Electrochemical stripping analysis of nanogold label-induced silver deposition for ultrasensitive multiplexed detection of tumor markers

A multiplexed electrochemical immunoassay method was developed for simultaneous ultrasensitive measurement of tumor markers based on electrochemical stripping analysis of silver nanoparticles (Ag NPs). The Ag NPs were deposited on a disposable immunosensor array with a reduction reaction catalyzed by nanogold labels. The immunosensor array was prepared by covalently immobilizing capture antibodies on chitosan modified screen-printed carbon electrodes. Through a sandwich-type immunoreaction, antibody-functionalized Au NPs were captured onto immunosensor surface to induce the silver deposition from a silver enhancer solution. The deposited Ag NPs could be directly measured by anodic stripping analysis in KCl solution. The catalytic deposition enhanced the analytical sensitivity for detection of protein markers. The interference of dissolved oxygen could be avoided as the detection was performed with positive stripping potential range. Using carcinoembryonic antigen and α-fetoprotein as model analytes, the proposed multiplexed immunoassay method showed wide linear ranges of three orders of magnitude with the detection limits down to 3.5 and 3.9 pg mL⁻¹, respectively. The localized silver deposition, as well as the stripping detection process, eliminated completely the electrochemical cross talk between adjacent immunosensors. The immunosensor array exhibited acceptable reproducibility, stability and accuracy, showing a promising potential in multianalyte determination for clinical application.     

A Label‐Free Amperometric Immunosensor Based on Redox‐Active Ferrocene‐Branched Chitosan/Multiwalled Carbon Nanotubes Conductive Composite and Gold Nanoparticles

A novel immunosensor has been developed by self‐assembling Au NPs onto a ferrocene‐branched chitosan/multiwalled carbon nanotubes (CS‐Fc/MWCNTs) modified electrode for the sensitive determination of hepatitis B surface antigen (HBsAg). The formation of CS‐Fc effectively avoids the leakage of Fc and retains its electrochemical activity. Incorporation of MWCNTs and Au NPs into CS‐Fc further increases the electrochemical active Fc in the CS films and provides interactive sites for the immobilization of HBsAb. The morphologies and electrochemistry of the formed biofilm were investigated by using scanning electron microscopy and electrochemical techniques. The immunosensor exhibits a specific response to HBsAg in the range of 1.0–420 ng mL^?1. Excellent analytical performance, fabrication reproducibility and operational stability of the proposed immunosensor indicated its promising application in clinical diagnostics.     

Comparison of Electrochemical and Surface Plasmon Resonance Immunosensor Responses on Single Thin Film

This paper reports results obtained when comparing an electrochemical enzyme immunosensor and a surface plasmon resonance (SPR) based immunosensor on the same gold surface installed in an electrochemical SPR flow cell. Simultaneous electrochemical and SPR measurements were performed on a gold surface modified with multilayers of poly‐L ‐lysine and poly‐styrenesulfonate assembled with the layer‐by‐layer method. First, we obtained the SPR response induced by the formation of an immunocomplex from the shift in the SPR angle by injecting an anti tumor necrosis factor‐α antibody solution labeled with alkaline phosphatase into the flow cell containing the multilayer modified with tumor necrosis factor‐α. Then we compared this SPR result with that obtained for the electrochemical oxidation current of p‐aminophenol catalyzed by alkaline phosphatase from p‐aminophenolphosphate on the same gold film. We compared the two immunosensor responses obtained using the different measurement principles and found that there was a high correlation efficient of 0.973 between them. This was because we were able to immobilize the immunoreagents with good stability and without losing the transport of the enzyme product in the multilayer whose thickness we easily controlled with nanometer scale accuracy. We also report that the detection limit of our electrochemical immunosensor after optimization was around 100 pg/mL (0.4 pM), which is one of the lowest values yet reported for an electrochemical immunosensor.     

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.     

