多层自组装硫脲和纳米金电流型萘免疫传感器的研究

建立了基于硫脲和纳米金层层自组装技术的无标记、高灵敏电流型免疫传感器,用于萘的检测。利用循环伏安法研究了修饰电极表面的电化学特性以及测试溶液的pH值、孵育时间和温度对免疫传感器性能的影响。实验表明,此免疫传感器在含不同浓度萘抗原的PBS溶液(pH7.4)中37℃下孵育30 min后,在pH7.4的测试底液中测定,响应电流与萘浓度在0.5～100μg/L范围内有良好的线性关系,r=0.9986,检出限为0.08μg/L。此传感器制备简单,灵敏度高,稳定性好,可以重复使用。应用于实际水样中萘的测定,回收率为94.3%～107.0%。     

石墨烯-壳聚糖复合物修饰电极构建电化学免疫传感器对1-芘丁酸的检测研究

采用石墨烯(GS)和壳聚糖(CS)复合膜修饰玻碳电极(GS-CS/GCE),利用1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐(EDC)和N-羟基丁二酰亚胺(NHS)(4∶1)活化GS-CS/GCE,共价固定多环芳烃抗体(anti-PAHs),构建灵敏度高、稳定性好的非标记电流型免疫传感器,用于1-芘丁酸(PBA)的检测。运用扫描电子显微镜对GS-CS复合膜的形貌进行表征。在pH 7.0含10 mmol/L K3Fe(CN)6和0.1 mmol/L KCl的磷酸盐溶液中,通过循环伏安法和示差脉冲伏安法研究修饰电极表面的电化学性质,并考察了免疫传感器的电化学性能。研究表明,由于石墨烯和壳聚糖的协同作用,GS-CS修饰的玻碳电极在Fe(CN)64-/3-溶液中的峰电流明显增大,有利于提高免疫传感器的灵敏度。在优化实验条件下,电极表面的anti-PAHs抗体固定量显著提高,增强了电极的分子识别性能。由于anti-PAHs抗体-抗原结合物的导电性较差,免疫传感器的峰电流随着待测溶液中PBA浓度的增大而减小,PBA浓度在0.1～80μg/L范围内呈良好的线性关系,检出限为0.03μg/L。该免疫传感器重现性好、特异性强,用于实际样品的测定,回收率为90%～105%。     

基于纳米金/壳聚糖/石墨烯的癌胚抗原阻抗型免疫传感器

研究了在PBS缓冲介质中,一种检测癌胚抗原的新型免标记阻抗型免疫传感器的制备及应用,基于石墨烯、纳米金在玻碳电极表面组装制备传感器,通过循环伏安法、交流阻抗法对制备的传感器进行表征。在优化的实验条件下,该免疫传感器的阻抗值随着检测溶液中癌胚抗原(CEA)浓度的增大而增大,并在0.1～85 ng/mL CEA范围内呈线性关系,回归方程为△Ret=1605.55+39.26ρ;检测限为0.04 ng/mL(R=0.9992)。该免疫传感器可用于临床上对CEA的检测。     

溶胶凝胶法固定抗体制备黄曲霉毒素免疫传感器

利用溶胶凝胶法,将正硅酸乙酯在HCl存在下水解形成的硅溶胶和黄曲霉毒素B1抗体的混合液涂于玻碳电极表面,制备非标记型电化学阻抗免疫传感器。以[Fe(CN)6]3-/4-的磷酸盐缓冲溶液为测试底液,分别研究传感器的循环伏安和交流阻抗行为。实验表明,电极因免疫反应所形成的复合物阻碍了[Fe(CN)6]3-/4-在电极表面的扩散,其氧化还原峰电流明显减小,电子转移阻抗随黄曲霉毒素浓度增加而线性增大。当介质pH=6.5和孵育时间为20 min时,免疫前后传感器的电子转移阻抗变化值最大。在此最佳条件下,传感器电子转移阻抗对黄曲霉毒素响应的线性范围为1.0～10μg/L;检出限为0.1μg/L(S/N=3)。此方法具有高的灵敏度和稳定性,可应用于食品中黄曲霉毒素的测定。     

氨基脲分子印迹电化学传感器的制备及应用

以氨基脲(SEM)为模板分子,邻苯二胺为功能单体,在0.1 mol/L NaAc-HAc缓冲液(pH 5.2)中,通过电聚合法制备分子印迹传感器.采用循环伏安法(CV)、差分脉冲法(DPV)、电化学阻抗谱(EIS)和扫描电镜表征了此分子印迹传感器的识别性能和表面形貌.结果表明,此传感器具有粗糙、多孔的形貌,对SEM具有良好的选择性识别.在0.5 mol/L KCl-0.005 mol/L K3[Fe(CN)6]溶液中,采用DPV方法考察了功能单体与模板分子的摩尔比、模板洗脱剂、洗脱时间和温育时间对传感器响应的影响.优化后的实验条件为:功能单体与模板分子的摩尔比1:2,洗脱液为0.2 mol/L NaOH,洗脱时间15 min,温育时间12 min.在优化的实验条件下,传感器的DPV峰电流差值与SEM的浓度在3.75～ 188 ng/L范围呈良好的线性关系,检出限为1.5 ng/L(S/N=3).将此传感器应用于市售新鲜虾肌肉及受污染虾肌肉中SEM含量测定,加标回收率为80％～97％.     

