Amperometric immunosensor based on toluidine blue/nano-Au through electrostatic interaction for determination of carcinoembryonic antigen

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.     

A novel label-free amperometric immunosensor for carcinoembryonic antigen based on redox membrane

A label-free immunosensor was developed to detect the presence of an antigen. This immunosensor was based on the modulation of the electrochemistry of the surface bound redox species K(3)Fe(CN)(6) (FC). The model antigen was carcinoembryonic antigen (CEA) and the model epitope was the antibody of CEA (anti-CEA). Glassy carbon (GC) electrode surfaces were first drop-coated with a mixture of FC and chitosan and air-dried. The electrode surface was then covered with nafion membrane, which contained gold nanoparticles. After binding with polyethyleneimine (PEI), glutaraldehyde (GA) was used to cross-link PEI and anti-CEA. Binding of CEA to the surface bound epitope resulted in attenuation of the FC electrochemistry. Under optimal conditions, the response of the label-free immunosensor had a linear range of 0.01-150 ng mL(-1) with a detection limit of 3 pg mL(-1) (S/N = 3). Its response was better than those of radioimmunoassays, enzyme-linked immunosorbent assays, and chemiluminescence assays.     

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

A sensitive amperometric immunosensor for carcinoembryonic antigen (CEA) was prepared. Firstly, a porous nano-structure gold (NG) film was formed on glassy carbon electrode (GCE) by electrochemical reduction of HAuCl₄ solution, then nano-Au/Chit composite was immobilized onto the electrode because of its excellent membrane-forming ability, and finally the anti-CEA was adsorbed onto the surface of the bilayer gold nanoparticles to construct an anti-CEA/nano-Au/Chit/NG/GCE immunosensor. The characteristics of the modified electrode at different stages of modification were studied by cyclic voltammetry (CV). The gold colloid, chitosan and nano-Au/Chit were characterized by transmission electron microscopy and UV–vis spectroscopy. In addition, the performances of the immunosensor were studied in detail. The resulting immunosensor offers a high-sensitivity (1310 nA/ng/ml) for the detection of CEA and has good correlation for detection of CEA in the range of 0.2 to 120.0 ng/ml with a detection limit of 0.06 ng/ml estimated at a signal-to-noise ratio of 3. The proposed method can detect the CEA through one-step immunoassay and would be valuable for clinical immunoassay.     

Study on immunosensor based on gold nanoparticles/chitosan and MnO<Subscript>2</Subscript> nanoparticles composite membrane/Prussian blue modified gold electrode

A novel and convenient immunosensor, based on the electrostatic adsorption characteristics between the positively charged MnO₂ nanoparticles (nano-MnO₂) and chitosan (CS) composite membrane (nano-MnO₂ + CS) and the negatively charged prussian blue (PB), was prepared for the detection of carcinoembryonic antigen (CEA). Firstly, PB was electro-deposited on the surface of the gold electrode in the constant potential, and then nano-MnO₂ + CS was adsorbed onto PB-modified electrode surface. Subsequently, Gold nanoparticles (nano-Au) were electro-deposited on the nano-MnO₂ + CS-modified electrode to immobilize antibody CEA (anti-CEA). Finally, bovine serum albumin (BSA) was employed to block sites against nonspecific binding. In our study, cyclic voltammetry (CV) and scanning electron microscopy (SEM) were used to characterize the fabricated process of the immunosensor. The immunosensor put up a rapid response time, high sensitivity and stability. Under the optimized conditions, cyclic voltammograms(CVs) determination of CEA displayed a broader linear response to CEA in two ranges, from 0.25 to 8.0 ng/mL, and from 8.0 to 100 ng/mL, with a relative low-detection limit of 0.083 ng/mL at three times the background and noise. The originality of the preparation of the immunosensor lies in not only using the synergistic effect of two kinds of nanomaterials (nano-MnO₂ and nano-Au) to immobilize anti-CEA, but also using nano-MnO₂ + CS to furnish a media transferring electron path. What is more, the researched methodology was efficient and potentially attractive for clinical immunoassays.     

Amperometric immunosensor for carcinoembryonic antigen detection with carbon nanotube-based film decorated with gold nanoclusters

A new amperometric immunosensor for the determination of carcinoembryonic antigen (CEA) was constructed. First, the uniform nanomultilayer film was fabricated via layer-by-layer (LBL) assembly of positively charged carbon nanotubes wrapped by poly(diallyldimethylammonium chloride) and negatively charged poly(sodium-p-styrene-sulfonate), which could provide a high accessible surface area and a biocompatible microenvironment. Subsequently, gold nanoclusters were electrodeposited on the electrode to immobilize anti-CEA. The fabricated process and electrochemical behaviors of the immunosensor were characterized by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and scanning electron microscopy (SEM). Under optimal conditions, the proposed immunosensor could detect CEA in two linear ranges from 0.1 to 2.0 ng mL⁻¹ and from 2.0 to 160.0 ng mL⁻¹, with a detection limit of 0.06 ng mL⁻¹.     