An ultrasensitive electrochemical immunosensor for CA72-4 based on a signal amplification strategy of MoS2 nanoflower-supported Au nanoparticles

Accurate detection of cancer antigen 72-4 (CA72-4), a tumor-associated glycoprotein, is of great significance for gastric cancer diagnosis and immunotherapy monitoring. Modification of noble metal nanoparticles on transition metal dichalcogenides can significantly enhance functions, such as electron transport. Molybdenum disulfide gold nanoparticles nanocomposites (MoS₂-Au NPs) were prepared in this study and a series of characterization studies were carried out. In addition, a label-free, highly sensitive electrochemical immunosensor molybdenum disulfide -Au nanoparticles/Glassy carbon electrode (MoS₂-Au NPs/GCE) was also prepared and used for the detection of CA72-4. The electrochemical performance of the immunosensor was characterized by electrochemical techniques, such as cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS). The results indicated that better MoS₂-Au NPs nanomaterials have been synthesized, and the prepared electrochemical immunosensor, MoS₂-Au NPs/GCE, showed excellent electrochemical performance. The sensor exhibited high detection sensitivity under optimal conditions, including an incubation time of 30 min, an incubation temperature of 25 °C, and a pH of 7.0. The electrochemical immunosensor also had a low detection limit of 2.0 × 10^?5 U/mL (S/N = 3) in a concentration range of 0.001–200 U/mL, with good selectivity, stability, and repeatability. In conclusion, this study provided a theoretical basis for the highly sensitive detection of tumor markers in clinical biological samples.     

A Label-Free Amperometric Immunoassay for Thrombomodulin Using Graphene/Silver-Silver Oxide Nanoparticles as a Immobilization Matrix

A simple, sensitive, and label-free electrochemical immunosensor has been developed for the measurement of serum thrombomodulin (TM), an endothelial glycoprotein which is associated with the progression and metastasis of tumors. At first, the graphene nanosheets, which were dispersed in Nafion solution, were used to coat the bare gold electrode. Then, silver-silver oxide nanoparticles (Ag-Ag₂O NPs) were immobilized on the graphene-modified electrode by a one-step electrochemical deposition method. Lastly, a thrombomodulin antibody (anti-TM) was attached via amido-Ag affinity. This strategy combines graphene/Ag-Ag₂O NPs hybrid materials as an immobilization matrix and Ag-Ag₂O NPs also as an electrochemical signal indication reagent. The main advantage of this strategy has two important aspects. One is the high stability and unique electronic properties of the graphene nanostructure. The other is the use of Ag-Ag₂O NPs as the immobilization matrix and redox probes, thus avoiding the laborious labeling protein operation. Using this strategy, the concentration of TM in the range of 0.1 to 20 ng/mL was detected, with a detection limit of 31.5 pg/mL (at 3σ). The proposed methodology demonstrates that the nanocomposite film composed of graphene and Ag-Ag₂O NPs is a potential for biosensor applications.     

Signal‐Enhanced Amperometric Immunosensor Based on Ferrocene‐Branched Poly(allylamine)/Multiwalled Carbon Nanotubes Redox‐Active Composite

A signal‐enhanced immunosensor has been developed by self‐assembling Au NPs onto a ferrocene‐branched poly(allylamine)/multiwalled carbon nanotubes (PAA‐Fc/MWNTs) modified electrode for the sensitive determination of hepatitis B surface antigen (HBsAg) as a model protein. The formation of PAA‐Fc/MWNTs composite not only effectively avoided the leakage of Fc and retained its electrochemical activity, but also enhanced the conductivity and charge‐transport properties of the composite. Further adsorption of Au NPs into the PAA matrix provided both the interactive sites for the immobilization of hepatitis B surface antibody (HBsAb) and a favorable microenvironment to maintain its activity. Tests performed with this immunosensor showed a specific response to HBsAg in the range of 0.1–350.0 ng mL^?1 with a detection limit of 0.03 ng mL^?1.     

