Regenerable Leptin Immunosensor Based on Protein G Immobilized Au‐Pyrrole Propylic Acid‐Polypyrrole Nanocomposite

A regenerable, labelless electrochemical immunosensor is investigated. In this work, pyrrole (Py) and pyrrole propylic acid (Pa) were co‐electropolymerized in the presence of gold nanoparticles to form a porous, conductive, stable and hydrophilic nanocomposite, followed by the covalent attachment of protein G to capture an antibody as the probe for the immunoassay. The regeneration of the sensor was achieved by rinsing the electrodes with 0.1 M glycine buffer (pH 2.7). The binding and dissociation of the antibody with protein G and optimization of the efficient immobilization were studied by impedance and optical measurements, respectively. The charge transfer resistance obtained from the impedance measurements is used to study the interaction between antibody‐protein G and antibody‐antigen. The immunosensor performance and its regenerability were evaluated by using anti‐leptin IgG as the probe protein to detect leptin in 0.01 M PBS, and its specificity was tested in 1% human serum. The leptin impedimetric immunosensor exhibits a detection dynamic range of 10–100 000 ng/mL with 10 ng/mL detection limit in 0.01 M PBS+1% serum solutions. This work proves the feasibility to make a sensitive, regenerative electrochemical immunosensor, which could be very useful for environmental control and food analysis.     

Chitosan/AuNPs Modified Graphene Electrochemical Sensor for Label‐Free Human Chorionic Gonadotropin Detection

A new immunosensor is presented for human chorionic gonadotropin (hCG), made by electrodepositing chitosan/gold‐nanoparticles over graphene screen‐printed electrode (SPE). The antibody was covalently bound to CS via its Fc‐terminal. The assembly was controlled by electrochemical Impedance Spectroscopy (EIS) and followed by Fourier Transformed Infrared (FTIR). The hCG‐immunosensor displayed linear response against the logarithm‐hCG concentration for 0.1–25 ng/mL with limit of detection of 0.016 ng/mL. High selectivity was observed in blank urine and successful detection of hCG was also achieved in spiked samples of real urine from pregnant woman. The immunosensor showed good detection capability, simplicity of fabrication, low‐cost, high sensitivity and selectivity.     

An Amperometric Immunosensor for IgG Based on Conducting Polymer and Carbon Nanotube‐Linked Hydrazine Label

An amperometric immunosensor for IgG was developed by covalently immobilizing anti‐IgG on multiwall carbon nanotube‐embedded conducting polymer, poly‐5,2′ : 5′′,2′′‐terthiophene‐3′‐carboxylic acid (MWCNT/pTTCA). The MWCNT/pTTCA modified electrode was characterized by SEM, EIS, and XPS. A hydrazine‐labeled secondary antibody‐MWCNT conjugate (Hyd‐MWCNT‐Ab₂) was applied for detection. Hydrazine was used as a catalyst for the reduction of hydrogen peroxide, which was monitored at ?0.3 V vs. Ag/AgCl. The calibration plots showed a linear range of 0.1–10 ng/mL with a detection limit of 0.084±0.004 ng/mL. The proposed immunosensor was evaluated for clinical applications in a rabbit serum sample.     

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.     

Biomolecule‐Doped Organic/Inorganic Hybrid Nanocomposite Film for Label‐Free Electrochemical Immunoassay of α‐1‐Fetoprotein

A new electrochemical immunosensor for the detection of α‐1‐fetoprotien (AFP) was developed based on AFP antibody (anti‐AFP)‐functionalized organic/inorganic hybrid nanocomposite membrane. To fabricate such a hybrid composite membrane, 3,4,9,10‐perylenetetracarboxylic acid‐bound thionine molecules (PTCTH) were initially doped into titania colloids (TiO₂), and then gold nanoparticles and anti‐AFP were immobilized onto the composite film in turn. Comparison with the electrode fabricated only with thionine not 3,4,9,10‐perylenetetracarboxylic acid, the immunosensor with PTCTH exhibited high sensitivity and fast electron transfer. The presence of gold nanoparticles provided a good microenvironment for the immobilization of biomolecules, enhanced the surface coverage of protein, and improved the sensitivity of the immunosensor. The modified process was characterized by scanning electron microscope (SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The surface topography of the membrane was investigated by scanning electron microscopy (SEM). Under optimal conditions, the proposed immunosensor exhibited a wide linear range from 2.5 to 200.0 ng/mL towards AFP with a detection limit of 0.5 ng/mL (S/N=3). The stability, reproducibility and precision of the immunosensor were acceptable. Comparison with the conventional enzyme‐linked immunosorbent assay (ELISA), the present method did not require more labeled procedures and washing steps. Significantly, the detection methodology provides a promising approach for other proteins or biosecurities.     

