Electrochemical Immunoassay Of Carcinoembryonic Antigen Research Articles

A bimetallic metal-organic framework with high enzyme-mimicking activity for an integrated electrochemical immunoassay of carcinoembryonic antigen.

Metal-organic frameworks (MOFs) show excellent catalytic activity and have been widely applied in diagnosis of diseases and tumors. However, current assay methods usually involve cumbersome configurations and complicated procedures, which inhibit their practical applications. Hence, a Cu-Ni MOF/carbon printed electrode (CPE)-based integrated electrochemical immunosensor was constructed for highly sensitive and efficient determination of carcinoembryonic antigen (CEA). First, highly conductive carbon ink was screen-printed onto a polyethylene terephthalate substrate to manufacture a CPE. Afterward, an aminated Cu-Ni MOF was prepared by a typical solvothermal strategy and modified on the CPE. Owing to its excellent peroxidase activity, the Cu-Ni MOF can catalytically oxidize hydroquinone using hydrogen peroxide, which greatly amplifies the peak current signal. Then the formation of an immune complex inhibited the catalytic activity of the MOF, thus enabling the quantitative determination of CEA content with a wide linear range of 0.5 pg mL-1-500 ng mL-1 and a low detection limit of 0.16 pg mL-1. Furthermore, the Cu-Ni MOF/CPE-based integrated portable electrochemical immunosensor also showed satisfactory performance in the detection of CEA in clinical serum samples with excellent accuracy, showing great potential for application in point-of-care disease diagnosis.

An immunosensor detects carcinoembryonic antigen by a double reduction strategy based on polyphenylamine as a sacrifice reducing agent

In this paper, a novel Ag NPs-PANI/MnO2 tag was synthesized for the first time, and the electrochemical immunoassay for carcinoembryonic antigen (CEA) was achieved. In this experiment, gold nanoparticles (Au NPs) were used as an electrochemical sensing platform to increase conductivity and improve electrode surface capture antibody (Ab1). We use PANI as a green sacrificial agent reduces Ag NPs and MnO2, while improving the conductivity and electrochemical stability of the immunosensor. MnO2 and Ag NPs effectively raise the sensitivity of the sensor by catalytically reducing hydrogen peroxide (H2O2) into water and oxygen to achieve a double amplified sensing signal. It is found that the surface plasmon resonance of Ag NPs has an interesting SERS characteristic structure, and the equivalent circuit simulation of the electrode assembly process results in an error of less than 10%, indicating that the electrochemical immunosensor is successfully prepared. Under optimal conditions, the electrochemical immunosensor has a linear range of 0.0005–80 ng/mL and a detection limit of 0.17 pg/mL (S/N = 3). The immunosensor is expected to be used in clinical laboratories to detect CEA and other tumor markers in human plasma for precancerous screening or surveillance.

A novel electrochemical immunoassay for carcinoembryonic antigen based on glucose oxidase-encapsulated nanogold hollow spheres with a pH meter readout.

A portable electrochemical immunosensing protocol was designed for the sensitive detection of a disease-related tumor biomarker (carcinoembryonic antigen, CEA, used in this case) on a pH meter using glucose oxidase (GOx)-encapsulated gold hollow microspheres (AuHMs) for signal amplification. The assay was carried out on a monoclonal anti-CEA capture antibody-coated microplate with a sandwich-type reaction mode. The GOx-entrapped AuHM was first synthesized using the reverse micelle method and then used as the signal-generation tag for the labeling of polyclonal anti-CEA detection antibody. Accompanying the formation of the sandwiched immunocomplexes, the loaded GOx molecules in the microsphere could catalyze glucose into gluconic acid and hydrogen peroxide. The as-produced gluconic acid changed the microenvironment of the detection solution, thus resulting in the shift of the pH value, which could be quantitatively determined on a portable pH meter. The use of gold hollow microspheres was expected to enhance the loaded amount of GOx for signal amplification. Two labeling protocols including GOx-labeled secondary antibody and GOx-AuHM-labeled secondary antibody were investigated for CEA detection, and improved analytical features were acquired with GOx-AuHM labeling. With the GOx-AuHM labeling strategy, the pH meter-based immunosensing device exhibited a good analytical performance for CEA detection within the dynamic linear range of 0.1-100 ng mL-1 at a detection limit of 0.062 ng mL-1. The strong attachment of anti-CEA antibodies to GOx-AuHM brought a good repeatability and intermediate precision down to 10%. Importantly, no significant differences at the 0.05 significance level were encountered in the analysis of 12 human serum specimens between the developed immunoassay and the commercialized electrochemiluminescent method for CEA determination.