Amplified inhibition of the electrochemical signal of ferrocene by enzyme-functionalized graphene oxide nanoprobe for ultrasensitive immunoassay

A nanoprobe-induced signal inhibition mechanism was designed for ultrasensitive electrochemical immunoassay at a chitosan-ferrocene (CS-Fc) based immunosensor. The nanoprobe was prepared by covalently loading signal antibody and high-content horseradish peroxidase (HRP) on the graphene oxide (GO) nanocarrier. The immunosensor was prepared through the stepwise assembly of gold nanoparticles (Au NPs) and capture antibody at a CS-Fc modified electrode. After sandwich immunoreaction, the GO-HRP nanoprobes were quantitatively captured onto the immunosensor surface and thus induced the production of a layer of insoluble film through the enzymatically catalytic reaction of the HRP labels. Both the dielectric immunocomplex formed on the immunosensor surface and the enzymatic precipitate with low electroconductivity led to the electrochemical signal decease of the Fc indicator, which was greatly amplified by the multi-enzyme signal amplification of the nanoprobe. Based on this amplified signal inhibition mechanism, a new ultrasensitive electrochemical immunoassay method was developed. Using carcinoembryonic antigen as a model analyte, this method showed a wide linear range over 5 orders of magnitude with a detection limit down to 0.54 pg/mL. Besides, the immunosensor showed good specificity, acceptable reproducibility and stability as well as satisfactory reliability for the serum sample analysis.     

Simultaneous electrochemical immunosensor based on water-soluble polythiophene derivative and functionalized magnetic material

A novel, sensitive electrochemical immunosensor for simultaneous determination of squamous cell carcinoma associated antigen (SCC-Ag) and carcinoembryonic antigen (CEA) for the combined diagnosis of cervical cancer was designed. The amplification strategy for electrochemical immunoassay was based on poly[3-(1,1′-dimethyl-4-piperidine-methylene) thiophene-2,5-diylchloride] (PDPMT-Cl) and functionalized mesoporous ferroferric oxide nanoparticles (Fe₃O₄ NPs). PDPMT-Cl dispersed in chitosan solution with enhanced electrical conductivity and solubility was used as matrices to immobilize the first antibodies. Different redox probes (thionine (Th) and ferrocenecarboxylic acid (Fca)) functionalized Fe₃O₄ NPs incubated with two kinds of secondary antibodies to fabricate the labels. Using an electrochemical analysis technique, two well-separated peaks were generated by Th and Fca, making the simultaneous detection of two analytes on the electrode possible. Under optimized conditions, this method showed wide linear ranges of three orders of magnitude with the detection limits of 4 pg mL⁻¹ and 5 pg mL⁻¹, respectively. The disposable immunosensor possessed excellent clinical value in cervical cancer screening as well as convenient point-of-care diagnostics.     

A competitive microcystin-LR immunosensor based on Au NPs@metal-organic framework (MIL-101)

A porous metal organic frameworks (MOFs) material (MIL-101) based on trivalent chromium skeleton were synthesized by hydrothermal synthesis method, and loaded with Au nanoparticles (Au NPs) to prepare Au NPs@MIL-101 composite materials which were used as a marker to label anti microcystin-LR (Anti-MC-LR). The composite materials have strong catalytic properties to the oxidation of ascorbic acid. Anti-MC-LR was immobilized on glassy carbon electrode surface using electrodeposition graphene oxide (GO) as a fixed matrix to construct a competitive microcystin-LR immunosensor.     

Nanoparticle-based electrochemical immunosensor for the detection of phosphorylated acetylcholinesterase: an exposure biomarker of organophosphate pesticides and nerve agents

