L-丝氨酸/壳聚糖/石墨烯/纳米金修饰电极的制备及应用

将L-丝氨酸(L-Serine)电聚合到裸金电极表面,再将壳聚糖(CS)、纳米金(Nano-Au)、石墨烯(GO)混合液滴涂在L-丝氨酸修饰的金电极上,制成L-Serine/GO/Nano-Au/CS/Au/CME电化学传感器.考察了胞嘧啶在该传感器上的电化学行为,优化了实验条件.结果表明,该传感器对胞嘧啶有良好的选择性和灵敏度,胞嘧啶的浓度在1.0×10~(-7)~1.0×10~(-3)mol/L范围内与峰电流的减小量呈现良好的线性关系,检出限为3.2×10~(-8)mol/L(S/N=3).将该传感器应用于实际样品中测定胞嘧啶,结果令人满意.     

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%。     

基于磁性Fe_3O_4/Au-Pt纳米复合物构建甲胎蛋白免疫传感器的研究

制备了磁性Fe3O4/Au-Pt纳米复合物,将该纳米复合物修饰在玻碳电极表面,用牛血清蛋白封闭电极表面非特异性吸附位点。通过循环伏安法对电极的制备过程以及响应性能进行表征。实验结果表明,该修饰电极对甲胎蛋白(AFP)有良好的电流响应,在甲胎蛋白浓度为0.05~1.0ng/mL和1.0~100.0ng/mL两个范围内,分别呈良好的线性,检出限为0.01ng/mL。该免疫传感器制备方法简单,灵敏度较高,有望应用于临床医学中检测人体血清中甲胎蛋白含量。     

基于纳米四氧化三铁-二茂铁构建甲胎蛋白免疫传感器的研究

用对交联剂1-乙基-3-(3-二甲基氨苪基)-碳化二亚胺/N-羟基琥珀酰亚胺将壳聚糖中的氨基与二茂铁甲酸中的羧基交联,并将制备好的纳米四氧化三铁加入其中反应形成富含氨基和羧基的磁性纳米复合物。该纳米复合物修饰到金电极表面,通过氨基及静电作用,吸附纳米金溶胶及甲胎蛋白抗体(anti-AFP)含量,从而成功制得高灵敏电流型甲胎蛋白免疫传感器。通过循环伏安法考察了电极表面的电化学特性。研究了孵育时间、温度、pH对免疫传感器的影响。在最优实验条件下,甲胎蛋白(AFP)质量浓度在0.01~10 ng/mL和10~120 ng/mL范围内,该免疫传感器的还原峰电流值与AFP的质量浓度有良好的线性关系,检出限为3 pg/mL。     

葡萄糖氧化酶在纳米金/壳聚糖/离子液体BMIMPF6修饰电极上的直接电化学及其应用

用滴涂法将葡萄糖氧化酶(GOD)修饰到纳米金(Nano-Au)/壳聚糖(CS)/1-丁基-3-甲基咪唑六氟磷酸盐(BMIMPF6)复合材料修饰的金电极表面,制备了GODNano-Au/CS/BMIMPF6/Au生物传感器。采用循环伏安法(CV)和扫描电子显微镜(SEM)对该生物传感器进行表征,并对其制备条件、电化学性质进行了较为详细的研究。结果表明,复合材料不仅为GOD提供了良好的微环境,而且通过纳米尺寸效应和离子液体的高导电性,促进电子转移,使GOD具有更高的活性。该修饰电极可作为葡萄糖生物传感器,在最优条件下,葡萄糖浓度在1.0×10-4~1.0×10-6 mol·L-1范围内呈良好的线性关系,其线性相关系数r=0.9995,检出限为3.85×10-8 mol·L-1。     