Robust enzyme-free electrochemical immunoassay of CEA enhanced by porous PdCu nanoparticles

The interferences of dissolved oxygen towards the electrochemical immunosensors occurred constantly, which will restrict the practical clinical application of electrochemical immunoassay. Herein, a new enzyme-free strategy has been proposed for ultrasensitive electrochemical immunoassay of carcinoembryonic antigen (CEA), which can completely avoid the interference of dissolved oxygen. Porous PdCu nanoparticles can catalyze aniline to produce polyaniline by the inherited peroxidase-like activity for generating electrochemical detection signal. More interesting, with the assistance of electropolymerization strategy, the electrochemical measurement signal could enhance sharply. Accordingly, we established a sandwich-type electrochemical immunosensor based on porous PdCu nanoparticles produced polyaniline with electropolymerization strategy for enzyme-free and dissolved oxygen interference-free detection of CEA, affording a satisfied performance with a broad linear detection range (0.1pg/mL∼10ng/mL) and low detection limit (0.08pg/mL). This method might be considered as a robust approach for clinical applications.

Three-dimensional gold nanoparticles/prussian blue-poly(3,4-ethylenedioxythiophene) nanocomposite as novel redox matrix for label-free electrochemical immunoassay of carcinoembryonic antigen

We present a novel label-free electrochemical immunosensor for detecting carcinoembryonic antigen (CEA) based on three-dimensional (3D) gold nanoparticles/prussian blue-poly(3,4-ethylenedioxythiophene) (AuNPs/PB-PEDOT) nanocomposite, which was firstly synthesized by a simple redox reaction between the PB precursors and EDOT in an aqueous solution, followed by the electrochemical reduction of HAuCl4. The 3D nanocomposite not only possessed large surface area and favorable microenvironment, but also exhibited remarkable conductivity, stability and excellent biocompatibility. In addition, PB showed excellent redox properties. Then AuNPs/PB-PEDOT was used as both electron mediators and 3D matrices in the fabrication of immunosensor. FT-IR spectra were employed to confirm the formation of PB, PEDOT and PB-PEDOT. Significantly, the AuNPs/PB-PEDOT exhibited a 3D and hierarchically porous nanostructure, while the PB-PEDOT showed core-shell structure. The AuNPs/PB-PEDOT modified immunosensor showed good linearity with the concentration of CEA ranging from 0.05 to 40ngmL−1, and the detection limit was 0.01ngmL−1. Moreover, the prepared electrode displayed good selectivity, high stability and good repeatability, and showed great potential for application in real sample analysis.

Ultrasensitive electrochemical immunoassay for carcinoembryonic antigen based on three-dimensional macroporous gold nanoparticles/graphene composite platform and multienzyme functionalized nanoporous silver label

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 10ngmL−1 with low detection limit of 0.35pgmL−1 and low limit of quantitation (LOQ) of 0.85pgmL−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

Electrochemical immunoassay for carcinoembryonic antigen based on signal amplification strategy of nanotubular mesoporous PdCu alloy

