Study on an immunosensor based on gold nanoparticles and a nano-calcium carbonate/Prussian blue modified glassy carbon electrode

An amperometric carcinoembryonic antigen (CEA) immunosensor was fabricated based on Prussian blue (PB), nano-calcium carbonate (nano-CaCO₃) and nano-gold modified glassy carbon electrode. First, PB as a mediator was deposited on glassy carbon electrode to obtain a negatively charged surface. Then, positive nano-CaCO₃ was adsorbed on the PB modified electrode through electrostatic interaction. Subsequently, gold nanoparticles were deposited on the nano-CaCO₃/PB modified electrode. The use of two kinds of nanomaterials (nano-CaCO₃ and nano-gold) with good biocompatibility as immobilization matrixes not only provides a biocompatible surface for protein loading but also avoids the leaking of PB. The size of nano-CaCO₃ was characterized by transmission electron microscopy (TEM). The factors influencing the performance of the immunosensor presented were studied in detail. Under the optimized conditions, cyclic voltammograms (CV) determination of CEA showed a specific response in two concentration ranges from 0.3 to 20 ng mL^?1 and from 20 to 100 ng mL^?1 with a detection limit of 0.1 ng mL^?1 at a signal-to-noise ratio of 3. The immunosensor presented exhibited high selectivity, sensitivity and good stability.     

Electrochemical Biosensor Based on Nanocomposites Film of Thiol Graphene‐Thiol Chitosan/Nano Gold for the Detection of Carcinoembryonic Antigen

In this paper, a thiol graphene‐thiol chitosan‐gold nanoparticles (thGP‐thCTS‐AuNPs) nanocomposites film with porous structure was fabricated by electrochemically depositing on glassy carbon electrode (GCE), which exhibited good biocompatibility and improved conductivity, to construct immunosensor free label for detection of carcinoembryonic antigen (CEA). The electrochemical behavior of this immunosensor was investigated by cyclic voltammetry. Under the optimum conditions, the immunosensor revealed a good amperometric response to CEA in two linear ranges (0.3–8.0 ng mL^?1 and 8.0–100 ng mL^?1) with a detection limit of 0.03 ng mL^?1. The results indicated that the immunosensor has the advantages of good selectivity, high sensitivity, and good stability for the determination of CEA.     

Ultrasensitive electrochemiluminescent immunosensor based on dual signal amplification strategy of gold nanoparticles-dotted graphene composites and CdTe quantum dots coated silica nanoparticles

A facile and ultrasensitive electrochemiluminescent (ECL) immunosensor for detection of prostate-specific antigen (PSA) was designed by using CdTe quantum dots coated silica nanoparticles (SiO₂@QDs) as bionanolabels. To construct such an electrochemiluminescence immunosensor, gold nanoparticles-dotted graphene composites were immobilized on the working electrode, which can increase the surface area to capture a large amount of primary antibodies as well as improve the electronic transmission rate. The as-prepared SiO₂@QDs used as bionanolabels, showed good ECL performance and good ability of immobilization for secondary antibodies. The approach provided a good linear response ranging from 0.005 to 10 ng?mL^?1 with a low detection limit of 0.0032 ng?mL^?1. Such immunosensor showed good precision, acceptable stability, and reproducibility. Satisfactory results were obtained for determination of PSA in human serum samples. Therefore, the proposed method provides a new promising platform of clinical immunoassay for other biomolecules.     

Disposable electrochemical immunosensor for myeloperoxidase based on the indium tin oxide electrode modified with an ionic liquid composite film containing gold nanoparticles, poly(o-phenylenediamine) and carbon nanotubes

A disposable electrochemical myeloperoxidase (MPO) immunosensor was fabricated based on the indium tin oxide electrode modified with a film composed of gold nanoparticles (AuNPs), poly(o-phenylenediamine), multi-walled carbon nanotubes and an ionic liquid. The composite film on the surface of the electrode was prepared by in situ electropolymerization using the ionic liquid as a supporting electrolyte. Negatively charged AuNPs were then adsorbed on the modified electrode via amine-gold affinity and to immobilize MPO antibody. Finally, bovine serum albumin was employed to block possible remaining active sites on the AuNPs. The modification of the electrode was studied by cyclic voltammetry and scanning electron microscopy. The factors affecting the performance of the immunosensor were investigated in detail using the hexacyanoferrate redox system. The sensor exhibited good response to MPO over two linear ranges (from 0.2 to 23.4 and from 23.4 to 300 ng.mL^?1), with a detection limit of 0.05 ng.mL^?1 (at an S/N of 3).