A nanoparticle-based electrochemical immunosensor has been developed for the detection of phosphorylated acetylcholinesterase (AChE), which is a potential biomarker of exposure to organophosphate (OP) pesticides and chemical warfare nerve agents. Zirconia nanoparticles (ZrO(2) NPs) were used as selective sorbents to capture the phosphorylated AChE adduct, and quantum dots (ZnS@CdS, QDs) were used as tags to label monoclonal anti-AChE antibody to quantify the immunorecognition events. The sandwich-like immunoreactions were performed among the ZrO(2) NPs, which were pre-coated on a screen printed electrode (SPE) by electrodeposition, phosphorylated AChE and QD-anti-AChE. The captured QD tags were determined on the SPE by electrochemical stripping analysis of its metallic component (cadmium) after an acid-dissolution step. Paraoxon was used as the model OP insecticide to prepare the phosphorylated AChE adducts to demonstrate proof of principle for the sensor. The phosphorylated AChE adduct was characterized by Fourier transform infrared spectroscopy (FTIR) and mass spectroscopy. The binding affinity of anti-AChE to the phosphorylated AChE was validated with an enzyme-linked immunosorbent assay. The parameters (e.g., amount of ZrO(2) NP, QD-anti-AChE concentration,) that govern the electrochemical response of immunosensors were optimized. The voltammetric response of the immunosensor is highly linear over the range of 10 pM to 4 nM phosphorylated AChE, and the limit of detection is estimated to be 8.0 pM. The immunosensor also successfully detected phosphorylated AChE in human plasma. This new nanoparticle-based electrochemical immunosensor provides an opportunity to develop field-deployable, sensitive, and quantitative biosensors for monitoring exposure to a variety of OP pesticides and nerve agents.     

Photoelectrochemical Immunosensor Array Based on Thioglycolic Acid Capped CdS Quantum Dots for Multiplexed Detection of Veterinary Drug Residues

A photoelectrochemical immunosensor based on multi‐electrode array was developed for simultaneous and sensitive determination of veterinary drug residues. In this system, poly(dimethyldiallylammonium chloride) (PDDA), Au nanoparticles (Au NPs) and thioglycolic acid (TGA)‐capped CdS quantum dots (QDs) were layer‐by‐layer assembled onto the home‐made Au electrode array. The assembling process of the (CdS/PDDA/Au NPs/PDDA)_n multilayer was characterized by electrochemical impedance spectroscopy. And then the antibodies for clenbuterol (CB), ractopamine (RAC) and chloramphenicol (CAP) were covalently immobilized onto the Au electrode array by 1‐ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide (EDC) coupling reaction, respectively. The concentrations of CB, RAC and CAP were measured based on the photoelectrochemical effects of CdS QDs. Under the optimal conditions, the limits of detection (LOD) for CB, RAC and CAP were 25, 50 and 2.2 pg/mL (3Δ), respectively, with acceptable recovery over the range of 95.40%–105.5% in pig liver samples. All results indicate that the immunosensor array system has potential application for practical, effective and high throughput analysis of veterinary drugs residues.     

Electrodeposited Cu‐Au Alloy Nanoparticles for Uric Acid Electrochemical Biosensor with Quick Response

A uric acid (UA) electrochemical biosensor based on the Cu‐Au alloy nanoparticles (NPs) and uricase was developed. The electrodeposition technique of Cu‐Au alloy NPs was selected to be a convenient potentiostatic method at –0.8 V in a single solution containing both Au(III) and Cu²⁺. Cyclic voltammetry and scanning electron microscopy proved the successful deposition of Cu‐Au alloy NPs. EIS demonstrated the good conductivity of Cu‐Au alloy NPs. The enzyme was immobilized on the surface of Cu‐Au alloy NPs modified electrode by casting with chitosan solution. The ultimate biosensor showed linear amperometric response towards UA in the concentration range of 3.0 to 26.0 μM with a detection limit of 0.8 μM. The main feature of the biosensor was its short response time, which was attributed to the good conductivity of Cu‐Au alloy NPs. Furthermore, the biosensor could avoid the interference of ascorbic acid and oxygen.     

