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1.
A label-free multiplexed immunoassay strategy was proposed for the simultaneous detection of two tumor markers, carcinoembryonic antigen (CEA) and α-fetoprotein (AFP). Monoclonal antibody of CEA was co-immobilized with ferrocenecarboxylic acid (FCA) inside the channels of mesoporous silica (MPS) to prepare the label-free probe for CEA. Also, monoclonal antibody of AFP was co-immobilized with horseradish peroxidase (HRP) inside the channels of MPS to prepare the label-free probe for AFP by using o-phenylenediamine (OPD) and H(2)O(2) as the electrochemical substrates. Thus, the multianalyte immunosensor was constructed by coating the probes of CEA and AFP respectively onto the different areas of indium-tin oxide (ITO) electrode. When the immunosensor was incubated with sample antigens, CEA and AFP antigens were introduced into the mesopores of MPS after the immunoassay reaction. Because all of the Si-OH groups on the external surface of MPS were blocked with Si(CH(3))(3), the proteins and substrates were limited to be embedded on the internal pore walls. Therefore, the electric response transfer was confined inside the pore channels. The nonconductive immunoconjugates blocked the electron transfer and the peak responses changed on the corresponding surface respectively. Then, the simultaneous detection of CEA and AFP achieved. The linear ranges of CEA and AFP were 0.5-45ngmL(-1) and 1-90ngmL(-1) with the detection limits of 0.2ngmL(-1) and 0.5ngmL(-1) (S/N=3), respectively. The fabricated immunosensor shows appropriate sensitivity and offers an alternative to the multianalyte detection of antigens or other bioactive molecules.  相似文献   

2.
In this article, a conspicuously simple and highly sensitive amperometric immunosensor based on the sequential electrodeposition of Prussian blue (PB) and gold nanoparticles (GNPs) on multiwalled carbon nanotube (MWCNT)-modified glassy carbon electrode (GCE) surface is proposed for the detection of α-fetoprotein (AFP). By comparison with PB, the MWCNT/PB composite film had been proven to show much better electrochemical stability and a larger response current. The electrodeposited GNP film can be used not only to immobilize biomolecules but also to avoid the leakage of PB and to prevent shedding of MWCNT/PB composite film from the electrode surface. The performance and factors influencing the performance of the immunosensor were investigated. Under optimal experimental conditions, the proposed immunosensor for AFP was observed with an ultralow limit of detection (LOD) equal to 3 pg/ml (at 3δ), and the linear working range spanned the concentrations of AFP from 0.01 to 300 ng/ml. Moreover, the immunosensor, as well as a commercially available kit, was examined for use in the determination of AFP in real human serum specimens. More significant, the assay mentioned here is simpler than the traditional enzyme-linked immunosorbent assay (ELISA), and an excellent correlation of levels of AFP measured was obtained, indicating that the developed immunoassay could be a promising alternative approach for detection of AFP and other tumor markers in the clinical diagnosis.  相似文献   

3.
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.  相似文献   

4.
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.  相似文献   

5.
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.  相似文献   

6.
Dai Y  Cai Y  Zhao Y  Wu D  Liu B  Li R  Yang M  Wei Q  Du B  Li H 《Biosensors & bioelectronics》2011,28(1):112-116
A sandwich electrochemical immunosensor for the sensitive determination of alpha fetoprotein (AFP) has been fabricated. Prussian blue modified hydroxyapatite (PB@HAP) was firstly prepared and used as electrochemical label due to the wonderful conductivity and good biocompatibility of HAP. The results proved that the immunosensor fabricated using the label based on PB@HAP loaded with horse radish peroxidase (HRP) and secondary anti-AFP antibody (Ab(2)) (PB@HAP-HRP-Ab(2)) had high sensitivity, and the sensitivity of the label PB@HAP-HRP-Ab(2) was much higher than labels of PB@HAP-Ab(2), PB-HRP-Ab(2) and HAP-HRP-Ab(2). The mixture of graphene sheet (GS) and thionine (TH) was not only used to immobilize anti-AFP antibody (Ab(1)) but also took part in the signal amplification. The amperometric signal increased linearly with AFP concentration in the range of 0.02-8 ng/mL with a low detection limit of 9 pg/mL. The immunosensor had the advantages of high sensitivity, good selectivity and good stability, and was applied to the analysis of AFP in serum sample with satisfactory results. Due to the low-cost and easy synthesis of PB@HAP, the screen-printed electrodes could be used instead of the bare glass carbon electrode in order to achieve mass production. In addition, it had potential application in the detection of other tumor markers.  相似文献   

