压电免疫传感器用于B因子的测定

B因子是一种不耐热的球蛋白,它参与机体的防御,在炎症过程、细胞和组织损伤中均起重要作用.B因子的检测方法常用的有单扩散法、火箭电泳法和溶血法,前者灵敏度不高,重复性差;后两者操作较复杂^[1,2].     

蛋白A定向固定抗体的纤维蛋白压电免疫传感器的研究

将9MHz双面镀金石英晶体浸入蛋白A溶液中,在晶体电极表面形成一层均匀的蛋白A薄层,用于定向固定人体纤维蛋白抗体.在蛋白A层上形成一层有序致密的自组装抗体分子膜,研制成一种新型的用于人体纤维蛋白检测的压电免疫传感器.比较了3种固定抗体方法的效果,从传感器的灵敏度、稳定性、重现性等考虑,蛋白A吸附法优于聚乙烯亚胺及牛血清白蛋白固定抗体的方法.研究了蛋白A浓度、抗体效价以及抗原抗体反应时间等对传感器灵敏度的影响,考察了电极的选择性和再生能力.纤维蛋白在1×10^-4～1×10^-2g/L浓度范围内有良好响应.     

基于新型聚钙羧酸修饰电极的甲胎蛋白电化学免疫传感器研究

通过电聚合制得新型聚钙羧酸修饰电极并用于构建检测甲胎蛋白（AFP）的高灵敏电化学免疫传感器. 采用扫描电镜（SEM）、电化学交流阻抗（EIS）观察、表征修饰电极和AFP单克隆抗体（Ab1）固定前后的差异. 固定Ab1的电极与一定浓度的AFP、辣根过氧化物酶联AFP单克隆抗体（HRP-Ab2）反应,形成夹心型免疫复合物. 辣根过氧化物酶（HRP）催化3,3',5,5'-四甲基联苯胺（TMB）底物产生电流信号,实现AFP浓度的测定. 本检测方法灵敏度高,重现性好.     

Screen-printed electrodes as impedimetric immunosensors for the detection of anti-transglutaminase antibodies in human sera

The concentration of anti-transglutaminase antibodies in human sera is an important analytical marker for the diagnosis of the autoimmune disorder celiac disease. In this work, an immunosensor for the electrochemical detection of anti-transglutaminase antibodies in human sera was developed. The immunosensor is based on the immobilization of transglutaminase onto screen-printed gold electrodes which were covered with a polyelectrolyte layer of poly (sodium-4-styrensulfonic acid). The antigen-antibody interaction was evaluated using an amplification step: incubation with peroxidase (POD)-labeled immunoglobulins and subsequent biocatalytic oxidation of 3-amino-9-ethylcarbazole (AEC). Changes in the interfacial properties of the sensor electrode were determined by electrochemical impedance spectroscopy (EIS). Impedance spectra could be fitted to a Randles equivalent circuit containing a constant phase element (CPE). Furthermore, it was shown that impedance measurements could be simplified by performing EIS at only two selected frequencies, without loss of reliability. Incubation of these disposable immunosensor chips with various anti-transglutaminase antibody concentrations resulted in changes in their charge transfer resistance (R_ct). Thereby, a calibration graph could be established. Finally, immunosensors were used for characterizing different human sera with respect to their anti-transglutaminase autoantibody concentration of the IgG and IgA type.     

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.     

Detection of Prunus Necrotic Ringspot Virus in Plant Extracts with Impedimetric Immunosensor based on Glassy Carbon Electrode

This paper concerns the development of an immunosensor for detection of Prunus necrotic ringspot virus (PNRSV) in plant extracts. The immunosensor fabrication consists of successive modification steps of glassy carbon electrodes: (i) creation of COOH groups, (ii) covalent immobilization of protein A with EDC/NHS coupling reaction, (iii) immobilization of anti‐PNRSV IgG polyclonal antibody, (iiii) filling free spaces with BSA. Each step was controlled with cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The immunosensor was applied for the determination of the PNRSV virus in extracts from cucumber leaves using EIS technique. It was capable of discriminating between samples consisting of extracts from healthy plants and consisting of leaf extracts from infected plants diluted 10 000 times with extract from healthy plants.     