Surface plasmon resonance immunosensor for the detection of Salmonella typhimurium

An immunosensor based on surface plasmon resonance (SPR) using protein G was developed for the detection of Salmonella typhimurium. A protein G layer was fabricated by binding chemically to self-assembly monolayer (SAM) of 11-mercaptoundecanoic acid (MUA) on gold (Au) surface. The formation of protein G layer on Au surface modified with 11-MUA and the binding of antibody and antigen in series were confirmed by SPR spectroscopy. The effect of detergent such as Tween-20 on binding efficiency of antibody and antigen was investigated by SPR. The binding efficiency of antigen to the antibody immobilized on Au surface was improved up to about 85% and 100% by using protein G and Tween-20, respectively. The surface morphology analyses of 11-MUA monolayer on Au substrate, protein G layer on 11-MUA monolayer and antibody layer immobilized on protein G layer were performed by atomic force microscope (AFM). Consequently, an immunosensor based on SPR for the detection of S. typhimurium using protein G was developed with a detection range of 10(2) to 10(9)CFU/ml. The current fabrication technique of a SPR immunosensor for the detection of S. typhimurium could be applied to construct other immnosensors or protein chips.     

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 (HSO(3)-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 HSO(3)-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 12 ng/mL with a low detection limit of 4.86 pg/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.     

Surface plasmon resonance immunosensor using self-assembled protein G for the detection of Salmonella paratyphi

A surface plasmon resonance (SPR) based immunosensor using self-assembled protein G was developed for the detection of Salmonella paratyphi. In order to endow a solid substrate binding affinity to protein G, the free amine (-NH2) of protein G was substituted into thiol (-SH) using 2-iminothiolane. Thus, self-assembled protein G was fabricated on gold (Au) substrate. The formation of protein G layer on Au surface, and the binding of antibody and antigen in series were confirmed by SPR spectroscopy. The surface morphology analysis of the protein G layer on Au surface was performed by atomic force microscope (AFM). Consequently, an immunosensor based on SPR for the detection of S. paratyphi using self-assembled protein G was developed with a detection range of 10(2)-10(7) CFU/ml. The current fabrication technique of a SPR immunosensor for the detection of S. paratyphi could be applied to construct other immnosensors or protein chips.     

Label-free electrochemical immunosensor based on Nile blue A-reduced graphene oxide nanocomposites for carcinoembryonic antigen detection

In this article, a novel, label-free, and inherent electroactive redox immunosensor for carcinoembryonic antigen (CEA) based on gold nanoparticles (AuNPs) and Nile blue A (NB) hybridized electrochemically reduced graphene oxide (NB–ERGO) is proposed. The composite of NB–graphene oxide (NB–GO) was prepared by π–π stacking interaction. Then, chronoamperometry was adopted to simultaneously reduce HAuCl₄ and nanocomposites of NB–GO for synthesizing AuNPs/NB–ERGO. The immunosensor was fabricated by capturing CEA antibody (anti-CEA) at this nanocomposite modified electrode. The immunosensor determination was based on the fact that, due to the formation of antigen–antibody immunocomplex, the decreased response currents of NB were directly proportional to the concentrations of CEA. Under optimal conditions, the linear range of the proposed immunosensor was estimated to be from 0.001 to 40 ng ml⁻¹ and the detection limit was estimated to be 0.00045 ng ml⁻¹. The proposed immunosensor was used to determine CEA in clinical serum samples with satisfactory results. The proposed method may provide promising potential application in clinical immunoassays with the properties of facile procedure, stability, high sensitivity, and selectivity.     

Immunosensor for detection of Legionella pneumophila using surface plasmon resonance

Immunosensor using surface plasmon resonance (SPR) onto self-assembled protein G layer was developed for the detection of Legionella pneumophila. A self-assembled protein G layer on gold (Au) surface was fabricated by adsorbing a mixture of 11-mercaptoundecanoic acid (MUA) and hexanethiol (molar ratio of 1:2) and the activation process for chemical binding between free amine (-NH(2)) of protein G and 11-(MUA) using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC) in series. The formation of self-assembled protein G layer on Au substrate and the binding of antibody and antigen in series were confirmed by SPR spectroscopy. The surface morphology analyses of self-assembled protein G layer on Au substrate and monoclonal antibody against L. pneumophila immobilized on protein G were performed by atomic force microscope (AFM). The immunosensor for detection of L. pneumophila using SPR was developed and its detection limit could find up to 10(5) cells/ml.     

Simultaneous determination of alpha-fetoprotein and carcinoembryonic antigen in human serum by time-resolved fluoroimmunoassay

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.     

