Sandwich-type electrochemiluminescence immunosensor based on N-(aminobutyl)-N-ethylisoluminol labeling and gold nanoparticle amplification

A novel electrochemiluminescence (ECL) sandwich-type immunosensor for human immunoglobulin G (hIgG) on a gold nanoparticle modified electrode was developed by using N-(aminobutyl)-N-ethylisoluminol (ABEI) labeling. The primary antibody, goat-anti-human IgG was first immobilized on a gold nanoparticle modified electrode, then the antigen (human IgG) and the ABEI-labeled second antibody was conjugated successively to form a sandwich-type immunocomplex. ECL was carried out with a double-step potential in carbonate buffer solution (CBS) containing 1.5 mM H₂O₂. The ECL intensity increased linearly with the concentration of hIgG over the range 5.0-100 ng/mL. The limit of detection was 1.68 ng/mL (S/N = 3). The relative standard deviation was 3.79% at 60 ng/mL (n = 9). The present immunosensor is simple and sensitive. It has been successfully applied to the detection of hIgG in human serums.     

Sandwich-type electrochemiluminescence immunosensor based on Ru-silica@Au composite nanoparticles labeled anti-AFP

A simple and sensitive sandwich-type electrochemiluminescence immunosensor for α-1-fetoprotein (AFP) on a gold nanoparticles (nano-Au) modified glassy carbon electrode (GCE) was developed by using Ru-silica (Ru(bpy)₃²⁺-doped silica) doped Au (Ru-silica@Au) composite as labels. The primary antibody, anti-AFP was first immobilized on the gold nanoparticles modified electrode due to the covalent conjugation, then the antigen and the Ru-silica@Au composite nanoparticles labeled secondary antibody was conjugated successively to form a sandwich-type immunocomplex through the specific interaction. The surfaces of Ru-silica nanoparticles were modified via the assemble of Au nanoparticles. The prepared Ru-silica@Au composite nanoparticles own the large surface area, good biocompatibility and highly effective electrochemiluminescence properties. The morphologies of the Ru-silica@Au composite nanoparticles were investigated by using transmission electronic microscope (TEM). The Ru-silica@Au composite nanoparticles labeled anti-AFP/AFP/bovine serum albumin (BSA)/anti-AFP/nano-Au modified GCE electrode was evaluated by means of cyclic voltammetry (CV) and electrogenerated chemiluminescence (ECL). The immunosensor performed high sensitivity and wide liner for detection AFP in the range of 0.05-50 ng/mL and the limit detection was 0.03 ng/mL (defined as S/N = 3).     

A novel amperometric immunosensor based on three-dimensional sol-gel network and nanoparticle self-assemble technique

A novel hepatitis B surface antigen (HBsAg) immunosensor has been developed by self-assembling gold nanoparticles to a thiol-containing sol-gel network. A cleaned gold electrode was first immersed in a hydrolyzed mercaptopropyltrimethoxysilane (MPS) sol-gel solution to assemble three-dimensional silica gel, and then gold nanoparticles were chemisorbed onto the thiol groups of the sol-gel network. Finally, hepatitis B surface antibody (HBsAb) was adsorbed onto the surface of the gold nanoparticles. Thus, an interfacial design of bare gold electrode (BGE)/MPS/Au/HBsAb was prepared to detect HBsAg in human serum based on the specific reaction of HBsAb and HBsAg. The electrochemistry of ferricyanide redox reaction was used as a marker to probe the interface and as a redox probe to determinate HBsAg. The main conditions of the assembly of MPS sol-gel, gold nanoparticles, the immobilization of HBsAb, and incubation time were investigated in detail. Compared with the glutaraldehyde binding approach, the antibodies immobilized by this method present larger amount and higher immunoactivity. The linearity of HBsAg in the range of 2-360 ng/mL with the correlation coefficient of 0.998 was obtained. This immunosensor system was evaluated on several clinical sample, the analytical results obtained by this method were in agreement with those detected by the enzyme-linked immunosorbent assay (ELISA) method, indicating a promising alternative tool for clinical diagnosis. Moreover, the studied immunosensor exhibited good reproducibility, long-term stability, high sensitivity and specificity.     