基于纳米金与碳纳米管-纳米铂-壳聚糖纳米复合物固定癌胚抗原免疫传感器的研究

采用纳米金(nano-Au)、多壁碳纳米管-纳米铂-壳聚糖的纳米复合物(MWNT-Pt-CS)及电子媒介体硫堇(Th)固载抗体制得高灵敏癌胚抗原免疫传感器．首先, 于壳聚糖溶液中用NaBH4还原H2PtCl6, 并将多壁碳纳米管分散于其中制得碳纳米管-纳米铂-壳聚糖纳米复合物, 并将其滴涂在玻碳电极上成膜; 然后, 吸附电子媒介体硫堇制得硫堇/碳纳米管-纳米铂-壳聚糖(Th/MWNT-Pt-CS)修饰电极．利用壳聚糖和硫堇分子中大量的氨基固定纳米金并吸附癌胚抗体(anti-CEA); 最后, 用辣根过氧化物酶(HRP)封闭活性位点从而制得高灵敏电流型免疫传感器．在优化的实验条件下, 该传感器响应的峰电流值与癌胚抗原(carcinoembryonic antigen)浓度在0.5～10和10～120 ng/mL的范围内保持良好的线性关系, 检测限为0.2 ng/mL．     

Ultrasensitive amperometric immunosensor for the determination of carcinoembryonic antigen based on a porous chitosan and gold nanoparticles functionalized interface

An immunosensor has been fabricated for direct amperometric determination of carcinoembryonic antigen. It is based on a biocompatible composite film composed of porous chitosan (pChit) and gold nanoparticles (GNPs). Firstly, a pChit film was formed on a glassy carbon electrode by means of electrodeposition. Then, thionine as a redox probe was immobilized on the pChit film modified electrode using glutaraldehyde as a cross-linker. Finally, GNPs were adsorbed on the electrode surface to assemble carcinoembryonic antibody (anti-CEA). The surface morphology of the pChit films was studied by means of a scanning electron microscope. The immunosensor was further characterized by cyclic voltammetry and electrochemical impedance spectroscopy. The electrochemical behaviors and factors influencing the performance of the resulting immunosensors were studied in detail. Results showed that the pChit films can enhance the surface coverage of antibodies and improve the sensitivity of the immunosensor. Under optimal conditions, the immunosensor was highly sensitive to CEA with a detection limit of 0.08 ng·mL^?1 at three times the background noise and linear ranges of 0.2～10.0 ng·mL^?1 and 10.0～160 ng·mL^?1. Moreover, the immunosensor exhibited high selectivity, good reproducibility and stability.     

Preparation of Fe3O4@C@CNC multifunctional magnetic core/shell nanoparticles and their application in a signal-type flow-injection photoluminescence immunosensor

We describe here the preparation of carbon-coated Fe₃O₄ magnetic nanoparticles that were further fabricated into multifunctional core/shell nanoparticles (Fe₃O₄@C@CNCs) through a layer-by-layer self-assembly process of carbon nanocrystals (CNCs). The nanoparticles were applied in a photoluminescence (PL) immunosensor to detect the carcinoembryonic antigen (CEA), and CEA primary antibody was immobilized onto the surface of the nanoparticles. In addition, CEA secondary antibody and glucose oxidase were covalently bonded to silica nanoparticles. After stepwise immunoreactions, the immunoreagent was injected into the PL cell using a flow-injection PL system. When glucose was injected, hydrogen peroxide was obtained because of glucose oxidase catalysis and quenched the PL of the Fe₃O₄@C@CNC nanoparticles. The here proposed PL immunosensor allowed us to determine CEA concentrations in the 0.005–50 ng?·?mL^-1 concentration range, with a detection limit of 1.8 pg?·?mL^-1.

Electrochemical Immunoanalysis for Carcinoembryonic Antigen Based on Multilayer Architectures of Gold Nanoparticles and Polycation Biomimetic Interface on Glassy Carbon Electrode

This paper describes a layer‐by‐layer (LBL) self‐assembly process of chitosan (CTS) and gold nanoparticles (Au) on the pretreated negatively charged glassy carbon (GC) electrode to fabricate electrochemistry immunosensor with a nontoxic biomimetic interface, which provided an environment similar to a native system and allowed more freedom in orientation for immobilization of carcinoembryonic antibody (anti‐CEA) to monitor carcinoembryonic antigen (CEA). UV‐vis spectroscope, atomic force microscopy (AFM), and cyclic voltammetric (CV) measurements were used to follow the multilayer film formation. The performance of the biominetic interface and factors influencing the assay system were investigated in detail. The differential pulse voltammetry (DPV) current response is used for the CEA concentration assay. The dynamic range was from 0.50 to 80.00 ng mL^?1 with a detection limit of 0.27 ng mL^?1 at 3σ. In addition, the experiment results indicate that immobilization described in this proposed method exhibits a good sensitivity, selectivity, and stability.     

