A doubly amplified electrochemical immunoassay for carcinoembryonic antigen

An ultrasensitive electrochemical immunoassay (EIA) for the detection of carcinoembryonic antigen (CEA) is described in this report. The assay involves utilizing enzyme-catalyzed deposition of a redox polymer and electrocatalytic oxidation of ascorbic acid (AA) by the deposited redox polymer, a dual-amplification scheme to enhance analytical signals. Briefly, CEA capturing antibody and redox polymer anchoring agent were covalently immobilized on a gold electrode. After incubating with CEA, the electrode was treated in detection antibody-glucose oxidase conjugate solution. Thereafter, it was dipped into the redox polymer solution. Upon the addition of glucose, the redox polymer was enzymatically reduced and deposited on the electrode surface. The deposited redox polymer exhibits excellent electrocatalytic activity towards the oxidation of AA. Consequently, CEA could be quantified amperometrically. This electrochemical immunoassay combines the specificity of the immunological reaction with the sensitivity of the doubly amplified electrochemical detection.     

Highly amplified electrochemiluminescence of peroxydisulfate using bienzyme functionalized palladium nanoparticles as labels for ultrasensitive immunoassay

An immunosensor based on the electrochemiluminescence (ECL) of peroxydisulfate was firstly proposed by coupling the cooperation of two enzymes to in situ generate coreactant with palladium nanoparticles (PdNPs) as catalyst for the ECL reaction. PdNPs were previously synthesized, which successfully attached to functional carbon nanotubes (FCNTs), to bind the secondary antibody and bienzyme (horseradish peroxidase and glucose oxidase). Then the prepared bioconjugates were introduced to the electrode via sandwich immunoreactions. Accordingly, a dramatically amplified ECL signal was obtained for that GOD catalyzed glucose to produce H(2)O(2) which was subsequently reduced by HRP to in situ generate O(2), then PdNPs as catalyst for the ECL reaction of peroxydisulfate/O(2). The present immunosensor was used to detect α-1-fetoprotein (AFP) and showed a wide linear range of 1×10(-5)-100ngmL(-1), with a low detection limit of 3.3fgmL(-1)(S/N=3). This new signal amplification strategy for preparation of the ECL immunosensor could be easily realized and has a potential application in ultrasensitive bioassays.     

A disposable electrochemical immunosensor for flow injection immunoassay of carcinoembryonic antigen

A new simple immunoassay method for carcinoembryonic antigen (CEA) detection using a disposable immunosensor coupled with a flow injection system was developed. The immunosensor was prepared by coating CEA/colloid Au/chitosan membrane at a screen-printed carbon electrode (SPCE). Using a competitive immunoassay format, the immunosensor inserted in the flow system with an injection of sample and horseradish peroxidase (HRP)-labeled CEA antibody was used to trap the labeled antibody at room temperature for 35 min. The current response obtained from the labeled HRP to thionine-H(2)O(2) system decreased proportionally to the CEA concentration in the range of 0.50-25 ng/ml with a correlation coefficient of 0.9981 and a detection limit of 0.22 ng/ml (S/N=3). The immunoassay system could automatically control the incubation, washing and current measurement steps with good stability and acceptable accuracy. Thus, the proposed method proved its potential use in clinical immunoassay of CEA.     

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.     

A novel impedimetric aptasensor,based on functionalized carbon nanotubes and prussian blue as labels

A simple and feasible electrochemical sensing protocol was developed for the detection of bisphenol A (BPA) by employing the gold nanoparticles (AuNPs), prussian blue (PB) and functionalized carbon nanotubes (AuNPs/PB/CNTs-COOH). An aminated complementary DNA as a capture probe and specific aptamer against BPA as a detection probe was immobilized on the surface of a modified glassy carbon (GC) electrode via the formation of covalent amide bond and hybridization, respectively. The proposed nanoaptasensor combined the advantages of the in situ formation of PB as a label, the deposition of neatly arranged AuNPs, and the covalent attachment of the capture probe to the surface of the modified electrode. Upon addition of target BPA, the analyte reacted with the aptamer and caused the steric/conformational restrictions on the sensing interface. The formation of BPA–aptamer complex at the electrode surface retarded the interfacial electron transfer reaction of the PB as a probe. Sensitive quantitative detection of BPA was carried out based on the variation of electron transfer resistance which relevant to the formation of BPA– aptamer complex at the modified electrode surface. Under the optimized conditions, the proposed aptasensor exhibited a high sensitivity, wide linearity to BPA and low detection limit. This aptasensor also displayed a satisfying electrochemical performance with good stability, selectivity and reproducibility.     