Metal Enhanced Electrochemical Cyclooxygenase‐2 (COX‐2) Sensor for Biological Applications

Pain measurement is commonly required in biomedical and other emergency situations, yet there has been no pain biosensor reported in literature. Conventional approaches for pain measurement relies on Wong‐Baker face diagrams, which are grossly inadequate for situations involving children or unconscious people. We report a label‐free immunosensor for monitoring the pain biomarker cylooxygenase‐2 (COX‐2) in blood. The sensor is based on the concept of metal‐enhanced detection (MED). MED relies on the idea that the immobilization of underpotential deposition (upd) metallic films deposited either as a monolayer or electrostatically held onto a solid gold substrate could significantly amplify bimolecular recognition such as involving antigen‐antibody (Ab‐Ag) interactions. The surface bound Ab‐Ag complex insulates the electrode; causing a decrease in concentration‐dependent redox signals. A linear detection range of (3.64–3640.00)×10^?4 ng/mL was recorded with a detection limit of 0.25×10^?4 ng/mL, which was 4 orders of magnitude lower than that reported for ELISA for the same biomarker. The immunosensor exhibited selectivity of less than 6 % for potential interferents.     

On‐Off PVC Membrane Based Potentiometric Immunosensor for Label‐Free Detection of Alpha‐Fetoprotein

A poly(vinylchloride) (PVC) membrane based potentiometric immunosensor for the direct detection of alpha‐fetoprotein (AFP) has been developed. First, Au colloid particle was chemisorbed upon amino groups of o‐phenylenediamine, which were dissolved in plasticized PVC membrane. Then alpha‐fetoprotein antibody (anti‐AFP) was immobilized upon the surface of the Au colloid particle to prepare a potentiometric AFP immunosensor. The Au colloid particle modified PVC membrane was characterized by digital photo and transmission electron microscope (TEM). The immunosensor exhibited fast potentiometric response (≤4 min) and showed specific response to AFP in the range of 4.9 to 158.5 ng/mL with a correlation coefficient of 0.9971 and a detection limit of 1.6 ng/mL. The factors influencing the performance of the immunosensor were also studied in detail. Moreover, the proposed method is economical and efficient as well as potentially attractive for clinical immunoassays.     

Double‐Layer Nanogold and Poly(amidoamine) Dendrimer‐ Functionalized PVC Membrane Electrode for Enhanced Electrochemical Immunoassay of Total Prostate Specific Antigen

A simple and portable electrochemical immunosensor for the detection of total prostate specific antigen (t‐PSA) in human serum was developed using a double‐layer nanogold particles and dendrimer‐functionalized polyvinyl chloride (PVC) membrane as immunosensing interface. To fabricate such a multifunctional PVC electrode, an o‐phenylenediamine‐doped PVC membrane was initially constructed, then nanogold particles and poly(amidoamine) G4‐dendrimer with a sandwich‐type format were assembled onto the PVC membrane surface, and then t‐PSA antibodies (anti‐PSA) were adsorbed on the nanogold surface. The detection principle of the immunosensor is based on the change in the electric potential before and after the antigen‐antibody interaction. The experimental conditions and the factors influencing the performance of the immunosensor were investigated. Under optimal conditions, the proposed immunosensor exhibits good electrochemical behavior in the dynamic range of 0.5–18 ng/mL relative to t‐PSA concentration with a relative low detection limit of 0.1 ng/mL (S/N=3). The precision, reproducibility, and stability of the immunosensor are acceptable. In addition, 43 serum specimens were assayed by the as‐prepared immunosensor, and consistent results were obtained in comparison with those obtained by the standard enzyme‐linked immunosorbent assay (ELISA). Compared with the conventional ELISAs, the developed immunoassay system was simple and rapid without labeling and separation steps. Importantly, the immobilization and detection methodologies could be extended for the immobilization and detection of other biomarkers.     

Electrochemical Immunoassay for Carbohydrate Antigen‐125 Based on Polythionine and Gold Hollow Microspheres Modified Glassy Carbon Electrodes

A new electrochemical immunosensor for the detection of carbohydrate antigen‐125 (CA125), a carcinoma antigen, was developed by immobilization CA125 antibody (anti‐CA125) on gold hollow microspheres and porous polythionine (PTH) modified glassy carbon electrodes (GCE). The gold hollow microspheres provided a biocompatible microenvironment for proteins, and greatly amplified the coverage of anti‐CA125 molecules on the electrode surface. The performance and factors influencing the immunosensor were investigated in detail. The detection is based on the current change before and after the antigen‐antibody interaction. Under optimal conditions, the amperometric changes were proportional to CA125 concentration ranging from 4.5 to 36.5 U/mL with a detection limit of 1.3 U/mL (at 3σ). The CA125 immunosensor exhibited good precision, high sensitivity, acceptable stability, accuracy and reproducibility. The as‐prepared immunosensors were used to analyze CA125 in human serum specimens. Analytical results suggest that the developed immunoassay has a promising alternative approach for detecting CA125 in the clinical diagnosis.     