Interests in using nanoporous metals for biosensing applications have been increasing. Herein, nanotubular mesoporous PdCu (NM-PdCu) alloy is used to fabricate a novel label-free electrochemical immunosensor for cancer biomarker carcinoembryonic antigen (CEA). It operates through physisorption of anti-CEA on NM-PdCu and the mixture of sulfonated graphene sheets (HSO3-GS) and thionine (TH) functionalized glassy carbon electrode interface as the detection platform. In this study, chitosan (CS)-PdCu is bound very strongly to carcinoembryonic antibody (anti-CEA), because of the good electron conductivity, high surface area, and good biocompatibility. CS-PdCu is immobilized on electrodes by electrostatic interactions between the negatively charged sulfo group of HSO3-GS and the abundant positively charged amino groups of chitosan. TH acts as the redox probe. Under the optimized conditions, the electrochemical immunosensor exhibits a wide working range from 0.01 to 12ng/mL with a low detection limit of 4.86pg/mL. The accuracy, reproducibility, and stability of the immunosensor are acceptable. The assay is evaluated for real serum samples, receiving satisfactory results. The nanoporous metal materials-based immunoassay provides a promising approach in clinical application and thus represents a versatile detection method.

Electrochemical immunosensor for carcinoembryonic antigen based on nanosilver-coated magnetic beads and gold-graphene nanolabels

A novel redox-active magnetic nanostructure was synthesized by using a wet chemical method for high-efficiency electrochemical immunoassay of carcinoembryonic antigen (CEA, as a model analyte). The nanostructures based on the combination of a magnetic nanocore, a layer of electroactive poly(o-phenylenediamine) (PPD), and a silver metallic shell displayed good adsorption properties for the attachment of anti-CEA antibody selective to CEA. The magnetic nanostructure presented good redox behaviors to facilitate and modulate the way it was integrated into a magnetic carbon paste electrode. The assay was based on a sandwich-type immunoassay protocol by using nanogold-patterned graphene oxide nanoscales (AuNP-GO), conjugated with horseradish peroxidase-labeled anti-CEA, as secondary antibodies and biofunctionalized magnetic nanostructures as immunosensing probes. Under optimal conditions, the nanoparticle-based immunocomposites exhibited good electrochemical responses for the determination of CEA, and allowed the detection of CEA at a concentration as low as 1.0pgmL−1 at a signal-to-noise ratio of 3. In addition, the magnetic immunosensing had good reproducibility, and acceptable accuracy, and could be successfully applied for the detection of CEA in the clinical serum specimens. Significantly, by controlling the target biomolecules, this assay can be easily extended for use with other immunosensings, and thus represents a versatile design routine.

Biofunctionalized dendritic polyaniline nanofibers for sensitive electrochemical immunoassay of biomarkers

Multi-armed dendritic polyaniline nanofibers (MPANFs) were first synthesized and functionalized with horseradish peroxidase (HRP) and carcinoembryonic antibody (anti-CEA) for highly efficient electrochemical immunoassay of carcinoembryonic antigen (CEA, as a model analyte here) in this work. Transmission electron microscope (TEM) and scanning electron microscope (SEM) techniques were employed to characterize the synthesized MPANFs. By using anti-CEA-conjugated core-shell gold-Fe(3)O(4) nanocomposites (GoldMag) as immunosensing probes and biofunctionalized MPANFs as molecular tags, a new sandwich-type homogeneous immunoassay strategy was developed for the determination of CEA by coupling with a home-made flow-through magneto-controlled microfluidic device. Under optimal conditions, the electrochemical immunoassay exhibited a wide dynamic range of four orders of magnitude from 1.0 pg mL(-1) to 50 ng mL(-1) with a low detection limit of 0.1 pg mL(-1) CEA at 3σ. Intra- and inter-assay coefficients of variation were below 10%. The assayed results for clinical serum specimens with the electrochemical immunoassay were received in good accordance with the results obtained from the referenced enzyme-linked immunosorbent assay (ELISA) method.

A Sandwich Electrochemical Immunosensor Using Magnetic DNA Nanoprobes for Carcinoembryonic Antigen

