab1/PSA/Ab2‐horseradish peroxidase (HRP) sandwich was formed on the electrode surface. The analytical signal was produced via electrochemical reduction of THox, generated by biocatalytic oxidation of the THred in the presence of HRP/H2O2. Under optimal conditions, the proposed sensor responded linearly to PSA in the range from 10.0 pg mL−1 to 25.0 ng mL−1, with a low detection limit of 6.7 pg mL−1 (S/N=3). Kinetic parameters of the interaction of β‐CD with Ab1 were also investigated. Finally, the applicability of the immunosensor was successfully investigated for the detection of PSA in human serum samples.  相似文献   

2.
A simple, highly sensitive and label‐free electrochemical impedance spectroscopy (EIS) immunosensor was developed using Nafion and gold nanoparticles (nano‐Au/Nafion) composites for the determination of 1‐pyrenebutyric acid (PBA). Under the optimal conditions, the amount of immobilized antibody was significantly improved on the nano‐Au/Nafion electrode due to the synergistic effect and biocompatibility of Nafion film and gold nanoparticles composites. The results showed that the sensitivity and stability of nano‐Au/Nafion composite electrode for PBA detection were much better than those of nano‐Au modified glassy carbon electrode (nano‐Au/GCE). The plot of increased electron transfer resistances (Rets) against the logarithm of PBA concentration is linear over the range from 0.1 to 150 ng·mL?1 with the detection limit of 0.03 ng·mL?1. The selectivity and accuracy of the proposed EIS immunosensor were evaluated with satisfactory results.  相似文献   

3.
Calmodulin (CaM) is an important intracellular calcium‐binding protein. It plays a critical role in a variety of biological and biochemical processes. In this paper, a new electrochemical immunosensing protocol for sensitive detection of CaM was developed by using gold‐silver‐graphene (AuAgGP) hybrid nanomaterials as protein immobilization matrices and gold nanorods (GNRs) as enhanced electrochemical labels. Electrode was first modified with thionine‐chitosan film to provide an immobilization support for gold‐silver‐graphene hybrid nanomaterials. The hybrid materials formed an effective matrix for binding of CaM with high density and improved the electrochemical responses as well. Gold nanorods were prepared for the fabrication of enhanced labels (HRP‐Ab2‐GNRs), which provided a large capacity for HRP‐Ab2 immobilization and a facile pathway for electron transfer. With two‐step immunoassay format, the HRP‐Ab2‐GNRs labels were introduced onto the electrode surface, and produced electrochemical responses by catalytic reaction of HRP toward enzyme substrate of hydrogen peroxide (H2O2) in the presence of thionine. The proposed immunosensor showed an excellent analytical performance for the detection of CaM ranging from 50 pg mL?1 to 200 ng mL?1 with a detection limit of 18 pg mL?1. The immunosensor has also been successfully applied to the CaM analysis in two cancer cells (HepG2 and MCF‐7) with high sensitivity, which has shown great potency for improving clinic diagnosis and treatment for cancer study.  相似文献   

4.
本文研制了一种用金胶壳聚糖仿生膜来同时固定四甲基联苯胺(TMB)和酶标抗体的新型电化学免疫传感器,用于检测血清肿瘤标志物前列腺特异性抗原(PSA)的含量。固定的TMB作为电子传递媒介体,在扫速小于45 mV/s时,电极表现为一个表面控制过程,而在扫速大于45 mV/s时则表现为一个扩散控制过程。将固定有酶标抗体和TMB的免疫传感器与待测PSA抗原一起培育,在该传感器上形成的免疫复合物通过TMB-H2O2-HRP电化学体系进行了测定。在优化实验条件下,PSA的线性检测范围为5-30 ng·mL-1,检测限为1.0 ng·mL-1。该PSA免疫传感器制备方法简单,成本低廉,具有较好的稳定性和重现性。  相似文献   