A Reagentless Amperometric Immunosensor for Alpha‐Fetoprotein Based on Gold Nanoparticles/TiO2 Colloids/Prussian Blue Modified Platinum Electrode

A novel reagentless amperometric immunosensor for the determination of alpha‐fetoprotein (AFP) was prepared by immobilizing TiO₂ colloids on Prussian blue (PB) modified platinum electrode, which yielded a positively charged interface with strong adsorption to deposit gold nanoparticles for immobilization of alpha‐fetoprotein antibody (anti‐AFP). The factors influencing the performance of the proposed immunosensors were studied in detail. Under the optimized conditions, cyclic voltammograms determination of AFP showed a specific response in two concentration ranges from 3.0 to 30.0 ng/mL and from 30.0 to 300.0 ng/mL with a detection limit of 1.0 ng/mL at a signal‐to‐noise ratio of 3. The proposed immunosensor exhibited high selectivity, good reproducibility, long‐term stability (>2 months) and good repeatability.     

Ultrasensitive potentiometric immunosensor based on SA and OCA techniques for immobilization of HBsAb with colloidal Au and polyvinyl butyral as matrixes

A novel potentiometric immunosensor for detection of hepatitis B surface antigen (HBsAg) has been developed by means of self-assembly (SA) and opposite-charged adsorption (OCA) techniques to immobilize hepatitis B surface antibody (HBsAb) on a platinum electrode. A cleaned platinum electrode was first pretreated in the presence of 10% HNO3 and 2.5% K2CrO4 solution and held at -1.5 V (vs SCE) for 1 min to make it negatively charged and then immersed in a mixing solution containing hepatitis B surface antibody, colloidal gold (Au), and polyvinyl butyral (PVB). Finally, HBsAb was successfully immobilized onto the surface of the negatively charged platinum electrode modified nanosized gold and PVB sol-gel matrixes. The modified procedure was characterized by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The immobilized hepatitis B surface antibody exhibited direct electrochemical behavior toward hepatitis B surface antigen (HBsAg). The performance and factors influencing the performance of the resulting immunosensor were studied in detail. More than 95.7% of the results of the human serum samples obtained by this method were in agreement with those obtained by enzyme-linked immunosorbent assays (ELISAs). The resulting immunosensor exhibited fast potentiometric response (<3 min) to HBsAg. The detection limit of the immunosensor was 2.3 ng.mL(-1), and the linear range was from 8 to 1280 ng.mL(-1). Moreover, the studied immunosensor exhibited high sensitivity, good reproducibility, and long-term stability (>6 months).     

A layer-by-layer assembly label-free electrochemical immunosensor for the detection of microcystin-LR based on CHIT/PAMAM dendrimer/silver nanocubes

Utilising the affinity and high combination ability between silver nanocubes and amino group (–NH₂), a novel electrochemical immunosensor was constructed for the ultrasensitive detection of microcystin-LR (MC-LR) based on G4-polyamidoamine (PAMAM) dendrimer and Ag nanocubes as immobilised substrate of anti-MC-LR. G4-PAMAM dendrimers were covalently bound on the chitosan (CHIT) – modified electrode by glutaraldehyde (GA), providing abundant amino groups to absorb much more Ag nanocubes comparing without using PAMAM. Subsequently, antibodies of MC-LR were immobilised with highly dense through Ag-NH₂. K₃Fe(CN)₆/K₄Fe(CN)₆ was used as electroactive redox probe. Ag nanocubes/PAMAM can enhance the antibody loading amount, which would bind more MC-LR and hinder the electron transfer of K₃Fe(CN)₆/K₄Fe(CN)₆. Differential pulse voltammetry (DPV) was employed to evaluate the analytical performance of the fabricated signal-off immunosensor. The response current had negative correlation with the concentration of MC-LR. The linear range covered was from 0.05 ng/mL to 25 μg/mL with detection limit (DL) of 0.017 ng/mL at 3σ. The proposed approach showed high specificity for the detection of MC-LR, with acceptable reproducibility, stability and reliability. Compared with the enzyme-linked immunoassay (ELISA) method by analyzing real water samples from Dian Lake, this immunosensor revealed acceptable accuracy with a relative error of 12.7%, indicating a potential alternative method for MC-LR detection in water sample.     