7.
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.  相似文献   

8.
A novel multi-array immunoassay device based on the insert-plug model of piezoelectric (Pz) immunosensor fabricated with the screw clamp apparatus has been developed for quantitative detection of tumor markers such as alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), prostate specific antigen (PSA), and carcinoma antigen 125 (CA125) in serum, in which single immunosensor can oscillate independently with the frequency stability of +/-1 Hz (hertz) in air phase and +/-2 Hz in liquid phase. These response characteristics of Pz tumor marker multi-array immunoassay device such as time-cost, reproducibility and specificity, etc. were also investigated, respectively. The detection range for AFP, CEA, PSA and CA125 obtained by multi-array Pz immunosensor were 20-640 ng/ml, 1.5-30 microg/ml, 1.5-40 ng/ml and 5-150 IU/ml, respectively, with the coefficient of variance (CV) less than 5% and no cross-reactivates with other tumor markers in serum were observed. Application of the multi-array immunosensor to clinical samples demonstrated that results were in good agreement with chemiluminescence immunoassay (CLIA). Moreover, the multi-array Pz immunosensor could be regenerated to be reused for three cycles without appreciable loss of response activity. Therefore, the proposed multi-array immunoassay device based on Pz immunosensor provides a rapid, sensitive, specific, reusable, convenient and reliable alternative for the detection of tumor markers in clinical laboratory.  相似文献   

9.
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.  相似文献   

10.
A novel immunoaffinity column used as an immunosensor for flow-injection chemiluminescent (CL) immunoassay was prepared by immobilizing antibody on carboxylic resin beads. The immunosensor could fast recognize and trap the immunocomplex of horseradish peroxidase (HRP)-labeled antibody and antigen, which was firstly formed with a micro-bubble accelerated pre-incubation process, to produce a sandwich immunocomplex. The HRP introduced in the immunoaffinity column could catalyze the CL reaction to produce enzyme-enhanced emission. With alpha-fetoprotein (AFP) as a mode, a flow-injection CL immunoassay was proposed. The whole assay for one sample, including the pre-incubation and the regeneration of immunoaffinity column, could be performed within 16min. The linear range was 1.0-80ng/ml with a correlation coefficient of 0.998 and a detection limit of 0.1ng/ml at a signal/noise ratio of 3. The intra- and inter-assay coefficients of variation at 20ng/ml AFP were 1.2% and 8.5%, respectively. The storage stability of the immunoaffinity column and the accuracy for sample detection were acceptable. This flexible, sensitive, low-cost, and rapid method is valuable for clinical immunoassay.  相似文献   

11.
A novel simultaneous measurement method for alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA) in human sera by time-resolved fluoroimmunoassay (TR-FIA) is described. The proposed approach combines the use of europium-labeled anti-AFP antibody for AFP TR-FIA and biotinylated anti-CEA antibody complexed to samarium-labeled streptavidin for CEA TR-FIA. A 96-well microtiter plate coated with a mixture of anti-AFP and anti-CEA monoclonal antibodies was used for the assay. After it was reacted with a solution containing AFP and CEA, a mixture of anti-AFP antibody labeled with BHHCT-Eu(3+) and biotinylated anti-CEA antibody was added. The AFP concentration was determined by measuring the solid-phase fluorescence of the europium-labeled anti-AFP antibody at 615 nm. Then a BHHCT-Sm(3+)-labeled streptavidin-bovine serum albumin conjugate (SA-BSA) was added to react with the biotinylated anti-CEA antibody. After the reaction, the unreacted SA-BSA was washed out, and a 0.1 M NaOH solution containing 1.0 x 10(-5) M TOPO and 0.05% SDS was added to dissociate the samarium-labeled SA-BSA in the immune complex on the surface of the well into the solution. The CEA concentration was determined by measuring the solution fluorescence of 643 nm from the samarium-labeled SA-BSA. The present method gives detection limits of 0.07 ng/ml for AFP and 0.3 ng/ml for CEA. The coefficient variations of the method are less than 7%, and the recoveries are in the range of 90-110% for serum samples. The AFP and CEA concentrations in 27 human serum samples were determined by the present method as well as by single assay for comparison. A good correlation was obtained with the correlation coefficients of 0.990 for AFP and 0.973 for CEA.  相似文献   