Sensitive electrochemical immunosensor array for the simultaneous detection of multiple tumor markers

A novel electrochemical immunosensor array for the simultaneous detection of multiple tumor markers was developed by incorporating electrochemically addressing immobilization and one signal antibody strategy. As a proof-of-principle, an eight-electrode array including six carbon screen-printed working electrodes was used as a base array for the analysis of two important tumor markers, carcinoembryonic antigen (CEA) and α-fetoprotein (AFP) and a horseradish peroxidase-labeled antibody was employed as a signal antibody. The immunosensor in the array was fabricated in sequence by covalently coupling the capture antibody onto the surface of the desired working electrode, which was firstly electrochemically addressably grafted with an aminophenyl group by reduction of in situ generated aminophenyl diazonium cation generated from p-phenylenediamine, using glutaraldehyde as cross-linker. This allowed the selective immobilization of the capture antibody at the desired position on a single array via an electrochemical operation. The immunoassay in sandwich mode was performed by specifically binding the targets, second antibodies and one signal antibody to the immunosensor array. The result showed that the steady current density was directly proportional to the concentration of target CEA/AFP in the range from 0.10 to 50 ng mL(-1) with a detection limit of 0.03 ng mL(-1) for CEA and 0.05 ng mL(-1) for AFP (S/N = 3), respectively. This work demonstrates that the employment of an electrochemically addressing method for the fabrication of an immunosensor array and one signal antibody is a promising approach for the determination of multiple tumor markers in clinical samples.     

Investigation of SPR and electrochemical detection of antigen with polypyrrole functionalized by biotinylated single-chain antibody: A review

An electrochemical label-free immunosensor based on a biotinylated single-chain variable fragment (Sc-Fv) antibody immobilized on copolypyrrole film is described. An efficient immunosensor device formed by immobilization of a biotinylated single-chain antibody on an electropolymerized copolymer film of polypyrrole using biotin/streptavidin system has been demonstrated for the first time. The response of the biosensor toward antigen detection was monitored by surface plasmon resonance (SPR) and electrochemical analysis of the polypyrrole response by differential pulse voltammetry (DPV). The composition of the copolymer formed from a mixture of pyrrole (py) as spacer and a pyrrole bearing a N-hydroxyphthalimidyl ester group on its 3-position (pyNHP), acting as agent linker for biomolecule immobilization, was optimized for an efficient immunosensor device. The ratio of py:pyNHP for copolymer formation was studied with respect to the antibody immobilization and antigen detection. SPR was employed to monitor in real time the electropolymerization process as well as the step-by-step construction of the biosensor. FT-IR demonstrates the chemical copolymer composition and the efficiency of the covalent attachment of biomolecules. The film morphology was analyzed by electron scanning microscopy (SEM).Results show that a well organized layer is obtained after Sc-Fv antibody immobilization thanks to the copolymer composition defined with optimized pyrrole and functionalized pyrrole leading to high and intense redox signal of the polypyrrole layer obtained by the DPV method. Detection of specific antigen was demonstrated by both SPR and DPV, and a low concentration of 1 pg mL⁻¹ was detected by measuring the variation of the redox signal of polypyrrole.     

Electrochemical Immunoassay for Tumor Marker CA19-9 Detection Based on Self-Assembled Monolayer

A CA19-9 electrochemical immunosensor was constructed using a hybrid self-assembled membrane modified with a gold electrode and applied to detect real samples. Hybrid self-assembled membranes were selected for electrode modification and used to detect antigens. First, the pretreated working electrodes were placed in a 3-mercaptopropionic acid (MPA)/β-mercaptoethanol (ME) mixture for 24 h for self-assembly. The electrodes were then placed in an EDC/NHS mixture for 1 h. Layer modification was performed by stepwise dropwise addition of CA19-9 antibody, BSA, and antigen. Differential pulse voltammetry was used to characterize this immunosensor preparation process. The assembled electrochemical immunosensor enables linear detection in the concentration range of 0.05–500 U/mL of CA19-9, and the detection limit was calculated as 0.01 U/mL. The results of the specificity measurement test showed that the signal change of the interfering substance was much lower than the response value of the detected antigen, indicating that the sensor has good specificity and strong anti-interference ability. The repeatability test results showed that the relative standard deviations were less than 5%, showing good accuracy and precision. The CA19-9 electrochemical immunosensor was used for the actual sample detection, and the experimental results of the standard serum addition method showed that the RSD values of the test concentrations were all less than 10%. The recoveries were 102.4–115.0%, indicating that the assay has high precision, good accuracy, and high potential application value.     

压电胰岛素-C肽微阵列免疫传感器研究

以AT切型、基频10MHz的镀金膜石英晶体作为换能器,将抗人胰岛素和C肽单克隆抗体固定在石英晶体电极表面,用2×5检测池固定夹具构建一种新型压电胰岛素-C肽微阵列免疫传感器.研究了抗体固定方法、抗体工作浓度、固定量、一致性以及传感器的响应参数如检测温度、时间和特异性等的影响.该微阵列传感器在胰岛素浓度为2.5～160.0mIU/L、C肽浓度为0.375～12.0ng/mL范围内响应特性良好,压电晶体频率偏移值与胰岛素和C肽浓度呈良好的线性关系.将此微阵列传感器用于人血清标本的测定,结果与放射免疫法符合(r为0.92和0.94).此微阵列传感器具有灵敏度高、特异性好,低密度阵列结构,检测通量较高,不需标记,操作简单、能实时在线检测和重复使用等优点,能用于临床实验诊断,具有临床推广应用价值.     