5.
We report on the construction of a label-free electrochemical immunosensor for detecting the core antigen of the hepatitis C virus (HCV core antigen). A glassy carbon electrode (GCE) was modified with a nanocomposite made from gold nanoparticles, zirconia nanoparticles and chitosan, and prepared by in situ reduction. The zirconia nanoparticles were first dispersed in chitosan solution, and then AuNPs were prepared in situ on the ZrO2-chitosan composite. In parallel, a nanocomposite was synthesized from AuNPs, silica nanoparticles and chitosan, and conjugated to a secondary antibody. The properties of the resulting nanocomposites were investigated by UV-visible photometry and transmission electron microscopy, and the stepwise assembly process was characterized by means of cyclic voltammetry and electrochemical impedance spectroscopy. An sandwich type of immunosensor was developed which displays high sensitivity to the HCV core antigen in the concentration range between 2 and 512?ng?mL?1, with a detection limit of 0.17?ng?mL?1 (at S/N?=?3). This immunosensor provides an alternative approach towards the diagnosis of HCV.
Fig
A sandwich-type immunosensor was constructed for the detection of HCV core Ag. AuNPs/ZrO2-Chits nanocomposites were prepared by in situ reduction method. AuNPs/SiO2-Chits nanocomposite integrated with secondary antibody (Ab2) without labeled HRP. The immunosensor displayed high sensitivity to HCV core antigen with a detection limit of 0.17?ng?mL?1 (S/N?=?3).  相似文献   

6.
In this study, a novel strategy to amplify electrochemical signals by mesoporous PdPt nanoparticles with core-shell structures anchored on a three-dimensional PANI@CNTs network as nanozyme labels (PdPt/PANI@CNTs) was proposed for the sensitive monitoring of α-fetoprotein (AFP, Ag). First, the mesoporous PdPt nanoparticles prepared by a facile chemical reduction method had excellent biocompatibility with biomolecules, which could capture a large amount of AFP-Ab2 (Ab2) and exhibit plentiful pores to entrap more thionine (Thi) into mesoporous PdPt nanoparticles with enhanced loading and abundant active sites. Furthermore, the resulting mesoporous PdPt nanoparticles were abundantly dotted on the surface of a three-dimensional PANI@CNTs network with excellent conductivity and a high specific surface area through the bonding of the amino group to form PdPt/PANI@CNTs nanozyme labels. Most importantly, the as-prepared PdPt/PANI@CNTs nanozyme labels exhibited unexpected enzyme-like activity towards the reduction of hydrogen peroxide owing to the highly indexed facets, enhancing the current response to realize signal amplification. In view of the advantages of nanozyme labels and the involvement of gold nanoparticles (AuNPs, which behave as electrode materials) for the sensitive determination of AFP, the as-developed immunosensor could obtain a dynamic working range of 0.001 ng mL−1–100.0 ng mL−1 at a detection limit of 0.33 pg mL−1 via DPV (at 3σ). Furthermore, the nanozyme-based electrochemical immunosensor exhibited remarkable analytical performance, which brought about feasible ideas for disease diagnosis in the future.  相似文献   

7.
α‐Synuclein (α‐SYN) is a very important neuronal protein that is associated with Parkinson’s disease. In this paper, we utilized Au‐doped TiO2 nanotube arrays to design a photoelectrochemical immunosensor for the detection of α‐SYN. The highly ordered TiO2 nanotubes were fabricated by using an electrochemical anodization technique on pure Ti foil. After that, a photoelectrochemical deposition method was exploited to modify the resulting nanotubes with Au nanoparticles, which have been demonstrated to facilitate the improvement of photocurrent responses. Moreover, the Au‐doped TiO2 nanotubes formed effective antibody immobilization arrays and immobilized primary antibodies (Ab1) with high stability and bioactivity to bind target α‐SYN. The enhanced sensitivity was obtained by using {Ab2‐Au‐GOx} bioconjugates, which featured secondary antibody (Ab2) and glucose oxidase (GOx) labels linked to Au nanoparticles for signal amplification. The GOx enzyme immobilized on the prepared immunosensor could catalyze glucose in the detection solution to produce H2O2, which acted as a sacrificial electron donor to scavenge the photogenerated holes in the valence band of TiO2 nanotubes upon irradiation of the other side of the Ti foil and led to a prompt photocurrent. The photocurrents were proportional to the α‐SYN concentrations, and the linear range of the developed immunosensor was from 50 pg mL?1 to 100 ng mL?1 with a detection limit of 34 pg mL?1. The proposed method showed high sensitivity, stability, reproducibility, and could become a promising technique for protein detection.  相似文献   