Reagentless amperometric carbohydrate antigen 19-9 immunosensor based on direct electrochemistry of immobilized horseradish peroxidase

A reagentless immunosensor for rapid determination of carbohydrate antigen 19-9 (CA19-9) in human serum was proposed. This strategy was based on the immobilization of antibody in colloidal gold nanoparticle modified carbon paste electrode and the direct electrochemistry of horseradish peroxidase (HRP) that was labeled to a CA19-9 antibody. The nanoparticles were efficient for preserving the activity of immobilized biomolecules. Thus, the immobilized HRP displayed its direct electrochemistry with a rate constant of 1.02 s⁻¹. The incubation of the immunosensor in phosphate buffer solution (PBS) including CA19-9 antigen leading to the formation of antigen-antibody complex, which made the block of electron transfer of HRP toward electrode and resulted in significant peak current decrease of HRP. Under the optimal conditions, the current decrease was proportional to CA19-9 concentrations ranging from 2 to 30 U/ml with a detection limit of 1.37 U/ml at a current decrease by 10%. The immunosensor showed an acceptable accuracy compared with those obtained from immunoradiometric assays, with intra-assay coefficient of 7.3 and 6.9% at CA19-9 concentrations of 5 and 15 U/ml, respectively, and inter-assay coefficient of 9.6% at a CA19-9 concentration of 20 U/ml. The storage stability was acceptable in a pH 7.0 PBS at 4 °C for 10 days. This method avoids the addition of electron transfer mediator, thus simplifies the immunoassay procedure and decreases the analytical time. It provides a new promising platform for clinical immunoassay.     

An electrochemical biosensor for α-fetoprotein based on carbon paste electrode constructed of room temperature ionic liquid and gold nanoparticles

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

Electrochemical immunosensor based on nanoporpus gold loading thionine for carcinoembryonic antigen

Nanoporous gold (NPG) has recently received considerable attention in analytical electrochemistry because of its good conductivity and large specific surface area. A facile layer-by-layer assembly technique fabricated NPG was used to construct an electrochemical immunosensor for carcinoembryonic antigen (CEA). NPG was fabricated on glassy carbon (GC) electrode by alternatively assembling gold nanoparticles (AuNPs) and silver nanoparticles (AgNPs) using 1,4-benzenedimethanethiol as a cross-linker, and then AgNPs were dissolved with HNO₃. The thionine was absorbed into the NPG and then gold nanostructure was electrodeposited on the surface through the electrochemical reduction of gold chloride tetrahydrate (HAuCl₄). The anti-CEA was directly adsorbed on gold nanostructure fixed on the GC electrode. The linear range of the immunosensor was from 10 pg mL⁻¹ to 100 ng mL⁻¹ with a detection limit of 3 pg mL⁻¹ (S/N = 3). The proposed immunosensor has high sensitivity, wide linear range, low detection limit, and good selectivity. The present method could be widely applied to construct other immunosensors.     

A sensitive impedance immunosensor based on functionalized gold nanoparticle-protein composite films for probing apolipoprotein A-I

A sensitive and label-free electrochemical impedance immunosensor via covalent coupling the antibody with functionalized gold nanoparticles (FAuNP) for probing apolipoprotein A-I was presented. The hybrid gold nanoparticles were prepared with a two-in-one strategy, i.e. via the stepwise employment of self-assembled monolayer (SAM) and sol-gel techniques, to improve the performance of such a label-free immunosensor, which was investigated by electrochemical impedance spectroscopy. It was found that this novel FAuNP immunosensor showed higher protein-loading capacity and better response properties (6-17 times) than that fabricated by normal SAM technique did. The remarkably improved properties of the immunosensor were ascribed to FAuNP with the larger surface-to-volume ratio, more free amino linkage groups, and the lower nonspecific protein adsorption. As a result, the thus-prepared antibody-modified immunosensor showed reproducible (R.S.D. = ±3.2%, n = 10) linear response to apolipoprotein A-I (Apo A-I) antigens in the range of 0.1-10 ng mL⁻¹. The detection limit of this immunosensor was 50 pg mL⁻¹ (corresponding to 1.8 pmol L⁻¹), which was two orders of magnitude lower than that of the traditional methods. These results exhibited the novel immunosensor had a high sensitivity, stability and selectivity for the determination of Apo A-I, especially in clinic microanalysis.     