Capacitance Polypyrrole‐based Impedimetric Immunosensor for Interleukin‐10 Cytokine Detection

In the present study, we developed a novel label‐free capacitance impedimetric immunosensor based on the immobilization of the human monoclonal antibody anti‐interleukin‐10 (anti‐IL‐10 mAb) onto polypyrrole (PPy)‐modified silicon nitride (Si₃N₄) substrates. The immunosensor was used for the detection of the recombinant interleukin‐10 antigen (rh IL‐10) that may be secreted in patients at the early stage of inflammation. The immunosensor was created by chemical deposition of PPy conducting layer on pyrrole?silane (SPy)‐treated Si/SiO₂/Si₃N₄ substrates (Si/SiO₂/Si₃N₄?SPy), followed by anti‐IL‐10 mAb immobilization through carboxyl‐functionalized diazonium (CMA) protocol and carbodiimide chemistry. The surface characterization and the biofunctionalization steps were characterized by SEM, FTIR and cyclic voltammetry (CV) while the detection process was carried out by using electrochemical impedance spectroscopy (EIS) analyses. The created immunosensor showed two linear fittings (R²=0.999) for the detection of rh IL‐10 within the concentration range from 1–50 pg/mL. It exhibited high sensitivity (0.1128 (pg/mL)^?1) with a very low limit of detection (LOD)=0.347 pg/mL, more particularly, at the low concentration range (1–10 pg/mL). Thus, this developed polypyrrole‐based immunosensor represents a promising strategy for creation of miniaturized label‐free, fast and highly sensitive biosensors for diagnosis of inflammation biomarkers at very low concentrations with reduced cost.     

A novel biosensing interfacial design based on the assembled multilayers of the oppositely charged polyelectrolytes

A novel biosensing interfacial design strategy has been produced by the alternate adsorption of the oppositely charged polyelectrolytes. A quartz-crystal microbalance (QCM) as a model transducer was modified by use of mercaptoacetic acid (MAA) self-assembled monolayer (SAM) and the adsorption multilayers of the oppositely charged polyelectrolytes. MAA-SAM was first applied to the gold electrode surface of the crystal, and the positively charged chitosan was used as a double-sided linker to attach the negatively charged alginate-HSA antibodies to the negatively charged MAA-SAM layer. The assembly process and conditions were studied using the real-time output device and the surface topologies of the resulting crystals were characterized by atomic force microscopy (AFM) imaging. It is discovered that the optimal pH of immobilizing antibodies was 7.2 and the suited dilution ratio of antibodies was 10:30. The proposed immunosensor in optimal conditions has a linear detection range of 12.3-184.5 μg/mL for HSA detection. Comparing with the direct immobilization method of antibodies, the immunosensor with the proposed immobilization procedure shows some advantages, such as improved sensitivity due to the well-retained antibody activity and the significantly extended detection range. In particular, the regeneration of the developed immunosensor was simple and fast. Analytical results indicate that the developed immobilization procedure is a promising alternative for the immobilization of biorecognition element on the electrode surface.     

Piezoelectric immunosensor based on magnetic nanoparticles with simple immobilization procedures

A novel method for immobilizing antibodies (antigens) based on magnetic nanoparticles has been proposed for piezoelectric immunoassay. The goat-anti-IgG antibody (IgGAb) as the model analyte was first covalently immobilized to magnetic nanoparticles, which were surface modified with amino-groups. The magnetic bio-nanoparticles (MBN-s) formed were attached to the surfaces of quartz crystal with the help of a permanent magnet. The detection of immunoglobulin G (IgG) was performed with the sensor prepared. The process of immobilization and immunoreaction was monitored by frequency recording. From the SEM images of the sensor surface before and after immobilization of MBN, one can see that the MBN was homogeneously adsorbed on sensor surface. The piezoelectric immunosensor can determine IgG in the range of 0.6-34.9 μg ml⁻¹ with a detection limit of 0.36 μg ml⁻¹. The MBN and immunocomplex layer can easily be removed simply by taking away the magnetic field, making the piezoelectric sensor easy to be regenerated.     