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

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

A novel electrochemical immunoassay based on diazotization-coupled functionalized bioconjugates as trace labels for ultrasensitive detection of carcinoembryonic antigen

In this article, a novel sandwich-type electrochemical immunosensor based on the signal amplification strategy of diazotization-coupling concept for ultrasensitive detection of carcinoembryonic antigen (CEA) was reported. It operates through physisorption of monoclonal anti-CEA on 4-aminothiophenol (4Atp) functionalized gold electrode interface as the detection platform. Diazo-4Atp-coupled-thionine (Thi)-conjugated gold nanoparticles (GNPs) were prepared for immobilization of horseradish peroxidase (HRP) and secondary anti-CEA to form core-shell bioconjugates that were used as electrochemical signal amplification reagent. The sensitivity of the immunosensor was greatly amplified by a dual amplification: one is that a large number of thionine and HRP was introduced on the electrode surface through sandwich immunoreaction, the other is that HRP as enhancer could catalyze the oxidation reaction of thionine by H(2)O(2), which results in great enhancement of the reduction peak current. Thus, the bioconjugates-based assay provided an amplification approach for detecting CEA at trace levels and led to a detection limit as low as 0.7 pg/mL (at a three times signal-to-noise ratio) that is well-below the threshold value of 2.5 ng/mL for clinical diagnosis. The assay was evaluated for clinical serum samples with various CEA concentrations and received in excellent accordance with the results obtained from the referenced enzyme-linked immunosorbent assay (ELISA).     

Fabrication of self-assembled protein A monolayer and its application as an immunosensor

The self-assembled layer of modified protein A was fabricated. In order to modify protein A, the surface group of protein A was substituted with thiol (-SH) functionality by using N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) and dithiothreitol (DTT). The formation of a self-assembled protein A layer on a Au substrate and its increased binding capacity to antibody were confirmed by surface plasmon resonance (SPR) spectroscopy. The surface structure of self-assembled protein A layer, and the binding status of anti-bovine serum albumin (anti-BSA) and BSA were determined by atomic force microscopy (AFM). Treatment on the self-assembled protein A layer with a detergent, such as Tween 20, increased the binding capacity of anti-BSA, because protein A aggregation was reduced significantly by the detergent; this was confirmed by SPR spectroscopy. The self-assembled layer of chemically modified protein A with enhanced binding capacity can be used for immunosensor fabrication.     

Radiolocalisation of an anti-CEA monoclonal antibody (FO23C5) and its fragments in a colon carcinoma xenograft model

A new monoclonal antibody designated FO23C5 against a protein component of carcinoembryonic antigen (CEA) has been developed. A xenograft system of human colon cancer was used to compare the intact monoclonal IgG with its fragments (Fab')2 and Fab) and with an established anti-CEA antibody (MAb35) and the antibody AUA1 raised against the colon carcinoma cell line. We demonstrate that FO23C5 compares well with the existing anti-CEA antibody and with AUA1, and that F(ab')2 fragments perform best in achieving optimal tumour to normal tissue ratios compared with intact IgG and Fab fragment.     

A novel multi-array immunoassay device for tumor markers based on insert-plug model of piezoelectric immunosensor

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.     

A highly sensitive method for the demonstration of carcinoembryonic antigen in normal and neoplastic colonic tissue

Summary A highly sensitive method for the immuno-histochemical localisation of carcinoembryonic antigen (CEA) is described. This method is based on the binding of a peroxidase-antiperoxidase complex (PAP) to anti-CEA antibodies by means of an anti-gamma-globulin which reacts with both the anti-CEA and the antiperoxidase antibodies. Using the technique described here, CEA was localised in conventionally processed normal and cancerous colonic tissue. In normal as well as in neoplastic tissues, a CEA-specific staining of cell membranes and cytoplasm was demonstrated. In frozen sections of normal colonic tissue CEA was found even at high dilutions of the first antibody; this indicates the high sensitivity of the method. The applicability of the method to conventionally processed and thereby well preserved tissue specimens opens the possibility to identify CEA by light microscopy even at very low concentrations.     

Electrochemical DNA base pairs quantification and endonuclease cleavage detection

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.     

Comparison of CEA distribution in lesions and tumors of salivary glands as determined with monoclonal and polyclonal antibodies

Immunohistochemical localization of carcinoembryonic antigen (CEA) with conventional antibody to CEA (anti-CEA), nonspecific crossreacting antigen (NCA)-absorbed polyclonal antibody to CEA (NCAa-CEA), and monoclonal antibody to CEA (Mono-CEA) have been compared in obstructive lesions and salivary gland tumors. Normal salivary glands gave strong staining of the luminal borders of acinar cells with anti-CEA, whereas no staining occurred with Mono-CEA. Obstructive lesions showed occasionally marked staining with anti-CEA in some acinar cells, but there was no reaction with Mono-CEA. Of 69 pleomorphic adenomas examined, 34 were positively stained with anti-CEA, 18 with NCAa-CEA and 8 with Mono-CEA along the luminal borders of the tumor cells. The frequency of positive staining of material within tubular lumina was similar with all three immunoreagents. Neoplastic cells were positive with Mono-CEA in only three cases, while eight cases were positive with NCAa-CEA and 11 cases with anti-CEA. In salivary gland tumors true CEA is be found mainly at the border of tumor cells, but the frequency of positive reactions is low.