石墨烯-壳聚糖修饰电极构建石杉碱甲免疫传感器

通过将石杉碱甲抗体(anti-HupA)固定到由还原态氧化石墨烯/壳聚糖(r GO/CS)复合膜修饰的玻碳电极表面,制备高灵敏度的电化学免疫传感器,利用石杉碱甲抗原抗体特异性反应可阻断[Fe(CN)6]3-/4-氧化还原反应体系电子转移的特点,建立了检测石杉碱甲(Hup A)的电化学免疫传感器分析方法。采用循环伏安法(CV)、交流阻抗法(EIS)及差示脉冲伏安法(DPV)研究Hup A在[Fe(CN)6]3-/4-氧化还原体系的电化学行为,利用DPV法测定Hup A浓度。在优化条件下,15 mmol/L K3[Fe(CN)6]+0. 1 mol/L KCl+0. 1 mol/L PBS(pH 6. 5)测试体系中,Hup A浓度的对数与峰电流差值在1. 0×10-13~1. 0×10-10mol/L范围内呈良好的线性关系,相关系数(r)为1. 000 0,检出限为3. 0×10-14mol/L,回收率为99. 4%~102%。该方法具有特异性强、灵敏度高、操作简单、绿色环保等特点,可用于中药材中石杉碱甲浓度的快速检测。     

高稳定性安培型葡萄糖生物传感器的研究

采用有机改性溶胶凝胶-聚乙烯醇(Ormosil-PVA)包埋葡萄糖氧化酶,制备普鲁士蓝修饰玻碳电极,并基于此制备了安培型葡萄糖生物传感器。以含有FeCl3,K3Fe(CN)6和EDTA的溶液为沉积液,通过循环伏安电沉积法制备了高稳定性的普鲁士蓝修饰电极。考察了扫描电位范围、外加电位、pH和温度等因素对传感器的影响。葡萄糖浓度在20μmol/L~2 mmol/L范围内与响应电流呈线性关系,相关系数R=0.9965,以3倍空白值的标准偏差计算此传感器的检出限为8.1μmol/L。其中,在低浓度(20~100μmol/L)范围内,电流与浓度也呈良好的线性关系(R=0.9938)。     

巯基β-环糊精/还原氧化石墨烯/金纳米粒子复合膜修饰电极间接检测3,3′,4,4′-四氯联苯

本文采用巯基β-环糊精(β-CD-SH)/还原氧化石墨烯(rGO)/金纳米粒子(AuNPs)复合膜对玻碳电极(GCE)进行修饰,构建了以K_3[Fe(CN)_6]为探针,间接检测3,3′,4,4′-四氯联苯(PCB77)的电化学传感器。通过循环伏安法(CV)与差分脉冲伏安法(DPV)对该传感器的电化学性质及电极反应机理进行了研究,并优化了电极的制备及其检测PCB77的条件。在最佳条件下,PCB77浓度在0.5～10.0μmol/L范围与峰电流变化值ΔI_p呈良好的线性关系,相关系数R=0.9954,检出限为0.081μmol/L。对实际样品的检测,加标回收率在91.2%～106.2%之间。     

用于高灵敏检测人肠道病毒71型的纳米金电化学免疫传感器

在玻碳电极表面滴加纳米金,通过纳米金(AuNPs)的生物相容性以固定人肠道病毒71型(EV71)抗体从而制备EV71电化学免疫传感器,该电化学免疫传感器可灵敏检测EV71。在pH 7.0的含有0.1mol·L-1氯化钾和5mmol·L-1 K3Fe(CN)6/K4Fe(CN)6的磷酸盐缓冲溶液中,通过循环伏安法考察了该免疫传感器的分析性能。当EV71的质量浓度在0.1~80μg·L-1范围内时,氧化还原探针[Fe(CN6)]3-/4-的氧化峰电流随EV71质量浓度的增大呈线性降低,检出限(3S/N)为0.025μg·L-1。对20μg·L-1的EV71抗原标准溶液测定5次,测定值的相对标准偏差为3.0%。以空白样品为基体进行加标回收试验,所得回收率在98.3%~100%之间。     

铁氰化钾分光光度法测定头孢他啶

建立了K3[Fe(CN)6]光度法测定头孢他啶的方法。研究表明:控制溶液pH 4.0左右,头孢他啶可以将Fe(III)还原为Fe(II),还原生成的Fe(II)与K3[Fe(CN)6]反应生成可溶性的普鲁士蓝,其最大吸收波长为735 nm。头孢他啶在0.05～7.2μg/mL范围内与吸光度呈良好的线性关系,线性回归方程A=0.03414+0.07334c(μg/mL),线性相关系数R=0.9992,表观摩尔吸光系数ε=4.0×104 L/(mol.cm),检出限为0.026μg/mL。本法能直接用于注射用头孢他啶的含量的测定,结果满意。     