An electrochemical immunosensor for the tumor marker α-fetoprotein using a glassy carbon electrode modified with a poly(5-formylindole), single-wall carbon nanotubes,and coated with gold nanoparticles and antibody

We report on a label-free electrochemical immunosensor for α-fetoprotein (α-FP). It is based on the use of a glassy carbon electrode that was first modified with conducting poly(5-formylindole) and single-walled carbon nanotubes (P5FIn/SWNTs), and then coated with gold nanoparticles and the respective antibody. The presence of aldehyde groups warrants direct immobilization of the antibody and results in a convenient method for fabricating of the immunosensor. Gold nanoparticles (GNPs) were deposited on the P5FIn/SWNTs composite material, and the modified electrode was applied to the detection of α-FP. The analytical signal is obtained by measuring the change of amperometric response at a typical working voltage of 100 mV before and after the immunoreaction. The detection limit is 200 fg mL^?1. The immunosensor is simple, sensitive, specific and reproducible. It has the potential for reliable point-of-care diagnosis of tumor or other diseases. Figure

A simple electrochemical immunosensor based on conducting poly(5-formylindole) and single-walled carbon nanotubes composite was fabricated to detect alpha-fetoprotein. The detection limit is 200 fg mL^?1. This immunosensor is simple, sensitive, specific and reproducible.     

A disposable immunosensor based on gold colloid modified chitosan nanoparticles-entrapped carbon paste electrode

A new strategy is described to construct disposable electrochemical immunosensors for the assay of human immunoglobulin. It is based on a carbon paste electrode constructed from chitosan nanoparticles modified with colloidal gold. The stepwise assembly process of the immunosensor was characterized by means of cyclic voltammetry and electrochemical impedance spectroscopy. Assay conditions that were optimized included the amount of chitosan nanoparticles in the preparation of carbon paste electrode, antibody concentration, and the incubation time of the antibody immobilization. Using hexacyanoferrate as a mediator, the current change increased with the concentration of human immunoglobulin G. A linear relationship in the concentration range 0.3 to 120 ng mL^?1 was achieved, with a detection limit of 0.1 ng mL^?1 (S/N?=?3). The method combines the specificity of the immunological reaction with the sensitivity of the gold colloid amplified electrochemical detection, and it has potential application in clinical immunoassay.     

A Sensitive Electrochemical Immunosensor for α‐Fetoprotein Detection with Colloidal Gold‐Based Dentritical Enzyme Complex Amplification

A sensitive and specific electrochemical immunosensor was developed with α‐fetoprotein (AFP) as the model analyte by using gold nanoparticle label for enzymatic catalytic amplification. A self‐assembled monolayer membrane of mercaptopropionic acid (MPA) was firstly formed on the electrode surface through gold‐sulfur interaction. Monoclonal mouse anti‐human AFP was covalently immobilized to serve as the capture antibody. In the presence of the target human AFP, gold nanoparticles coated with polyclonal rabbit anti‐human AFP were bound to the electrode via the formation of a sandwiched complex. With the introduction of goat anti‐rabbit IgG conjugated with alkaline phosphatase, the dentritical enzyme complex was formed through selective interaction of the secondary antibodies with the colloidal gold‐based primary antibody at the electrode, thus affording the possibility of signal amplification for AFP detection. Current response arising from the oxidation of enzymatic product was significantly amplified by the dentritical enzyme complex. The current signal was proportional to the concentration of AFP from 1.0 ng mL^?1 to 500 ng mL^?1 with a detection limit of 0.8 ng mL^?1. This system could be extended to detect other target molecules with the corresponding antibody pairs.     

基于金纳米颗粒负载的金属有机骨架材料的癌胚抗原传感器的研制

以负载Au的金属有机骨架材料(AuNPs/Cu-TPA)标记CEA抗体(Ab2)为信号探针,通过电还原的方法将氧化石墨烯还原到电极上,研制了一种捕获CEA抗体(Ab1)的电化学免疫传感器,并将其应用于癌胚抗原(CEA)检测.所合成的MOFs材料中含有大量Cu2+,且电化学信号比较稳定,因此可以通过检测MOFs材料中Cu2+的信号实现对CEA的检测.此信号探针不需要预处理和酸处理,易负载贵金属从而固定抗体,大大简化了检测步骤并缩短了检测时间.此传感器对CEA的检测灵敏度好,操作简便.在最优实验条件下,此传感器的线性范围为0.1～ 80 ng/mL,检出限为0.03 ng/mL,线性相关系数为0.9887,可用于真实样品中CEA的测定.     