12.
A novel amperometric immunosensor based on chitosan–gold nanoparticles (Chit–GNPs) composite film and thionine (Thi) was prepared for the determination of α-1-fetoprotein (AFP). The immunosensor was prepared by electro-depositing a Chit–GNPs composite matrix on the surface of the glass carbon electrode, then Thi was immobilized onto the Chit–GNPs film using glutaraldehyde as a cross-linker. Furthermore, the GNPs were chemisorbed onto Thi film for immobilization of α-1-fetoprotein antibody. The procedure of the immunosensor was characterized by means of cyclic voltammograms. The performance and influencing factors of the resulting immunosensor were studied in details. Under optimal conditions, the immunosensor was highly sensitive to AFP and the linear range covered from 0.40 to 200.0 ng mL−1 with a detection limit of 0.24 ng mL−1 at three times background noise. Moreover, the simple and controllable electro-deposition method overcame the irreproducibility for preparing Chit-based immunosensor systems and the proposed immunosensor displayed a satisfactory reproducibility and stability.  相似文献   

13.
A novel, label-free amperometric immunosensor has been developed for the rapid detection of heat-killed Escherichia coli O157:H7 (E. coli O157:H7). This immunosensor was prepared as follows. First, the long-chain, amine-terminated alkanethiol 11-amino-1-undecanethiol hydrochloride (AUT) was self-assembled onto a gold electrode surface to form an ordered, oriented, compact, and stable monolayer possessing -NH(2) functional groups that could immobilize massive gold nanoparticles (GNPs). Next, chitosan-multiwalled carbon nanotubes-SiO(2)/thionine (CHIT-MWNTs-SiO(2)@THI) nanocomposites and GNPs multilayer films were prepared via layer-by-layer (LBL) assembly. The surface area enhancement from the LBL assembly of the multilayer films improves the stability of the immobilized CHIT-MWNTs-SiO(2)@THI. More important, the sensitivity and stability of the immunosensor can be enhanced proportionally to the quantity of the THI mediator immobilized on the electrode surface. Finally, the E. coli O157:H7 antibody (anti-E. coli O157:H7) was covalently bound to the GNP monolayer and its bioactivity was measured by enzyme-linked immunosorbent assay (ELISA). Transmission electron microscopy (TEM) was employed to characterize the morphology of the MWNTs, CHIT-MWNTs, and CHIT-MWNTs-SiO(2)@THI. Under optimal conditions, the calibration curve for heat-killed E. coli O157:H7 has a working range of 4.12×10(2)-4.12×10(5) colony-forming units (CFU)/ml, and the total assay time was less than 45 min.  相似文献   

14.
A novel electrochemiluminescence (ECL) immunosensor for ultrasensitive detection of α-1-fetoprotein (AFP) was designed based on the in situ bi-enzymatic reaction to generate coreactant of peroxydisulfate for signal amplification. In this work, AuNPs were electrodeposited on the glassy carbon electrode (GCE) surface, which promoted the electron transfer. Then, L-cysteine and another layer of AuNPs were, respectively assembled onto the modified electrode surface, which formed the multilayer films for amplifying the ECL signal of peroxydisulfate and immobilizing antibody. At last, glucose oxidase (GOD) and horseradish peroxidase (HRP) were employed to block the nonspecific binding sites. When proper amounts of glucose were added in the detection solution, GOD catalyzed the oxidation of glucose to generate H(2)O(2), which could be further catalyzed by HRP to generate O(2) for the signal amplification. The linear range for AFP detection was 0.001-100 ng mL(-1), with a low detection limit of 3.3 × 10(-4) ng mL(-1). The novel strategy has the advantages of simplicity, sensitivity, good selectivity and reproducibility which might hold a new promise for highly sensitive bioassays applied in clinical detection.  相似文献   