A Renewable Potentiometric Immunosensor Based on Fe3O4 Nanoparticles Immobilized Anti‐IgG

A renewable potentiometric immunosensor for detection of immunoglobulin G (IgG) has been developed by magnetic force attraction of Fe₃O₄ nanoparticles immobilized goat‐anti‐human IgG antibody. For preparing sensitive film of the sensor, cysteine was bonded on the nano‐Fe₃O₄ particles surface. The cysteine functionalized magnetic nanoparticles was attracted on a solid paraffin carbon paste electrode surface to covalently immobilize of anti‐immunoglobulin G (anti‐IgG) by employing a conventional glutaraldehyde‐crosslinking method. The immunosensor showed a specific response to human immunoglobulin G in the range of 0.1–1.2 ng/mL with a detection limit of 0.023 ng/mL. The immunosensor based on the magnetic nanoparticles was made easily by this method. It can be used expediently, renewed easily and low‐cost relatively. The renewable potentiometric immunosensor with better stability and higher sensitivity can be employed extensively in clinical diagnosis, monitoring of disease and environmental studies and etc.     

Amperometric Immunosensor Based on L-Cysteine/Gold Colloidal Nanoparticles for Carbofuran Detection

In this study, anti-carbofuran monoclonal antibodies (Ab) were immobilized onto a gold electrode surface modified with multilayers of L-cysteine and gold colloidal nanoparticles (GNPs). Furthermore, horseradish peroxidase (HRP) as enzyme membrane was used for blocking unspecific sites and amplifying signal. The conformational properties of the immunosensor were characterized using electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The concentration of antibody solution, pH of working buffer and incubation time were studied in detail for optimization of analytical performance. Under optimal conditions, the variation of current response was proportional to the concentration of carbofuran which ranged from 0.01 ng/mL to 50 ng/mL with a correlation coefficient of 0.9912. The detection limit was 0.01 ng/mL (S/N = 3). The proposed immunosensor exhibited good reproducibility and stability and it can be used for the rapid detection of carbofuran pesticide.     

基于双层纳米金修饰的高灵敏电位型乙肝表面抗原免疫传感器研究

基于电沉积和层层自组装技术，提出了一种新的生物分子固定化方法，研制成一种高灵敏电位型乙肝表面抗原免疫传感器。利用L-半胱胺酸（LCys）的双官能团结合双层纳米金，从而通过比表面积大，生物相容性好的纳米金胶吸附大量抗体，同时用聚乙烯醇缩丁醛（PVB）薄膜的笼效应把乙肝表面抗体（HBsAb）和纳米金固定在玻碳电极上，从而制得了高灵敏度、高稳定性的电位型免疫传感器。采用循环伏安法（CV）对电极的层层自组装过程进行了考察，并对该免疫传感器的性能进行了详细的研究。该免疫传感器线性范围是8．5～256．0ng／mL，线性相关系数为0．9978，灵敏度为89．0，检出限为3．1ng／mL。已用于病人的血清样品分析。     

Amperometric Immunosensor for the Determination of α‐1‐Fetoprotein Based on Core‐Shell‐Shell Prussian Blue‐BSA‐Nanogold Functionalized Interface

In the present work, a newly functional nanoparticle has been prepared to immobilize the protein for the detection of α‐1‐fetoprotein (AFP). Prussian blue (PB) nanoparticle was initially synthesized under ultrasonic condition, then bovine serum albumin (BSA) was used to coat the PB nanoparticle to improve the stability of the PB nanoparticle as well as functionalize the surface of PB nanoparticle, and then gold colloids were loaded on the BSA‐coated PB nanoparticle to construct a core‐shell‐shell nanostructure via the conjunction of thiolate linkages or alkylamines of the BSA. Finally, a convenient, effective and sensitivity amperometric immunosensor for the detection of α‐1‐fetoprotein (AFP) was constructed by the employment of these functional core‐shell‐shell microspheres. The preparation of the nanoparticle (Au‐BSA‐PB NPs) was characterized by transmission electron microscopy (TEM), and the assembly of the biosensor was characterized with cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The dynamic range of the resulted immunosensor for the detection of AFP is from 0.02 ng/mL to 200.0 ng/mL with a detection limit of 0.006 ng/mL (S/N=3). Moreover, this biosensor displays good selectivity, stability and reproducibility.     