Ag(I)-cysteamine complex based electrochemical stripping immunoassay: ultrasensitive human IgG detection

An ultrasensitive electrochemical immunosensor for a protein using a Ag (I)-cysteamine complex (Ag-Cys) as a label was fabricated. The low detection of a protein was based on the electrochemical stripping of Ag from the adsorbed Ag-Cys complex on the gold nanoparticles (AuNPs) conjugated human immunoglobulin G (anti-IgG) antibody (AuNPs-anti-IgG). The electrochemical immunosensor was fabricated by immobilizing anti-IgG antibody on a poly-5,2':5',2'-terthiophene-3'-carboxylic acid (polyTTCA) film grown on the glassy carbon electrode through the covalent bond formation between amine groups of anti-IgG and carboxylic acid groups of polyTTCA. The target protein, IgG was sandwiched between the anti-IgG antibody that covalently attached onto the polyTTCA layer and AuNPs-anti-IgG. Using square wave voltammetry, well defined Ag stripping voltammograms were obtained for the each target concentration. Various experimental parameters were optimized and interference effects from other proteins were checked out. The immunosensor exhibited a wide dynamic range with the detection limit of 0.4 ± 0.05 fg/mL. To evaluate the analytical reliability, the proposed immunosensor was applied to human IgG spiked serum samples and acceptable results were obtained indicating that the method can be readily extended to other bioaffinity assays of clinical or environmental significance.     

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.     

Strategically functionalized carbon nanotubes as the ultrasensitive electrochemical probe for picomolar detection of sildenafil citrate (Viagra)

The present work demonstrates the utility of the functionalized carbon nanotubes, poly(4-aminobenzene sulfonic acid) (PABS) grafted multiwalled carbon nanotubes, MWNT-g-PABS, as an electrode modifier towards achieving ultrasensitive detection of a model drug, sildenafil citrate (SC). PABS units in MWNT-g-PABS interact with SC, pre-concentrate and accumulate at the surface. The electron transduction from SC to electrode is augmented via MWNT-g-PABS. As a result, the MWNT-g-PABS modified electrode exhibited ultrasensitive (57.7 μA/nM) and selective detection of SC with a detection limit of 4.7 pM. The present work provides scope towards targeting ultrasensitivity for the detection of biomolecules/drug through rational design and incorporation of appropriate chemical components to carbon nanotubes.     

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.     

Horseradish peroxidase functionalized gold nanorods as a label for sensitive electrochemical detection of alpha-fetoprotein antigen

In this study, a novel tracer, horseradish peroxidase (HRP) functionalized gold nanorods (Au NRs) nanocomposites (HRP–Au NRs), was designed to label the signal antibodies for sensitive electrochemical measurement of alpha-fetoprotein (AFP). The preparation of HRP–Au NRs nanocomposites and the labeling of secondary antibody (Ab₂) were performed by one-pot assembly of HRP and Ab₂ on the surface of Au NRs. The immunosensor was fabricated by assembling carbon nanotubes (CNTs), Au NRs, and capture antibodies (Ab₁) on the glassy carbon electrode. In the presence of AFP antigen, the labels were captured on the surface of the Au NRs/CNTs via specific recognition of antigen–antibody, resulting in the signal intensity being clearly increased. Differential pulse voltammetry (DPV) was employed to record the response signal of the immunosensor in phosphate-buffered saline (PBS) containing hydrogen peroxide (H₂O₂) and 3,3′,5,5′-tetramethylbenzidine (TMB). Under optimal conditions, the signal intensity was linearly related to the concentration of AFP in the range of 0.1–100 ng ml⁻¹, and the limit of detection was 30 pg ml⁻¹ (at signal/noise [S/N] = 3). Furthermore, the immunoassay method was evaluated using human serum samples, and the recovery obtained was within 99.0 and 102.7%, indicating that the immunosensor has potential clinical applications.     

A novel microfluidic immunoassay system based on electrochemical immunosensors: an application for the detection of NT-proBNP in whole blood

Electrochemical immunosensors have attracted great interest in the search for a selective, simple and reliable system for molecular recognition. Presently, electrochemical immunosensors have been widely studied for biomedical molecular's detection, but the regeneration of these immunosensors has restricted their wide application. To prepare a regeneration-free immunosensor, which may be more suitable for clinical determination, a repeatable immunoassay system was developed based on an electrochemical immunosensor with magnetic nanoparticles, biotin-avidin system (BAS) and Fab antibodies for the heart failure markers aminoterminal pro-brain natriuretic peptides (NT-proBNP). At the same time, a microfluidic system was combined into the proposed system, which enabled continuous determination. Using NT-proBNP as a model system, the proposed immunosensor exhibited rapid and sensitive amperometric response to NT-proBNP with good selectivity, stability, and a wide linear range (0.005-1.67 ng/mL and 1.67-4 ng/mL with a detection limit of 0.003 ng/mL under optimal conditions). Importantly, the proposed immunosensor was also suitable for the detection of other proteins and provided new opportunities for disease diagnosis.     