15.
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.  相似文献   

16.
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.  相似文献   

17.
A direct electrochemical immunoassay system based on the immobilization of α-1-fetoprotein antibody (anti-AFP), as a model system, on the surface of core-shell Fe2O3/Au magnetic nanoparticles (MNP) has been demonstrated. To fabricate such an assay system, anti-AFP was initially covalently immobilized on to the surface of core-shell Fe2O3/Au MNP. Anti-AFP-modified MNP (bio-nanoparticles) were then attached to the surface of carbon paste electrode with the aid of a permanent magnet. The performance and factors influencing the performance of the resulting immunosensor were studied. α-1-Fetoprotein antigen was directly determined by the change in current or potential before and after the antigen–antibody reaction versus saturated calomel electrode. The electrochemical immunoassay system reached 95% of steady-state potential within 2 min and had a sensitivity of 25.8 mV. The linear range for AFP determination was from 1 to 80 ng AFP ml−1 with a detection limit of 0.5 ng AFP ml−1. Moreover, the direct electrochemical immunoassay system, based on a functional MNP, can be developed further for DNA sensor and enzyme biosensor. Revisions requested 2 November 2005; Revisions received 17 January 2006  相似文献   

18.
A non-competitive immunoassay based on micellar electrokinetic capillary chromatography (MECC) with laser-induced fluorescence (LIF) detection has been developed for the determination of alpha-fetoprotein (AFP). The anti-AFP antibody was labeled with fluorescein isothiocyanate (FITC) and the product was used as a fluorescent tracer, then AFP was mixed with the labeled antibody. After incubation, the immune AFP-antibody complex was separated from labeled free antibody by MECC. The parameters affecting separation such as the concentration of sodium dodecyl sulfate (SDS), the buffer pH and separation voltage were investigated and the following conditions were selected: 20 mM tetraborate containing 100 mM SDS at pH 9.50, and 20 kV separation voltage. The detection limit of this assay was 0.1 ng/ml with a linear range spanning two orders of magnitude. This method was applied to determine AFP in human serum.  相似文献   

19.
A new electrochemical sensor based on a novel organic–inorganic material (PNFCTs) was proposed for detection of paracetamol in this paper. First, PNFCTs were prepared with multi-walled carbon nanotubes (MWNTs) and a derivative of 3,4,9,10-perylenetetracarboxylic dianhydride (PTC-NH2) via cross-linking method. Then, PNFCTs were coated onto the surface of the glassy carbon electrode (GCE) to form porous organic conducting polymer films (PNFCTs/GCE), which could not only increase the loading of paracetamol efficiently but also provide an interface with exceptional electrical conductivity for paracetamol. Finally, gold nanoparticles (GNPs) were attached to the electrode surface through electrodepositing method, which obtained GNPs/PNFCTs/GCE electrode. The electrochemical behavior of paracetamol on GNPs/PNFCTs/GCE was explored by cyclic voltammetrys (CVs) and differential pulse voltammograms (DPVs). The results showed that the GNPs/PNFCTs/GCE exhibited excellent electrocatalytic activity to paracetamol, which should be attributed to remarkable properties of the new composite nanomaterials with porous nanostructure and exceptional electrical conductivity. The wide liner range and detection limit were 0.3–575 and 0.1 μM, respectively. Finally, it was successfully used to detect paracetamol in dilution human serum and commercial tablets. The sensor shows great promise for simple, sensitive, and selective detection paracetamol and provides a promising approach in paracetamol clinical research and overdose diagnostic applications.  相似文献   

20.
We have fabricated a label-free electrochemical immunosensor using microelectrode arrays modified with single-walled carbon nanotubes (SWNTs). Label-free detection of a cancer marker, total prostate-specific antigen (T-PSA), was carried out using differential pulse voltammetry (DPV). The current signals, derived from the oxidation of tyrosine (Tyr), and tryptophan (Trp) residues, increased with the interaction between T-PSA on T-PSA-mAb covalently immobilized on SWNTs. The selectivity of our biosensor was challenged using bovine serum albumin (BSA) as the target protein. The detection limit for T-PSA was determined as 0.25 ng/mL. Since the cut-off limit of T-PSA between prostate hyperplasia and cancer is 4 ng/mL, the performance of our label-free electrochemical immunosensor seems promising for further clinical applications.  相似文献   

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