An ultrasensitive luminol cathodic electrochemiluminescence immunosensor based on glucose oxidase and nanocomposites: Graphene–carbon nanotubes and gold-platinum alloy

In the present study, a novel and ultrasensitive electrochemiluminescence (ECL) immunosensor based on luminol cathodic ECL was fabricated by using Au nanoparticles and Pt nanoparticles (nano-AuPt) electrodeposited on graphene–carbon nanotubes nanocomposite as platform for the detection of carcinoembryonic antigen (CEA). For this introduced immunosensor, graphene (GR) and single wall carbon nanotubes (CNTs) dispersed in chitosan (Chi-GR-CNTs) were firstly decorated on the bare gold electrode (GE) surface. Then nano-AuPt were electrodeposited (DpAu-Pt) on the Chi-GR-CNTs modified electrode. Subsequently, glucose oxidase (GOD) was employed to block the non-specific sites of electrode surface. When glucose was present in the working buffer solution, GOD immediately catalyzed the oxidation of glucose to in situ generate hydrogen peroxide (H₂O₂), which could subsequently promote the oxidation of luminol with an amplified cathodic ECL signal. The proposed immunosensor was performed at low potential (−0.1 to 0.4 V) and low concentration of luminol. The CEA was determined in the range of 0.1 pg mL⁻¹ to 40 ng mL⁻¹ with a limit of detection down to 0.03 pg mL⁻¹ (S N⁻¹ = 3). Moreover, with excellent sensitivity, selectivity, stability and simplicity, the as-proposed luminol-based ECL immunosensor provided great potential in clinical applications.     

Study on the immobilization of anti-IgG on Au-colloid modified gold electrode via potentiometric immunosensor, cyclic voltammetry, and electrochemical impedance techniques

The immobilization of anti-IgG on Au-colloid modified gold electrodes has been investigated. A cleaned gold electrode was first immersed in a mercaptoethylamine (AET) solution, and then gold nanoparticles were chemisorbed onto the thiol groups of the mercaptoethylamine. Finally, anti-IgG was adsorbed onto the surface of the gold nanoparticles. Potentiometric immunosensor, cyclic voltammetry, and electrochemical impedance techniques were used to investigate the immobilization of anti-IgG on Au colloids. In the impedance spectroscopic study, an obvious difference of the electron transfer resistance between the Au-colloid modified electrode and the bare gold electrode was observed. The cyclic voltammogram tends to be more irreversible with increased anti-IgG concentration. Using the potentiometric immunosensor, the proposed technique is based on that the specific agglutination of antibody-coated gold nanoparticles, averaging 16 nm in diameter, in the presence of the corresponding antigen causes a potential change that is monitored by a potentiometry. It is found that the developed immunoagglutination assay system is sensitive to the concentration of IgG antigen as low as 12 ng mL(-1). Experimental results showed that the developed technique is in satisfactory agreement with the ELISA method, and that gold nanoparticles can be used as a biocompatible matrix for antibody or antigen immobilization.     