Gold Nanoparticle‐Based Redox Signal Enhancement for Sensitive Detection of Human Chorionic Gonadotropin Hormone

A sensitive immunosensor for the detection of pregnancy marker, human chorionic gonadotropin hormone (hCG), was developed using the direct electrical detection of Au nanoparticles. We utilized disposable screen‐printed carbon strips (SPCSs) for the development of our immunosensor, which provided cost‐effective tests with the required antigen sample volume as small as 2 μL. After the recognition reaction between the surface‐immobilized primary antibody and hCG, the captured antigen was sandwiched with a secondary antibody that was labeled with Au nanoparticles. Au nanoparticles were exposed to a preoxidation process at 1.2 V for 40 s, which was subsequently followed with a reduction scan on the same surface using differential pulse voltammetry (DPV). We could observe Au nanoparticle‐labeled antigen‐antibody complexes immobilized on the surface of SPCS using scanning electron microscopy (SEM). Additionally, the number of Au nanoparticles on the immunosensor was determined using SEM images, and showed a linear relationship with the current intensity obtained from the DPV measurements with a detection limit of 36 pg/mL hCG (612 fM, 3.6×10^?4 IU/mL). Our immunosensor system, a combination of the screen‐printing technology with Au nanoparticles provides a promising biosensor for various applications in life sciences.     

基于AuNPs/PDDA-GO纳米复合物的电化学免疫传感器的构建及对SirT1蛋白的检测

基于AuNPs/PDDA-GO纳米复合物制备了一种新型电化学免疫传感器, 并将其用于SirT1的检测. 首先, 在电极表面修饰复合材料AuNPs/PDDA-GO, 然后将目标蛋白SirT1固定到修饰了AuNPs/PDDA-GO的电极表面, 再通过特异性免疫反应结合一抗(Ab₁)和辣根过氧化酶标记的二抗分子(HRP-Ab₂), 最后用示差脉冲伏安法检测电流信号, 实现了对SirT1蛋白水平的测定. 在优化的实验条件下, SirT1蛋白的浓度在0.1~100 ng/mL范围内与响应电流呈良好线性关系, 检出限为0.029 ng/mL.     

The Signal‐Enhanced Label‐Free Immunosensor Based on Assembly of Prussian Blue‐SiO2 Nanocomposite for Amperometric Measurement of Neuron‐Specific Enolase

A signal‐enhanced label‐free electrochemical immunosensor was constructed by the employment of Prussian blue doped silica dioxide (PB‐SiO₂) nanocomposite. At first, PB‐SiO₂ nanocomposite which was produced by using a microemulsion method was used to obtain a nanostructural monolayer on a glassy carbon electrode (GCE) surface. Next amino‐functionalized interface were prepared by self‐assembling 3‐aminopropyltriethoxy silane (APTES) on the PB‐SiO₂ nanoparticle surface. Then chitosan stabled gold nanoparticle (CS‐nanoAu) was subsequently attached, while the entire surface was finally loaded with neuron‐specific enolase antibody (anti‐NSE) via the adsorption of gold nanoparticle. The sensitivity of the proposed immunosensor has greatly improved as the PB‐SiO₂ nanostructural sensing film provides plenty of active sites which might catalyze the reduction of H₂O₂. The immunosensor exhibited good linear behavior in the concentration range from 0.25–5.0 and 5.0–75 ng/mL for the quantitative analysis of neuron‐specific enolase (NSE), a putative serum marker of small‐cell lung carcinoma (SCLC), with a limit of detection of 0.08 ng/mL. The resulting NSE immunosensor showed high sensitivity and long‐term lifetime which can be attributed to the extremely high catalytic activity and biocompatibility of CS‐nanoAu/APTES/PB‐SiO₂ nanostructural multilayers.     

QCM‐Based Immunosensor for the Determination of Ochratoxin A

Abstract

A piezoelectric immunosensor based on a competitive format was developed for determination of ochratoxin A (OTA) concentration. Surface modifications via two self‐assembled monolayers (SAMs) were investigated respectively and a better result was obtained with the SAM of 16‐mercaptohexadecanoic acid (16‐MHDA). The quartz crystal microbalance (QCM)‐based immunosensor was fabricated by immobilizing anti‐OTA antibodies onto the surface of the 16‐MHDA‐modified electrode, and allowing competition between free OTA and that conjugated with BSA to occur. The assay exhibited a working range of 50–1000 ng/mL and a detection limit of 16.1 ng/mL. Studies of interference and matrix effects were performed to evaluate the feasibility of the developed immunosensor for the direct analysis of OTA in real samples. Recoveries were conducted at 50, 200, and 1000 ng/g and were determined to be in the range of 142%–76%. The OTA assay is specific. No cross‐reactivates were observed with citrinin.