Cu@Au alloy nanoparticle as oligonucleotides labels for electrochemical stripping detection of DNA hybridization

Synthesis of the novel Cu@Au alloy nanoparticle and its application in an electrochemical DNA hybridization detection assay is described in this article. We report a low-temperature method for generating core-shell particles consisting of a core of Cu and a thin layer of Au shell that can be readily functionalized with oligonucleotides. Core-shell Cu@Au particles were successfully labeled to a 5'-alkanethiol capped oligonucleotides probe that is related to the colitoxin gene. The DNA genetic sensing assay relies on the electrostatic adsorption of target oligonucleotides onto conducting polypyrrole (PPy) surface at the glassy carbon electrode (GCE), and its hybridization to the alloy particle-oligonucleotides DNA probe. Hybridization events between probe and target were monitored by the release of the copper metal atoms anchored on the hybrids by oxidative metal dissolution and the indirectly determination of the solubilized Cu2+ ions by sensitive anodic stripping voltammetry (ASV). The detection limit is 5.0 pmol l(-1) of target oligonucleotides. The Cu@Au core-shell nanoparticles combining the surface modification properties of Au with the good electrochemical activity of Cu core shows their perspective application in the electrochemical DNA hybridization analysis assay.     

An enhanced chemiluminescent enzyme immunoassay for serum carcinoembryonic antigen based on a modification of a commercial kit

A conventional colorimetric peroxidase end-point (ortho-phenylenediamine substrate), used in an enzyme immunoassay for carcinoembryonic antigen, employing plastic beads as solid support, has been replaced by a much faster (30 seconds versus 30 minutes) enhanced chemiluminescent assay for the peroxidase label. Para-iodophenol was used to enhance the light emission from the peroxidase catalysed chemiluminescent reaction between luminol and hydrogen peroxide. Values for precision and carcinoembryonic antigen concentration obtained with the chemiluminescent and colorimetric versions of the immunoassay on 62 serum specimens were in good agreement.     

Study on an amperometric immunosensor based on Nafion-cysteine composite membrane for detection of carcinoembryonic antigen

In this work, protonated l-cysteine was entrapped in Nafion (Nf) membrane by cation exchange function, forming Nf-Cys (cysteine) composite membrane, which was more stable, compact, biocompatible, and favorable for mass and electron transfer compared with Nf film solely. Then gold (Au) nanoparticles were adsorbed onto the electrode surface by thiol groups on the composite membrane. After that, nano-Au monolayer was formed, onto which carcinoembryonic antibody was loaded to prepare carcinoembryonic antigen (CEA) immunosensor. The results indicated that the immunosensor had good current response for CEA using potassium ferricyanide as the redox probe. A linear concentration range of 0.01 to 100 ng/ml with a detection limit of 3.3 pg/ml (signal/noise = 3) was observed. Moreover, the morphology of the modified Au substrates was investigated with atomic force microscopy, and the electrochemical properties and performance of modified electrodes were investigated by cyclic voltammograms and electrochemical impendence spectroscopy. The results exhibited that the immunosensor has advantages of simple preparation, high sensitivity, good stability, and long life expectancy. Thus, the method can be used for CEA analysis.     

A simple electrochemical aptasensor for ultrasensitive protein detection using cyclic target-induced primer extension

A simple electrochemical aptasensor was developed for ultrasensitive protein detection by combining a novel strategy of cyclic target-induced primer extension (CTIPE) with an aptamer-hairpin probe and enzyme-amplified electrochemical readout. In the presence of protein target, the immobilized aptamer-hairpin probe recognized the protein to trigger primer extension reaction by target-induced conformational transition, which released the protein from replicated DNA duplex. The released target could cyclically bind with other aptamer-hairpin probes and trigger new primer extension, leading to formation of numerous biotin-tagged DNA duplex, which significantly amplified the protein recognition event and facilitated the subsequent enzymatic signal enhancement, leading to an ultrasensitive electrochemical aptasensor. Using human vascular endothelial growth factor as a model protein, the designed aptasensor could detect protein down to 0.82 pg mL(-1) with a linear range from 1 pg mL(-1) to 1 ng mL(-1). The proposed aptasensor was amenable to quantification of protein in complex biological matrixes, and would become a simple and powerful tool for bioanalysis and clinic diagnostic application.     

Visual detection of carcinoembryonic antigen on surfaces.

An attempt is made to detect carcinoembryonic antigen (CEA) at clinically interesting concentrations by using a simple immunologic surface test. Antibodies to CEA are detected in a direct test at concentrations below 1 ng/ml. The sensitivity of this assay is mainly limited by diffusion to the reacting surface. CEA is detected in an inhibition test at concentrations down to 20 ng/ml. The sensitivity of this inhibition test is limited by the average equilibrium constant in solution of the antibody-antigen reaction.     