8.
A sensitive and specific electrochemical immunosensor was developed with α‐fetoprotein (AFP) as the model analyte by using gold nanoparticle label for enzymatic catalytic amplification. A self‐assembled monolayer membrane of mercaptopropionic acid (MPA) was firstly formed on the electrode surface through gold‐sulfur interaction. Monoclonal mouse anti‐human AFP was covalently immobilized to serve as the capture antibody. In the presence of the target human AFP, gold nanoparticles coated with polyclonal rabbit anti‐human AFP were bound to the electrode via the formation of a sandwiched complex. With the introduction of goat anti‐rabbit IgG conjugated with alkaline phosphatase, the dentritical enzyme complex was formed through selective interaction of the secondary antibodies with the colloidal gold‐based primary antibody at the electrode, thus affording the possibility of signal amplification for AFP detection. Current response arising from the oxidation of enzymatic product was significantly amplified by the dentritical enzyme complex. The current signal was proportional to the concentration of AFP from 1.0 ng mL?1 to 500 ng mL?1 with a detection limit of 0.8 ng mL?1. This system could be extended to detect other target molecules with the corresponding antibody pairs.  相似文献   

9.
A signal‐enhanced immunosensor has been developed by self‐assembling Au NPs onto a ferrocene‐branched poly(allylamine)/multiwalled carbon nanotubes (PAA‐Fc/MWNTs) modified electrode for the sensitive determination of hepatitis B surface antigen (HBsAg) as a model protein. The formation of PAA‐Fc/MWNTs composite not only effectively avoided the leakage of Fc and retained its electrochemical activity, but also enhanced the conductivity and charge‐transport properties of the composite. Further adsorption of Au NPs into the PAA matrix provided both the interactive sites for the immobilization of hepatitis B surface antibody (HBsAb) and a favorable microenvironment to maintain its activity. Tests performed with this immunosensor showed a specific response to HBsAg in the range of 0.1–350.0 ng mL?1 with a detection limit of 0.03 ng mL?1.  相似文献   

10.
A approach was successfully employed for constructing a solid‐state electrochemiluminescence (ECL) immunosensor by layer‐by‐layer self‐assembly of multiwall carbon nanotubes (MWCNTs)‐Nafion composite film, Ru(bpy)32+/nano‐Pt aggregates (Ru‐PtNPs) and Pt nanoparticles (PtNPs). The influence of Pt nanoparticles on the ECL intensity was quantitatively evaluated by calculating the electroactive surface area of different electrodes with or without PtNPs to immobilize Ru(bpy)32+. The principle of ECL detection for target α‐fetoprotein antigen (AFP) was based on the increment of resistance after immunoreaction, which led to a decrease in ECL intensity. The linear response range was 0.01–10 ng mL?1 with the detection limit of 3.3 pg mL?1. The immunosensor exhibited advantages of simple preparation and operation, high sensitivity and good selectivity.  相似文献   

11.
Present work demonstrates the fabrication of new and facile sandwich‐type electrochemical immunosensor based on palladium nanoparticles (PdNPs), polyaniline (PANI) and fullerene‐C60 nanocomposite film modified glassy carbon electrode (PdNP@PANI‐C60/GCE) for ultrasensitive detection of Prostate‐specific antigen (PSA) biomarker. PdNP@PANI‐C60 was electrochemically synthesized on GCE and used as an electroactive substrate. PdNP@PANI‐C60 was characterized by scanning electron microscopy (SEM), energy‐dispersive X‐ray spectroscopy (EDS), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Primary antibody anti‐PSA (Ab1) was covalently immobilized on PdNP@PANI‐C60/GCE using NHS/EDC linkers. In the presence of PSA antigen, horseradish peroxidase secondary antibody (HRP‐Ab2) was brought into the surface of the electrode, developing stable amplified signals of H2O2 reduction. Under the optimal conditions, a linear curve for determination of PSA at the proposed immunosensor was 1.6×10?4 ng.mL?1 to 38 ng.mL?1 with a limit of detection (LOD) of 1.95×10?5 ng.mL?1. The proposed immunosensor was successfully validated in serum and urine samples towards PSA detection with satisfactory and acceptable results.  相似文献   

12.
In this paper, a thiol graphene‐thiol chitosan‐gold nanoparticles (thGP‐thCTS‐AuNPs) nanocomposites film with porous structure was fabricated by electrochemically depositing on glassy carbon electrode (GCE), which exhibited good biocompatibility and improved conductivity, to construct immunosensor free label for detection of carcinoembryonic antigen (CEA). The electrochemical behavior of this immunosensor was investigated by cyclic voltammetry. Under the optimum conditions, the immunosensor revealed a good amperometric response to CEA in two linear ranges (0.3–8.0 ng mL?1 and 8.0–100 ng mL?1) with a detection limit of 0.03 ng mL?1. The results indicated that the immunosensor has the advantages of good selectivity, high sensitivity, and good stability for the determination of CEA.  相似文献   