Dual‐Amplification of Antigen–Antibody Interactions via Backfilling Gold Nanoparticles on (3‐Mercaptopropyl) Trimethoxysilane Sol‐Gel Functionalized Interface

A new dual‐amplification strategy of electrochemical signaling from antigen–antibody interactions was proposed via backfilling gold nanoparticles on (3‐mercaptopropyl) trimethoxysilane sol‐gel (MPTS) functionalized interface. The MPTS was employed not only as a building block for the electrode surface modification but also as a matrix for ligand functionalization with first amplification. The second signal amplification strategy introduced in this study was based on the backfilling immobilization of nanogold particles to the immunosensor surface. Several coupling techniques, such as with nanogold but not MPTS or with MPTS but not nanogold, were investigated for the determination of carcinoembryonic antigen (CEA) as a model, and a very good result was obtained with nanogold and MPTS coupling immunosensor. With the noncompetitive format, the formation of the antigen–antibody complex by a simple one‐step immunoreaction between the immobilized anti‐CEA and CEA in sample solution introduced membrane potential change before and after the antigen–antibody interaction. Under optimal conditions, the proposed immunosensor exhibited a good electrochemical behavior to CEA in a dynamic concentration range of 4.4 to 85.7 ng/mL with a detection limit of 1.2 ng/mL (at 3 δ). Moreover, the precision, reproducibility and stability of the as‐prepared immunosensor were acceptable. Importantly, the proposed methodology would be valuable for diagnosis and monitoring of carcinoma and its metastasis.     

Electrochemical Signal Tracing by Glucose Oxidase and Ferrocene Dually Functionalized Gold Nanoprobe for Ultrasensitive Immunoassay

A glucose oxidase (GOD) and ferrocene (Fc) dually functionalized gold nanoprobe was simply prepared for electrochemical immunoassay. By combination with sandwich immunoreaction at a carbon nanotube (CNT)‐based immunosensor and signal tracing of the nanoprobe through the Fc‐mediated GOD‐catalytic reaction, a new electrochemical immunoassay method was successfully developed. Both the multi‐enzyme signal amplification of the nanoprobe and the electron transfer promotion of the CNTs modified on the immunosensor greatly enhanced the signal response. Thus this method showed excellent analytical performance including ultrahigh sensitivity, wide linear range as well as good specificity, reproducibility, stability and reliability for human IgG measurement.     

苯并噻唑和铁氧化物纳米粒子修饰的磁性棒碳糊电极上肼的电催化氧化反应：同时检测肼和苯酚

开发了一种磁性Fe3O4纳米粒子和2-(3,4-二羟苯基)苯并噻唑(DPB)修饰的磁性棒碳糊电极(MBCPE)用于电化学检测肼.首先将DPB自组装在Fe3O4纳米粒子上,然后将此复合物吸附于设计的MBCPE上. MBCPE电极将磁性纳米粒子吸引到电极表面.所得新型电极具有高的导电性和大的有效比表面积,因而对肼的电催化氧化反应有非常大的电流响应.采用伏安法、扫描电镜、电化学阻抗谱、红外光谱和紫外-可见光谱对修饰电极进行了表征.采用伏安法研究了在磷酸盐缓冲溶液(pH=7.0)中MBCPE/Fe3O4NPs/DPB电极上肼的电化学行为.作为电化学传感器, MBCPE/Fe3O4NPs/DPB电极对肼氧化反应表现出极高的电催化活性.在DPB存在下,肼的氧化电势下降,但其催化电流增加.电催化电流与肼浓度在0.1–0.4和0.7–12.0μmol/L二个区间内表现出线性关系,检测限为18.0 nmol/L.另外,研究了MBCPE/Fe3O4NPs/DPB电极同时检测肼和苯酚的性能.伏安实验结果显示,苯酚的线性区域为100–470μmol/L,检测限为24.3μmol/L.采用此电极检测了水样品中的肼和苯酚.     