A Novel Electrochemical Immunosensor for Prostate-Specific Antigen Based on Noncovalent Nanocomposite of Ferrocene Monocarboxylic Acid with Graphene Oxide

A novel electrochemical immunosensor was developed for the determination of prostate-specific antigen based on immobilization of appropriate antibodies on gold nanoparticles and a poly-(2,6-pyridinediamine) modified electrode. The nanocomposite of ferrocene monocarboxylic acid hybridized graphene oxide was prepared by a π-π stacking interaction and was used as the electrochemical probe. A sandwich-type complex immunoassay was applied with polyclonal prostate-specific antigen antibodies labeled with the nanocomposite of ferrocene monocarboxylic acid hybridized graphene oxide. In order to improve the sensitivity, a potentiostatic method was used to reduce graphene oxide. Cyclic voltammetry and differential pulse voltammetry were employed to characterize the assembly process and the performance of the immunosensor. Under optimal conditions, the peak current of the immunosensor increased with concentration, showing a linear relationship between the peak current and the logarithm of the prostate-specific antigen concentrations in a wide range of 2.0 pg mL^?1 to 10.0 ng mL^?1 with a low detection limit of 0.5 pg mL^?1. The immunosensor was used for the determination of prostate-specific antigen in serum.     

Electrochemiluminescence immunoassay for the carcinoembryonic antigen using CdSe:Eu nanocrystals

CdSe:Eu nanocrystals were successfully synthesized and characterized by transmission electron microscopy, X-ray powder diffraction, and X-ray photoelectric spectroscopy. The CdSe:Eu nanocrystals showed enhanced green electrochemiluminescence (ECL) intensity when compared to pure CdSe nanocrystals. Further, the nanocrystals were used to design an ECL immunosensor for the detection of carcinoembryonic antigen (CEA) that has a linear response over the 1.0 fg·mL^?1 to 100 ng·mL^?1 CEA concentration range with a 0.4 fg·mL^?1 detection limit. The assay was applied to the determination of CEA in human serum samples.

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Graphical abstract Schematic of the assay: GCE-glassy-carbon electrode, Ab- Antibody, BSA- Bovine serum albumin, Ag- Antigen. CdSe:Eu nanocrystals were used to design an ECL immunosensor for the detection of carcinoembryonic antigen.

     

Electrochemical immunosensor for the carcinoembryonic antigen based on a nanocomposite consisting of reduced graphene oxide,gold nanoparticles and poly(indole-6-carboxylic acid)

We describe a label-free electrochemical immunosensor for the carcinoembryonic antigen (CEA). It is based on a nanocomposite consisting of electrochemically reduced graphene oxide, gold nanoparticles (AuNPs), and poly(indole-6-carboxylic acid). Coupled to nanoparticle-amplification techniques and modified with ionic liquid (IL), this immunoassay shows high sensitivity and good selectivity for CEA. At the best working voltage of 0.95 V (vs. Ag/AgCl), the lower detection limit is 0.02 ng·mL^?1, and the response to CEA is linear in the range from 0.02 to 90 ng·mL^?1. The method was applied to the determination of CEA in spiked serum samples and gave recoveries in the range from 98.5 % to 102 %.

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Graphical abstract A label-free electrochemical immunosensor was fabricated for the carcinoembryonic antigen (CEA) with a detection limit of 0.02 ng·mL^?1. It is based on a nanocomposite consisting of electrochemically reduced graphene oxide (erGO), gold nanoparticles (Au NP), and poly(indole-6-carboxylic acid) (PICA).

     

Determination of carcinoembryonic antigen using a novel amperometric enzyme-electrode based on layer-by-layer assembly of gold nanoparticles and thionine

Electrochemical sensing of carcinoembryonic antigen(CEA)on a gold electrode modified by the se- quential incorporation of the mediator,thionine(Thi),and gold nanoparticles(nano-Au),through co- valent linkage and electrostatic interactions onto a self-assembled monolayer configuration is de- scribed in this paper.The enzyme,horseradish peroxidase(HRP),was employed to block the possible remaining active sites of the nano-Au monolayer,avoid the non-specific adsorption,instead of bovine serum albumin(BSA),and amplify the response of the antigen-antibody reaction.Electrochemical ex- periments indicated highly efficient electron transfer by the imbedded Thi mediator and adsorbed nano-Au.The HRP kept its activity after immobilization,and the studied electrode showed sensitive response to CEA and high stability during a long period of storage.The working range for the system was 2.5 to 80.0 ng/mL with a detection limit of 0.90 ng/mL.The model membrane system in this work is a potential biosensor for mimicking the other immunosensor and enzyme sensor.     