A label-free electrochemical immunosensor based on multi-functionalized graphene oxide for ultrasensitive detection of microcystin-LR

In this paper, a label-free electrochemical immunosensor for ultrasensitive detection of microcystin-leucine-arginine (MC-LR) based on multi-functionalized graphene oxide was constructed. The graphene oxide has a large surface area for the immobilization of the antibody. Meanwhile, the introduction of the AuNPs and 1-butyl-3-methylimidazolium hexafluorophosphate could enhance the response of the current by improving the electrical conductivity. Thus the electrochemical immunosensor could be prepared through a one-step process and differential pulse voltammetry was employed to detect sensitively MC-LR. Under optimal conditions, the current response of the immunosensor decreased proportionally to the logarithmic concentrations of MC-LR in the range of 0.1–1000 ng/mL with a detection limit of 0.1 ng/mL (S/N = 3). This one-step label-free electrochemical immunosensor showed good performance in specificity, stability, reproducibility, and application.     

双重信号放大的竞争型免疫传感器的制备及其应用于瘦肉精检测的研究

制备了葡萄糖氧化酶(GOD)-克伦特罗(Clenbuterol,CB)功能纳米复合物,并采用共价键合和温育组装等方法构建了双重信号放大的竞争型免疫传感器.研究了GOD催化氧化葡萄糖和普鲁士蓝(PB)催化还原H2O2双重信号放大的反应机理和传感器检测CB的作用机制.用扫描电子显微镜(SEM)等方法表征了纳米复合材料的形貌和复合物中GOD的活性,复合物中的GOD保持了良好的电催化性能和酶动力学响应,并且符合米氏动力学方程.最佳实验条件下,该免疫传感器对盐酸克伦特罗的检测线性范围为0.01～100 ng/mL,检测限达4.50 pg/mL.实验结果表明,该传感器对瘦肉精克伦特罗的检测具有灵敏度高,特异性强,重现性好,线性范围宽和检测限低等优点.将该方法用于猪肝样品的分析,加标样品回收率在97.5%～102%之间.该研究为瘦肉精及β-受体兴奋剂的分析提供了一种新方法.     

A novel impedance immunosensor based on O-phenylenediamine modified gold electrode to analyze abscisic acid

<正>An impedance immunosensor based on O-phenylenediamine modified gold electrode for the determination of phytohormone abscisic acid(ABA) was proposed.The operating pH,absorption time,absorption temperature and concentration of anti-ABA antibody were investigated to optimize the analytical performance.The calibration curve for the determination of ABA was obtained from this impedance immunosensor under optimal conditions.The results showed that the detection limit at about 1 ng/mL in the range of 10-5000 ng/mL.     

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.     

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.     

聚多巴胺仿生纳米微球用于构建电化学免疫传感器

利用多巴胺仿生聚合方法制备了具有良好生物相容性的聚多巴胺纳米微球,并在其表面原位合成银纳米颗粒.复合物微球具有良好的催化还原H2O2的性能以及良好的结合生物分子的能力.将制备的复合物微球作为标记物,将氨基化石墨烯作为基底材料,构建了检测人免疫球蛋白(Ig G)的夹心型电化学免疫传感器.运用循环伏安法和计时电流法对构建的电化学免疫传感器进行了性能分析,并对实验条件进行了考察优化.在最佳的实验条件下,免疫传感器的线性范围是0.1 pg/m L~15 ng/m L,检出限为0.025 pg/m L.     

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.     

Fabrication of an electrochemical immunosensor for α-fetoprotein based on a poly-L-lysine-single-walled carbon nanotubes/Prussian blue composite film interface

Single-walled carbon nanotubes functionalized with poly-L-lysine (PLL-SWCNTs) were successfully prepared and were used as a biocompatible platform to immobilize α-fetoprotein antibody (anti-AFP) which was labeled with horseradish peroxidase (HRP). Then, anti-AFP-HRP/PLL-SWCNT nanocomposites were coated onto a Prussian blue (PB) film-modified glassy carbon electrode surface. Glutaraldehyde was used to further stabilize the biosensing interface through a cross-linking step. All unspecific sites were blocked by bovine serum albumin to fabricate a novel electrochemical immunosensor for α-fetoprotein determination. The immunosensor was characterized by voltammetry and electrochemical impedance spectroscopy. Based on the catalytic current response of H₂O₂, the experimental conditions for α-fetoprotein determination were optimized. Under optimal conditions, the current response was linearly related to α-fetoprotein concentration in the range of 0.05～10.0 and 10.0～50.0 ng/mL with a detection limit of 0.011 ng/mL. The immunosensor was successfully used for the determination of α-fetoprotein in human blood plasma. The results were satisfied with that obtained with ELISA, demonstrating a good accuracy of the immunosensor.     

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。     

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.