Heparin‐gold Nanoparticles and Liquid Crystal Applied in Label‐free Electrochemical Immunosensor for Prostate‐specific Antigen

A label‐free electrochemical immunosensor based on the liquid crystal (E)‐1‐decyl‐4‐[(4‐decyloxyphenyl)diazenyl]pyridinium bromide (Br−Py), together with heparin‐stabilized gold nanoparticles (AuNP‐Hep) and Nafion is proposed for the determination of prostate‐specific antigen (PSA). The Br−Py liquid crystal presented redox properties and good film‐forming abilities on the electrode surface, and thus it is a suitable alternative as a redox probe for a label‐free electrochemical immunosensor, which could simplify the analysis methodology. The stepwise construction of the immunosensor and the incubation process (immunocomplex formation) were characterized by voltammetry and electrochemical impedance spectroscopy. The proposed immunosensor could directly detect PSA concentrations in the incubation samples, based on the suppression of the Br−Py redox peak (‘base peak’) current. After optimization, the immunosensor exhibited a linear response to PSA concentrations in the range of 0.1 to 50 ng mL⁻¹, with a calculated detection limit of 0.08 ng mL⁻¹. The reproducibility (coefficient of variance less than 3.0 %), selectivity and accuracy of the methodology were adequate. The immunosensor was satisfactorily applied in the quantification of PSA in human blood plasma samples.     

A Multi‐electrochemical Competitive Immunosensor for Sensitive Cocaine Determination in Biological Samples

A multi‐electrochemical competitive immunosensor for the rapid determination of unmetabolized cocaine (COC) in urine, saliva and human serum matrices is reported. Anti‐cocaine polyclonal antibodies were immobilized in an oriented way onto protein‐G functionalized magnetic beads. The immunosensor is based on an array of eight carbon‐based screen‐printed electrodes for simultaneous electrochemical determinations. The treatments of the biological samples were simplified and optimized for avoiding matrix interferences. The immunosensor was sensitive (EC₅₀≈2.92–3.88 ng mL^?1 COC), required a very small volume of sample (200 µL), was reproducible (%RSD was lesser than about 18 %), and accurate (recovery percentages ranged 88–117 %).     

石墨烯复合材料电化学免疫传感器在肿瘤标志物检测中的应用

监测肿瘤标志物水平变化是评估肿瘤治疗效果的重要方法.基于石墨烯复合材料构建的电化学免疫传感器可实现对肿瘤标志物的检测,检测灵敏度高、特异性好,是快速、准确分析肿瘤标志物含量的理想检测工具.本文重点阐述了石墨烯复合材料的电化学免疫传感器在肿瘤标志物检测中的应用进展.总结了其在肿瘤标志物检测中应用的优势和不足,最后对基于石...     

Direct Electrochemical Nano‐immunosensor for Microcystin‐LR in Seawater

Microcystins are potent hepatotoxins produced by cyanobacteria, which proliferate in wastewaters with high nutrient content. Due to their high toxicity and potential risk to human health, even at low concentrations, the development of a sensitive and rapid method for the monitoring of microcystin‐LR (MC‐LR) in water samples is of great importance. In this context, a new direct electrochemical nano‐immunosensor for MC‐LR detection using the liquid crystal (E)‐1‐decyl‐4‐[(4‐decyloxyphenyl)diazenyl]pyridinium bromide (Br‐Py) as a redox probe and gold nanoparticles stabilized in bovine serum albumin (AuNP‐BSA) is described herein. The microcystin‐LR antibody (anti‐MC‐LR) was covalently immobilized using N‐(3‐dimethylaminopropyl)‐N‐ethylcarbodiimide hydrochloride (EDC) and N‐hydroxysuccinimide (NHS) on an AuNP‐BSA/BrPy film. The proposed sensor response is based on the inhibition of the Br‐Py electrochemical signal after the specific interaction of MC‐LR with immobilized anti‐MC‐LR on the electrode surface. The electrochemical behavior of the immunosensor was studied by square‐wave voltammetry (SWV) and electrochemical impedance spectroscopy (EIS). Under optimized conditions, using SWV and an incubation time of 15 min, the immunosensor exhibits a linear response to MC‐LR concentrations of 0.05 to 500.0 ng mL⁻¹ with a detection limit of 0.05 ng mL⁻¹. The anti‐MC‐LR/AuNP‐BSA/Br‐Py/GCE was successfully applied in the determination of MC‐LR in spiked seawater samples.