A Label-Free Electrochemical Immunosensor for Clostridium Difficile Toxin B Based on One-Step Immobilization of Thionine in a Silica Matrix

A label-free amperometric immunosensor was fabricated to test clostridium difficile toxin B. Multi-walled carbon nanotubes were modified on the surface of a glassy carbon electrode by electrodeposition. A sol-gel method was developed to encapsulate thionine in an electrochemically induced three-dimensional porous silica matrix by a one-step process. Gold nanoparticle layers were constructed by covalent bonds and electrostatic adsorption with thionine. The clostridium difficile toxin B antibody was immobilized on the gold nanoparticles to construct the immunosensor. Cyclic voltammetry and differential pulse voltammetry demonstrated that the formation of antibody-antigen complexes decreased the peak current of thionine. The morphologies of the nanocomposites were investigated by scanning electron microscopy and ultraviolet-visible spectrometry. The electrode was shown to be sensitive and specific to detect clostridium difficile toxin B from 1.0 to 80.0 ng/mL with a limit of detection of 0.3 ng/mL.     

一种基于静电吸附作用的直接检测补体C3新型可再生电容型免疫传感器

通过壳聚糖/褐藻酸钠体系实现抗体在电极上的固定,制得可多次再生的电容型免疫传感器,用于补体C₃的检测.先在金电极表面上组装一层半胱胺单分子层,通过戊二醛把壳聚糖修饰在金电极上,再用十二硫醇封闭电极,最后,利用壳聚糖与褐藻酸钠之间的强静电相互作用实现抗体的固定.使用交流阻抗法研究了溶液pH值和离子强度对电极膜层稳定性及对抗体固定性的影响.结果表明,所制备的传感器操作简便,易于再生,电容响应与补体C₃浓度在18.2～292.5ng/mL范围内呈线性关系,检出限为9.1ng/mL.     

A reusable capacitive immunosensor for detection of Salmonella spp. based on grafted ethylene diamine and self-assembled gold nanoparticle monolayers

Fabrication of a novel capacitive immunosensor based on grafted ethylene diamine and self-assembled gold nanoparticle monolayer on glassy carbon electrode for the detection of Salmonella spp. is described for the first time. In the present study, the Salmonella spp. monoclonal antibodies (denoted as McAbs) was immobilized on gold nanoparticles. Interaction of McAbs and Salmonella spp. was detected directly using the electrochemical impedance spectroscopy (EIS) technique. The experimental results showed that the concentration of antigen was measured through the relative change in capacitance in the corresponding specific binding of Salmonella spp. and McAbs. Under the optimized conditions, the relative changes in capacitance were proportional to the logarithmic values of Salmonella spp. concentrations in the range of 1.0 × 10² to 1.0 × 10⁵ CFU mL⁻¹ (r = 0.991) with the detection limit of 1.0 × 10² CFU mL⁻¹. The stability of proposed immunosensor could be estimated by determining the relative change in capacitance, which remained almost the same in two months and decreased gradually to 85.3% of initial value after four months’ storage. The used immunosensor could be regenerated repeatedly by immersing in glycine-HCl buffer solution (pH 2.8). Finally, the proposed immunosensor was successfully used for the detection of Salmonella spp. in lab-processed commercial pork samples.     

基于自组装电化学免疫传感器对污泥中PCB77的检测

构建了一种基于L-半胱氨酸、 壳聚糖、 戊二醛和纳米金层层自组装技术的新型无标记、 高灵敏电流型免疫传感器, 并用于检测3,3',4,4'-四氯联苯(PCB77). 利用示差脉冲法研究了修饰电极表面的电化学特性以及测试溶液的pH值、 孵育时间和温度对免疫传感器性能的影响. 实验结果表明, 该免疫传感器在含不同浓度PCB77的磷酸盐缓冲溶液(PBS, pH=7.4)中于35 ℃下孵育30 min后, 在含有5 mmol/L K₃Fe(CN)₆/K₄Fe(CN)₆(摩尔比1:1)和0.1 mol/L KCl的PBS溶液(pH=6.0)中测定, 响应电流与PCB77浓度在0.1～160 ng/mL范围内呈良好的线性关系, R=0.9964, 检出限为0.01 ng/mL. 该传感器制备简单、 灵敏度高、 稳定性好, 可以重复使用. 将其用于检测实际污泥样品中的PCB77, 回收率为95%～112%.     