13.
基于多层酶/纳米金固定甲胎蛋白免疫传感器的研究   总被引:22,自引:1,他引:21  
>利用自组装技术和静电吸附作用, 将甲胎蛋白抗体(anti-AFP)固定在多层辣根过氧化物酶/纳米金及L-半胱胺酸修饰的金电极表面, 制备出用于检测甲胎蛋白抗原(AFP)的无试剂型免疫传感器. 通过交流阻抗技术、循环伏安法和计时电流法考察了电极的电化学特性, 并对该免疫传感器的作用机理及性能进行了详细的研究. 用计时电流法测得AFP的线性范围为1.0~10.0和10~200 ng•mL-1, 检出限为0.5 ng•mL-1. 实验结果表明, 该方法提高了抗体的固定量, 增强了传感器的灵敏度和稳定性, 且该传感器响应迅速、选择性好, 血清中常见抗原不干扰测定. 将其用于临床血清检验, 与放射免疫测定法(RIA)的符合率为86.7%.  相似文献   

14.
《Electroanalysis》2017,29(9):2098-2105
An ultrasensitive electrochemiluminescence (ECL) immunosensor for the detection of tetrodotoxin (TTX) is proposed, which are composed of the branched poly‐(ethylenimine) (BPEI) functionalized graphene (BGNs)/Fe3O4‐Au magnetic capture probes and luminol‐capped gold nanocomposites (luminol‐AuNPs) as the signal tag. Herein, a typical sandwich immunecomplex was constructed on the glassy carbon electrode. The BGNs/Fe3O4‐Au hybrids could efficiently conjugate primary antibody via the Au−S chemical bonds or Au−N chemical bonds and rapidly separate under external magnetic field. The introduction of BPEI to GO could enhance the luminol‐ECL intensity. Meanwhile, the multifunctional nanocomposites have been proved with good water‐solubility, excellent electron transfer, outstanding stability, etc. The luminescent luminol‐AuNPs, a high efficient electrochemiluminescence marker, can be assembled on the second antibody, which can produce the ECL signal to achieve the determination of TTX. This proposed ECL immunosensor with a linear range from 0.01–100 ng/mL can be applied in the detection of TTX in real samples with satisfactory results.  相似文献   

15.
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   

16.
A novel electrochemical magnetoimmunosensor for fast and ultrasensitive detection of H9N2 avian influenza virus particles (H9N2 AIV) was designed based on the combination of high‐efficiency immunomagnetic separation, enzyme catalytic amplification, and the biotin–streptavidin system. The reusable, homemade magneto Au electrode (M‐AuE) was designed and used for the direct sensing. Immunocomplex‐coated magnetic beads (IMBs) were easily accumulated on the surface of the M‐AuE to obtain the catalytically reduced electrochemical signal of H2O2 after the immunoreaction. The transducer was regenerated through a simple washing procedure, which made it possible to detect all the samples on a single electrode with higher reproducibility. The magnetic‐bead‐based electrochemical immunosensor showed better analytical performance than the planar‐electrode‐based immunosensor with the same sandwich construction. Amounts as low as 10 pg mL?1 H9N2 AIV could be detected even in samples of chicken dung. This electrochemical magnetoimmunosensor not only provides a simple platform for the detection of the virus with high sensitivity, selectivity, and reproducibility but also shows great potential in the early diagnosis of diseases.  相似文献   

17.
Caifeng Ding  Fei Zhao  Jin-Ming Lin 《Talanta》2009,78(3):1148-4751
A novel and effective electrochemical immunosensor for the rapid determination of α-fetoprotein (AFP) based on carbon paste electrode (CPE) consisting of room temperature ionic liquid (RTIL) N-butylpyridinium hexafluorophosphate (BPPF6) and graphite. The surface of the CPE was modified with gold nanoparticles for the immobilization of the α-fetoprotein antibody (anti-AFP). By sandwiching the antigen between anti-AFP on the CPE modified with gold nanoparticles and the secondary antibody, polyclonal anti-human-AFP labeled with horseradish peroxidase (HRP-labeled anti-AFP), the immunoassay was established. The concentration of AFP was determined based on differential pulse voltammetry (DPV) signal, which was generated in the reaction between O-aminophenol (OAP) and H2O2 catalyzed by HRP labeled on the sandwich immunosensor. AFP concentration could be measured in a linear range of 0.50-80.00 ng mL−1 with a detection limit of 0.25 ng mL−1. The immunosensor exhibited high sensitivity and good stability, and would be valuable for clinical assay of AFP.  相似文献   