A sensitive amperometric immunosensor for alpha-fetoprotein based on carbon nanotube/DNA/Thi/nano-Au modified glassy carbon electrode

A novel amperometric immunosensor for the determination of alpha-fetoprotein (AFP) was constructed using films of multi-wall carbon nanotubes/DNA/thionine/gold nanoparticles (nano-Au). Firstly, multi-wall carbon nanotubes (MWCNT) dispersed in poly(diallydimethlammonium chloride) (PDDA) were immobilized on the nano-Au film which was electrochemically deposited on the surface of glassy carbon electrode. Then a negatively charged DNA film was absorbed on the positively charged PDDA. Subsequently, thionine was attached to the electrode via the electrostatic interaction between thionine and the DNA. Finally, the nano-Au was retained on the thionine film for immobilization of AFP antibody (anti-AFP). The modification process was characterized by cyclic voltammetry (CV) and scanning electron microscope (SEM). The factors possibly influenced the performance of the proposed immunosensors were studied in detail. Under optimal conditions, the proposed immunosensor exhibited good electrochemical behavior to AFP in a two concentration ranges: 0.01–10.0 and 10.0–200.0 ng/mL with a relatively low detection limit of 0.04 ng/mL at three times the background noise. Moreover, the selectivity, repeatability and stability of the proposed immunosensor were acceptable.     

Disposable immunoassay for hepatitis B surface antigen based on a graphene paste electrode functionalized with gold nanoparticles and a Nafion-cysteine conjugate

We report on the modification of a graphene paste electrode with gold nanoparticles (AuNPs) and a Nafion-L-cysteine composite film, and how this electrode can serve as a platform for the construction of a novel electrochemical immunosensor for the detection of hepatitis B surface antigen (HBsAg). To obtain the immunosensor, an antibody against HBsAg was immobilized on the surface of the electrode, and this process was followed by cyclic voltammetry and electrochemical impedance spectroscopy. The peak currents of a hexacyanoferrate redox system decreased on formation of the antibody-antigen complex on the surface of the electrode. Then increased electrochemical response is thought to result from a combination of beneficial effects including the biocompatibility and large surface area of the AuNPs, the high conductivity of the graphene paste electrode, the synergistic effects of composite film, and the increased quantity of HBsAb adsorbed on the electrode surface. The differential pulse voltammetric responses of the hexacyanoferrate redox pair are proportional to the concentration of HBsAg in the range from 0.5–800?ng?mL^?1, and the detection limit is 0.1?ng?mL^?1 (at an S/N of 3). The immunosensor is sensitive and stable.

Electroanalytical Determination of Carcinoembryonic Antigen at a Silica Nanoparticles/Titania Sol–Gel Composite Membrane‐Modified Gold Electrode

A highly hydrophilic and nontoxic colloidal silica nanoparticle/titania sol–gel composite membrane was prepared on a gold electrode via a chemical vapor deposition method. With carcinoembryonic antigen (CEA) as a model antigen and encapsulation of carcinoembryonic antibody (anti‐CEA) in the composite architecture, this membrane could be used for reagentless electrochemical immunoassay. The presence of silica nanoparticles provided a congenial microenvironment for adsorbed biomolecules. The formation of immunoconjugate by a simple one‐step immunoreaction between CEA in sample solution and the immobilized anti‐CEA introduced the change in the potential. The modified procedure was further characterized by electrochemical impedance spectroscopy and cyclic voltammetry. Compared to the commonly applied methods, i.e., the TiO₂ direct embedding procedure, this strategy could allow for antibodies immobilized with higher loading amount and better retained immunoactivity. The resulting immunosensor exhibited high sensitivity, good precision, acceptable stability, accuracy, reproducibility and wide linear range from 1.5 to 240 ng mL^?1 with a detection limit of 0.5 ng mL^?1 at 3σ. Analytical results of clinical samples show that the developed immunoassay is comparable with the enzyme‐linked immunosorbent assays (ELISAs) method, implying a promising alternative approach for detecting CEA in the clinical diagnosis. Furthermore, this composite membrane could be used efficiently for the entrapment of other biomarkers and clinical applications.     

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).