Electrochemical immuno-bioanalysis for carcinoma antigen 125 based on thionine and gold nanoparticles-modified carbon paste interface

A novel electrochemical immunosensor for the determination of carcinoma antigen 125 (CA125) was developed by means of immobilizing CA125 antibody (anti-CA125) on gold nanoparticles (Au) and thionine (Thi)-modified carbon paste interface. To avoid the leak of hydrophilic gold nanoparticles and thionine from carbon paste interface, the Au-Thi-modified carbon paste electrodes (CPEs) were first treated in the mixture solution containing 10% HNO₃ and 2.5% K₂Cr₂O₇ for 1.5 min at +1.5 V to make the carbon surface with -COOH groups, which can react with -NH₂ groups on the thionine molecule, in the meantime, gold nanoparticles were absorbed on the thionine surface. Subsequently, CA125 antibodies were assembled onto the surface of gold nanoparticles. The fabrication process of the immunosensor was characterized by fourier transform infrared spectroscopy (FTIR) and UV-vis absorption spectroscopy. The performance and factors influencing the performance of the immunosensor were studied in detail. A direct electrochemical immunoassay format was employed to detect CA125 antigen based on the current change before and after the antigen-antibody reaction. The current change was proportional to CA125 concentration ranging from 10 to 30 U/ml with a detection limit of 1.8 U/ml (at 3δ). The immunosensors were used to analyze CA125 in human serum specimens. Analytical results of clinical samples show that the developed immunoassay has a promising alternative approach for detecting CA125 in the clinical diagnosis.     

MCM-41 mesoporous material modified carbon paste electrode for the determination of cardiac troponin I by anodic stripping voltammetry

An anodic stripping voltammetric method for the determination of cardiac troponin I (cTnI) at a MCM-41 mesoporous material modified carbon paste electrode (MCM-MCPE) was investigated. The test was based on the dual monoclonal antibody “sandwich” principle using colloidal gold as a labeled substrate. Four main steps were carried out to obtain the analytical signal, i.e. electrode preparation, immunoreaction, silver enhancement, and anodic stripping voltammetric detection. The anodic stripping peak current increased linearly with the concentration of cTnI over the range of 0.8-5.0 ng/ml. A detection limit of 0.5 ng/ml was obtained. The established method was applied to detect cTnI in acute myocardial infarction (AMI) samples using routine enzyme-linked immunoadsorbent assay (ELISA) for comparison analysis, and good results were obtained.     

A new antibody immobilization strategy based on electrodeposition of nanometer-sized hydroxyapatite for label-free capacitive immunosensor

A new antibody immobilization strategy was proposed for the fabrication of a label-free capacitive immunosensor based on electrodeposition of nanometer-sized bioactive hydroxyapatite (HAP). By a procedure of constant current cathodal electrodeposition, a nano-HAP film with bioactivity was formed on a self-assembled β-mercaptoethanol monolayer-modified gold electrode. A suitable amount of chitosan was added into the electrodeposition solution with the aim of obtaining a strong and homogeneous HAP-coating film. After blocking with long-chain alkylthiol and then embedding antibody by coupling with divinylsulphone, the electrode was possessed of a higher initial capacitance value, which was suitable for capacitive transduction. The sensitive layer was characterized by Fourier transform infrared spectrum, scanning electron microscopy and electrochemical method. Human transferrin immunoassay was selected as the testing system. The linear response range of the sensor for transferrin was between 1 and 100 ng/mL with a detection limit of 0.15 ng/mL. After simply rinsing with subacidity solution, the regenerated sensor achieved up to 10 assay cycles without significant loss of sensitivity.     

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 12 ng mL⁻¹ in pH 5.5 HAc-NaAc containing 5 mM H₂O₂. At a signal-to-noise ratio of 3, the detection limit (LOD) is 5 pg mL⁻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.     