18.
《Electroanalysis》2018,30(3):402-414
A sensitive electrochemical immunosensor for Hepatitis B virus surface antigen (HBsAg) detection was fabricated based on hemin/G‐quadruplex interlaced onto Fe3O4‐AuNPs or hemin ‐amino‐reduced graphene oxide nanocomposite (H‐amino‐rGO‐Au). G‐quadruplex DNAzyme, which is composed of hemin and guanine‐rich nucleic acid, is an effective signal amplified tool for its outstanding peroxidase activity and Fe3O4‐AuNPs or (H‐amino‐rGO‐Au) nanocomposites with quasi‐enzyme activity provide appropriate support for the immobilization of hemin/G‐quadruplex. The target protein was sandwiched between the primary antibody immobilized on the GO and secondary antibody immobilized on the Fe3O4‐AuNPs or (H‐amino‐rGO‐Au) nanocomposites and glutaraldehyde was used as linking agent for the immobilization of primary antibody on the surface of GO. Both Fe3O4‐AuNPs and H‐amino‐rGO‐Au nanocomposite and also hemin/G‐quadruplex can cooperate the electrocatalytic reduction of H2O2 in the presence of methylene blue as mediator. The proposed immunosensor has a wide linear dynamic range of 0.1 pg/ml to 300 pg/ml with a detection limit of 60 fg/ml when Fe3O4‐AuNPs was used for immobilization of hemin/G‐quadruplex, while the dynamic range and DL were 0. 1–1000 pg/mL and 10 fg/mL, respectively in the presence of H‐amino‐rGO‐ Au nanocomposite as platform for immobilizing of hemin/G‐quadruplex. The proposed immunosensor was also used for analysis of HBsAg in spiked human serum samples with satisfactory results.  相似文献   

19.
《Electroanalysis》2017,29(3):861-872
We report here a new electrochemical probe for the development of a sensitive, and selective sandwich‐type electrochemical immunosensor for the detection of epidermal growth factor receptor (EGFR). The probe is a newly synthesized bovine serum albumin (BSA)‐templated Pb nanocluster (PbNC@BSA). For fabrication of the immunosensor, we employed streptavidin‐coated magnetic beads (MB) as a platform for immobilization of the biotinylated primary antibody (Ab1), and utilized the PbNC@BSA conjugated to secondary antibody (Ab2) as a signaling probe. After sandwiching the target protein between Ab1 and Ab2, we dissolved PbNC@BSA into an acid, and recorded square wave anodic stripping voltammetric (SWASV) signal of the Pb ions as an analytical signal for quantification of the EGFR. The immunosensor responded linearly towards EGFR within the range of 0.4 ng/mL to 35 ng/mL, with a detection limit of 8 pg/mL. The immunosensor displayed good sensitivity, selectivity, stability, and reproducibility, and proved suitable for direct measurement of EGFR in human serum samples. Moreover, we used the as‐synthesized PbNC@BSA as a fluorescence label for in vitro cell viability analysis as well as bioimaging of cancerous HeLa and non‐cancerous HUVEC cells. PbNC@BSA exhibited low cytotoxicity and high biocompatibility in living cells, and was a suitable fluorescent probe for live cell imaging, with potential therapeutic applications.  相似文献   

20.
A novel sandwich-type electrochemical immunosensor for human immunoglobulin G (hIgG) was developed using Au/SiO2 nanoparticles (NPs) with adsorbed horseradish peroxidase-anti-hIgG as the secondary antibody layer. The signal readout is based on the amperometric response to the catalytic reduction of hydrogen peroxide at an AuNPs-polythionine modified glassy carbon electrode. Under optimized conditions, the linear range is from 0.1 to 200 ng·mL?1, with a detection limit of 0.035 ng·mL?1 (at an S/N of 3). The immunosensor exhibited a performance that is better than that based on Au/SiO2NPs-excluded secondary antibody.  相似文献   

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