A piezoelectric immunosensor for the detection of cortisol

A piezoelectric crystal immunosensor has been developed for the detection and determination of cortisol. Cortisol antibody was layered onto the gold electrodes of a 10 MHz piezoelectric crystal which was pre-coated with either protein A or gluteraldehyde. Crystals pre-coated with protein A showed the best results with respect to stability and sensitivity. The sensor was successfully used for the determination of cortisol in standard solutions from 36-3628 micrograms/L (part per billion). The advantages of the proposed sensor include simplicity, short analysis time, cost effectiveness and selectivity. The results demonstrate the feasibility of cortisol assay in clinical testing and in drug monitoring.     

基于丝网印刷电极的甲胎蛋白电化学免疫传感器

报道了一种基于金纳米粒子(AuNPs)双重信号放大的高灵敏电化学免疫传感器,并应用于肝癌标志物甲胎蛋白(AFP)的检测。通过在丝网印刷电极(SPE)表面电沉积AuNPs提高电极的重现性,利用AuNPs的吸附作用固定AFP抗体,用于捕获样品中的待测AFP抗原,并进一步与固定了辣根过氧化物酶(HRP)标记检测抗体的纳米金免疫探针发生特异性结合,所形成的夹心免疫复合物可以催化底物得到响应电流。用扫描电镜(SEM)和微分脉冲伏安法(DPV)等技术研究电极组装过程以及电极的化学性质,讨论了影响免疫传感器性能的因素。在最优实验条件下,传感器的峰电流信号与AFP浓度在2.5~30ng/mL范围内呈良好的线性关系,检出限为0.16ng/mL。该传感器具有灵敏度高、成本低、仪器体积小的优点,具有较好的应用前景。     

纳米金修饰电流型CA19-9免疫传感器的制备及应用

通过固定辣根过氧化酶（HRP）标记CA19-9抗体在纳米金修饰的碳糊电极制备了CA19-9安培免疫传感器。该免疫传感器在舍有CA19-9抗原的磷酸盐缓冲溶液中培育后,溶液中CA19-9抗原分子和HRP标记CA19-9抗体分子免疫结合导致了传感器电流的降低。在优化的实验条件下,样品中CA19-9浓度在2～30U／mL范围内与电流降低成线性关系,方法的检出限为1．4U／mL。该免疫传感器表现出较好的稳定性、准确性和重现性,为临床免疫分析提供了一种快速便捷方法。     

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.     

Detection of complement C1-inhibitor with a piezoelectric immunosensor

A novel piezoelectric immunosensor has been developed for the detection of human complement C1-inhibitor. Anti-C1-inhibitor antibody was immobilized onto the gold electrodes of a 9 MHz AT-cut piezoelectric crystal. Coating the crystal with polyethyleneimine adhesion, followed by a glutaraldehyde cross-linking method to immobilize antibody showed better results than the physical adsorption method with respect to sensitivity and reproducibility. Under the optimized experimental conditions, the sensor showed good response to the C1-inhibitor in the range from 2.0 x 10(-8) to 1.2 x 10(-6) g. Other proteins in human serum did not remarkably interfere with the detection. The crystals could be regenerated 5 times, when bound materials on the crystal surface were eluted by strong acid and strong alkali solution and subsequently cleaned in an ultrasonic cleaner.     

Surface plasmon resonance aided electrochemical immunosensor for CK-MB determination in undiluted serum samples

This article presents a simple chronoamperometric immunosensor for the quantitative assessment of creatine kinase MB (CK-MB) in 50 μL undiluted serum samples. The immunosensor consists of gold working and counter electrodes patterned onto a glass chip by thin-film photolithography and an external Ag|AgCl reference electrode. The detection limit (DL) of the chronoamperometric method is 13 ng mL⁻¹ (DL = 2×RMSD/S, where RMSD is the residual mean standard deviation of the measured points around a calibration curve with a slope of S). In spiked serum samples, the response was linear up to 300 ng mL⁻¹ of CK-MB. A surface plasmon resonance (SPR) system with simultaneous electrochemical detection (EC-SPR) aided the development of the sandwich immunoassay. Real-time monitoring of the SPR signal was used to optimize the capture antibody immobilization, CK-MB and detection antibody binding, as well as to minimize the nonspecific adsorption of serum proteins to the sensor surface. The detection antibody has been labeled with alkaline phosphatase (ALP) enzyme for sensitive electrochemical detection. ALP catalyzes the hydrolysis of ascorbic acid phosphate and generates ascorbic acid, which is measured chronoamperometrically. The electrochemical immunoassay for CK-MB was less sensitive to nonspecific adsorption related interferences, had a better detection limit, and required a lower volume of sample than the SPR method.     

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.