A novel electrochemical immunosensor based on colabeled silica nanoparticles for determination of total prostate specific antigen in human serum

A novel electrochemical immunosensor using functionalized silica nanoparticles (Si NPs) as protein tracer has been developed for the detection of prostate specific antigen (PSA) in human serum. The immunosensor was carried out based on a heterogeneous sandwich procedure. The PSA capture antibody was immobilized on the gold electrode via glutaraldehyde crosslink. After reaction with the antigen in human serum, Si NPs colabeled with detection antibody and alkaline phosphatase (ALP) was sandwiched to form the immunocomplex on the gold electrode. ALP carried by Si NPs convert nonelectroactive substrate into the reducing agent and the latter, in turn, reduce metal ions to form electroactive metallic product on the electrode. Linear sweep voltammetry (LSV) was used to quantify the amount of the deposited silver and give the analytical signal for PSA. The parameters including the concentration of the ALP used to functionalize the Si NPs and the enzyme catalytic reaction time have been studied in detail and optimized. Under the optimum conditions of immunoreaction and electrochemical detection, the electrochemical immunosensor was able to realize a reliable determination of PSA in the range of 1–35 ng/mL with a detection limit of 0.76 ng/mL. For six human serum samples, the results performed with the electrochemical immunosensor were in good agreement with those obtained by chemiluminescent microparticle immunoassay (CMIA), indicating that the electrochemical immunosensor could satisfy the need of practical sample detection.     

A novel piezoelectric immunosensor for the detection of malarial Plasmodium falciparum histidine rich protein-2 antigen

A novel piezoelectric (PZ) immunosensor for the direct detection of malarial Plasmodium falciparum histidine rich protein-2 (PfHRP-2) antigen was developed. The mixed self-assembled monolayers (SAMs) of thioctic acid and 1-dodecanethiol were formed on gold surface of quartz crystal. Cyclic voltammetry, electrochemical impedance spectroscopy and surface Raman spectroscopy techniques were used to characterize the mixed SAMs. The rabbit anti-PfHRP-2 antibodies were coupled on mixed SAM modified gold surface of quartz crystal via NHS/EDC activation method. The PZ immunosensor was applied to detect PfHRP-2 in the linear range of 15-60 ng/ml with a detection limit of 12 ng/ml. It was also found that even after 14 days of storage, 50% of the activity still remained. Clinical human serum samples were tested with this method, and the results were in agreement with those obtained from commercially available ICT kit (NOW^® Malaria).     

Highly conducting gold nanoparticles-graphene nanohybrid films for ultrasensitive detection of carcinoembryonic antigen

A new label-free amperometric immunosensor was developed for detection of carcinoembryonic antigen (CEA) based on chitosan-ferrocene (CS-Fc) and nano-TiO₂ (CS-Fc + TiO₂) complex film and gold nanoparticles-graphene (Au-Gra) nanohybrid. CS-Fc + TiO₂ composite membrane was first modified on a bare glass carbon electrode. Then Au-Gra nanohybrid was formed on the CS-Fc + TiO₂ membrane by self-assembly strategy. Next, further immobilization of anti-CEA was constructed according to the strong interaction between Au-Gra and the amido groups of anti-CEA. Since Au-Gra nanohybrid films provided a congenial microenvironment for the immobilization of biomolecules, the surface coverage of antibody protein could be enhanced and the sensitivity of the immunosensor has been improved. The good electronic conductive characteristic might be attributed to the synergistic effect of graphene nanosheets and Au NPs. The modified process was characterized by scanning electron microscope (SEM) and cyclic voltammetry (CV). Under optimized conditions, the resulting biosensor displayed good amperometric response to CEA with linear range from 0.01 to 80 ng/mL and a detection limit of 3.4 pg/mL (signal/noise = 3). The results demonstrated that the immunosensor has advantages of high conduction, sensitivity, and long life time. This assay approach showed a great potential in clinical applications and detection of low level proteins.