A novel magnetic nanoparticle-based electrochemical immunoassay of carcinoembryonic antigen (CEA) was designed as a model using CEA antibody-functionalized magnetic beads [DNA/Fe3O4/ZrO2; Fe3O4 (core)/ZrO2 (shell) nano particles (ZMPs)] as immunosensing probes. To design the immunoassay, the CEA antibody and O-phenylenediamine (OPD) were initially immobilized on a chitosan/nano gold composite membrane on a glassy carbon electrode (GCE/CS-nano Au), which was used for CEA recognition. Then, horseradish peroxidase (HRP)-labeled anti-CEA antibodies (HRP-CEA Ab2) were bound to the surface of the synthesized magnetic ZMP nanoparticles as signal tag. Thus, the sandwich-type immune complex could be formed between secondary antibody (Ab2) modified DNA/ZMPs nanochains tagged by HRP and GCE/CS-nano Au. Unlike conventional nanoparticle-based electrochemical immunoassays, the recognition elements of this immunoassay included both electron mediators and enzyme labels, which obviously simplifies the electrochemical measurement process. The sandwich-type immunoassay format was used for online formation of the immunocomplex of CEA captured in the detection cell with an external magnet. The electrochemical signals derived from HRP during the reduction of H2O2 with OPD as electron mediator were measured. The method displayed a high sensitivity for CEA detection in the range of 0.008–200 ng/mL, with a detection limit of 5 pg/mL (estimated at a signal-to-noise ratio of 3). The precision, reproducibility, and stability of the immunoassay were good. The use of the assay was evaluated with clinical serum samples, and the results were in excellent accordance with those obtained using the standard enzyme-linked immunosorbent assay (ELISA) method. Thus, the magnetic nanoparticle-based assay format is a promising approach for clinical applications, and it could be further developed for the detection of other biomarkers in cancer diagnosis.

Carbon nanotube-based symbiotic coaxial nanocables with nanosilica and nanogold particles as labels for electrochemical immunoassay of carcinoembryonic antigen in biological fluids

A feasible and practicable amperometric immunoassay strategy for sensitive screening of carcinoembryonic antigen (CEA) in human serum was developed using carbon nanotube (CNT)-based symbiotic coaxial nanocables as labels. To construct such a nanocable, a thin layer of silica nanoparticles was coated on the CNT surface by sonication and sol–gel methods, and then colloidal gold nanoparticles were assembled on the amino-functionalized SiO2/CNTs, which were used for the label of horseradish peroxidase-anti-CEA conjugates (HRP-anti-CEA-Au/SiO2/CNT). In the presence of analyte CEA, the sandwich-type immunocomplex was formed on an anti-CEA/Au/thionine/Nafion-modified glassy carbon electrode by using HRP-anti-CEA-Au/SiO2/CNTs as detection antibodies. To embody the advantages of the protocol, the analytical properties of variously modified electrodes were compared in detail on the basis of different nanolabels. Under optimal conditions, the cathodic peak currents of the electrochemical immunosensor were proportional to the logarithm of CEA concentration over the range from 0.01 to 12ngmL−1 in pH 5.5 HAc-NaAc containing 5mM H2O2. At a signal-to-noise ratio of 3, the detection limit (LOD) is 5pgmL−1 CEA. Intra- and inter-assay coefficients of variation were below 9.5%. Meanwhile, the selectivity and stability of the immunosensor were acceptable. In addition, the technique was evaluated by spiking CEA standards in pH 7.4 PBS and with 35 clinical serum specimens, receiving excellent accordance with results from commercially available electrochemiluminescent enzyme-linked immunoassay.

Graphene-Prussian blue/gold nanoparticles based electrochemical immunoassay of carcinoembryonic antigen

A new electrochemical immunosensor for the determination of carcinoembryonic antigen (CEA) was developed with a Graphene (Gr)-Prussian blue (PB)/gold nanoparticles (GNP) immobilization platform. HRP-anti-CEA was immobilized on the platform and with the immunoreactions, the formed immunocomplex inhibited partly the bioactive center of the immobilized HRP, and decreased the HRP toward the reduction of H2O2, which was the base of the detection of CEA. The Gr-PB/GNP can be used not only as the electrochemical redox mediator but also to enhance the sensitivity. Under optimal experimental conditions, the proposed immunosensor for CEA was observed with an ultra low limit of detection equal to 3 pg mL−1 (at 3σ), and the linear working range spanned from 0.01 to 1.0 and 1.0 to 60 ng mL−1. Moreover, the immunosensor was examined for use in the determination of CEA in real human serum specimens. The assayed results of serum samples with the sensor received an acceptable agreement with the reference values. Importantly, the methodology provides a promising ultra sensitive assay strategy for clinical applications.