Novel immunoassay for carcinoembryonic antigen based on protein A-conjugated immunosensor chip by surface plasmon resonance and cyclic voltammetry

In this study, an immunosensor chip utilizing surface plasmon resonance (SPR) and cyclic voltammetry (CV) was fabricated for detecting carcinoembryonic antigen (CEA). Specifically, we applied in parallel an SPR instrument and a CV device to monitor the assembly of carcinoembryonic antibody (anti-CEA) on a protein A-conjugated surface and the subsequent ligand reaction. The immunosensor chips were constructed by various concentrations of protein A. To determine the surface characteristics of different self-assembly monolayers (SAMs), several quantitative and kinetic measurements were carried out. The extent of immobilization of anti-CEA and the immune response of anti-CEA antibody against CEA were measured using the SPR instrument and CV device. The terminal functional groups of protein A have different effects on the adsorption and covalent binding of immunoprotein depending on the steric hindrance. Through the parallel measurements, we demonstrate that SPR and CV are sensitive to measure the antigen–antibody binding capacity.     

Bimetallic AuPt nanochains: Synthesis and their application in electrochemical immunosensor for the detection of carcinoembryonic antigen

The recent development in the nanotechnology has paved the way for a large number of alloyed nanomaterials and devices of desirable properties which have useful functions for electrochemical sensor and biosensor applications. In this paper, bimetallic AuPt nanochains were synthesized through a mild chemical method, with which anti-horseradish peroxidase-conjugated anti-carcinoembryonic antigen (HRP-anti-CEA-NCAuPt) was developed for electrochemical detection of carcinoembryonic antigen (CEA) in a sandwich-type immunoassay format. The alloyed nanocrystals exhibit not only sound signal amplification effect of Au nanoparticles, but also further new combination of interfacial, electrical and structural properties arising from the disparate AuPt components. As a result, the electrochemical signal was significantly amplified by using the HRP-anti-CEA-NCAuPt as tracer and hydrogen peroxide as enzyme substrate. The linear range of the developed immunosensor is 0.01-200ng/mL and the detection limit is 0.11pg/mL of CEA, which makes the biometallic nanochains promising candidates for the next-generation sandwich-type electrochemical immunoassays.     

Sandwich electrochemical immunoassay for the detection of Staphylococcal enterotoxin B based on immobilized thiolated antibodies

A new approach for the sensitive detection of Staphylococcal enterotoxin B (SEB) is presented based upon an electrochemical enzymatic immunoassay that utilizes thiolated antibodies immobilized on a gold surface. This method relies on the use of amine- or sulfhydryl-reactive heterobifunctional cross-linkers for the introduction of 2-pyridyl-disulfide groups to the antibody. The disulfide-containing linkages are subsequently cleaved with a suitable reducing agent, such as dithiothreitol (DTT), and the thiolated antibody–gold bond is covalently formed on a gold working electrode. Various cross-linking agents for immobilization of the capture antibody onto the gold electrode were investigated and compared. Factors influencing the thiolation and immobilization were investigated and optimized. The feasibility of such antibody immobilization and the subsequent sandwich enzyme immunoassay is demonstrated for the sensitive detection of SEB. The detection limit estimated from a representative dose–response curve is 1 ng/mL, corresponding to 5 pg in a 5-μL sample. Coupling the specificity of immunoassays with the sensitivity and low detection limits of electrochemical detection shows real promise for future sensing technology in enabling the development of single-use disposable devices.     

A disposable electrochemical immunosensor based on carbon screen-printed electrodes for the detection of prostate specific antigen

A novel screen-printed electrode (SPEs) on sheets of vegetable parchment was prepared. The obtained SPEs were stable, convenient, inexpensive and suitable for large-area screen-printing. With these SPEs, we explored the fabrication of a novel, disposable and highly sensitive electro-analytical immunosensor using graphene nanosheets (GS) and horseradish peroxidase (HRP)-labeled signal antibody functionalized with gold nanoparticles (HRP-Ab(2)/Au NPs). GS was used to increase the conductivity and stability of this immunosensor due to its fast electron transportation and good biocompatibility. Au NPs could not only provide a large surface area for the immobilization of HRP-Ab(2) but also enhance the electroreduction between HRP and H(2)O(2) to amplify the electrochemical signal on the sandwich immuno-complexes modified SPEs. The proposed SPEs were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and electrochemical methods involving cyclic voltammetry (CV), and electrochemical impedence method. Using prostate specific antigen (PSA) as a model analyte, this immunosensor showed a wide linear range over 6 orders of magnitude with the minimum value down to 2pgmL(-1). In addition, this immunosensor could avoid the need of deoxygenation for the electrochemical immunoassay. Thus, it provided a promising potential in clinical applications.