Nanogold-actuated biomimetic peroxidase for sensitized electrochemical immunoassay of carcinoembryonic antigen in human serum

A new electrochemical immunoassay protocol for the determination of carcinoembryonic antigen (CEA) in human serum was developed by means of immobilizing horseradish peroxidase-labeled anti-CEA antibodies (HRP- anti-CEA) on the nanogold/nano-Fe 3O 4 particles-functionalized polyion complex (PIC) membrane. With a one-step immunoassay format, the formed immunocomplex inhibited partly the bioactive center of the immobilized HRP, and decreased the HRP toward the reduction of H 2O 2. Nanogold-actuated nano-Fe 3O 4 mimetic peroxidase was used for the sensitivity improvement of the electrochemical immunosensor with Prussian blue (PB) as mediators. Under optimal conditions, the electrochemical immunosensor displayed a wide dynamic range of 0.1–220 ng mL −1 with a relative low detection limit (LOD) of 10 pg mL −1 CEA (at 3 σ). Intra- and inter-assay show good precisions with a coefficient of variation (CV) lower than 7.8% and 7.3%, respectively. No significant difference at the 95% confidence level was encountered in the analysis of 17 human serum specimens between the electrochemical immunosensor and automatic electrochemiluminescent (ECL) analyzer for the determination of CEA.

Label-free electrochemical immunoassay of carcinoembryonic antigen in human serum using magnetic nanorods as sensing probes

A new potentiometric immunoassay protocol for the determination of carcinoembryonic antigen (CEA) in human serum was designed by using a novel transparent immunoaffinity reactor. To construct such an affinity reactor, Fe3O4 nanorods with average diameter of 40 nm and length of 1.0 μm were initially synthesized through hydrolysis of FeCl3 and FeSO4 in the urea solution, and then the synthesized nanorods were used as the immobilized affinity support for the conjugation of anti-CEA antibodies (bionanorods). With an external magnet, the magnetic bionanorods were attached to the surface of a self-made carbon paste electrode. The performance and factors influencing the immunosensors were evaluated. Under optimal conditions, the binding of CEA in the samples onto the immobilized anti-CEA changed the potential response, a dynamic range from 1.5 to 80 ng·mL−1, and a detection limit of 0.9 ng·mL−1 CEA (at 3σ). The immunosensors show good precision, high sensitivity, acceptable stability and reproducibility. The CEA concentrations of the clinical serum specimens assayed by the immunosensor show consistent results in comparison with those obtained by commercially available enzyme-linked immunosorbent assay.

Electrochemical immunoassay of carcinoembryonic antigen based on a lead sulfidenanoparticle label

We describe a lead sulfide nanoparticle (PbS NP)-based electrochemical immunoassay todetect a tumor biomarker, carcinoembryonic antigen (CEA). Cubic PbS NPswere prepared and functionalized with thioglycolic acid (TGA), which stabilizedthe formed NPs and offered carboxyl groups to conjugate with CEA antibodies.PbS NP conjugated with monoclonal CEA antibody was used as a label in animmunorecognition event. After a complete sandwich immunoreaction among the primaryCEA antibody (immobilized on the carboxyl-modified magnetic beads), CEA and thePbS-labeled secondary antibody (PbS-anti-CEA), PbS labels were captured to themagnetic-bead (MB) surface through the antibody–antigen immunocomplex.Electrochemical stripping analysis of the captured PbS was used to quantify theconcentration of CEA after an acid-dissolution step. The MBs and the magneticseparation platform were used to integrate a facile antibody immobilization withimmunoreactions and the isolation of immunocomplexes from reaction solutions inthe immunoassay. The voltammetric response is highly linear over the range of1–50 ng ml−1 CEA, and the limit of detection is estimated to be0.5 ng ml−1. The performance of this nanoparticle-based electrochemical immunoassay was successfullyevaluated with human serum spiked with CEA, indicating that this convenient andsensitive technique offers great promise for rapid, simple and cost-effective analysis oftumor biomarkers in biological fluids.

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

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