爆米花型金纳米用于信号放大构建夹心型免疫传感器测定甲胎蛋白

通过原位合成法以金纳米种子作为母核诱导纳米金在其表面原位生长生成爆米花型金纳米粒子,标记甲胎蛋白抗体(anti-AFP)和辣根过氧化物酶(HRP)结合形成免疫复合物(antiAFP@popcorn-shaped GNP@HRP)作为检测抗体。氧化石墨烯-亚甲基蓝-金纳米粒子(GOM B-AuNPs)纳米复合材料滴涂到玻碳电极(GCE)表面,形成纳米复合物用于捕获甲胎蛋白抗体构建了免疫传感平台(anti-AFP/GO-MB-AuNPs/GCE)。实验采用夹心型免疫分析模式,功能化爆米花型金纳米材料与石墨烯纳米复合物呈现出良好的性能,建立了一种可行的电流型免疫分析法用于灵敏分析血清样品中甲胎蛋白AFP。在优化的实验条件下,免疫传感器DPV电流响应与AFP浓度的对数值呈正比,线性范围为0.001~20 ng/mL,检测限为0.31 pg/mL(S/N=3)。制备的免疫传感器有良好的精密度,选择性和稳定性,可初步应用于临床检验中AFP测定。     

基于纳米金/硫堇层层自组装的新型电流型酶-癌胚抗原免疫传感器

将Nafion 膜固定在金电极(Au)表面, 通过静电吸附和共价键合作用将硫堇(Thi)和纳米金颗粒(nano-Au)层层自组装到Nafion膜修饰的金电极表面. 再通过形成的纳米金单层吸附癌胚抗体(anti-CEA), 最后用辣根过氧化物酶(HRP)代替牛血清白蛋白(BSA)封闭电极上的非特异性吸附位点, 并同时起到放大响应电流信号的作用, 从而制得高灵敏、高稳定电流型酶-癌胚抗原(CEA)免疫传感器. 通过循环伏安和交流阻抗考察了电极表面的电化学特性, 并对该免疫传感器的性能进行了详细的研究. 该传感器对CEA检测的线性范围为2.5~80.0 ng/mL, 检测限为0.90 ng/mL.     

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。     

基于PoPD-MWCNTs-离子液体/纳米金的髓过氧化物酶免疫传感器

在离子液体([EMIM]Br)中, 将聚邻苯二胺(PoPD)-多壁碳纳米管(MWCNTs)复合物原位电聚合到金电极(Au)表面, 利用纳米金固载髓过氧化物酶(MPO)抗体, 构建了一种用于MPO检测的无需标记的电流型免疫传感器. 采用循环伏安法(CV)和扫描电子显微镜(SEM)对修饰过程进行表征. 探讨了邻苯二胺单体浓度、pH、孵育时间和孵育温度对该免疫传感器性能的影响. 实验结果表明, 该传感器在最适条件下对MPO响应良好, 其线性范围为0.25～350 ng/mL, 线性相关系数为0.9985, 检出限为0.07 ng/mL. 该电流型免疫传感器具有稳定性好、灵敏度较高、特异性好、结果准确可靠和可再生等优点, 可应用于临床检测.     

基于石墨烯/纳米金/壳聚糖复合膜固定化酶的有机磷生物传感器

将乙酰胆碱酯酶(AChE)固定到还原态氧化石墨烯(rGO)/纳米金(Au NPs)/壳聚糖(CS)复合膜修饰的玻碳电极表面,制备了高灵敏度的电化学生物传感器,用于有机磷农药的检测。由于rGO具有较大的比表面积和良好的导电性,复合材料不仅为保持AChE的生物活性提供了良好的生物相容环境,而且rGO/Au NPs的协同作用也提高了传感器的灵敏度。CS/SiO2复合溶胶-凝胶网格状的结构为酶的固定化提供了良好的载体。农药抑制率与乐果浓度的负对数在0.1~10.0ng/mL范围内呈线性关系,检出限为0.02ng/mL(S/N=3)。     

An Immunosensor for Ultrasensitive Detection of 1‐Pyrenebutyric Acid with Enhanced Electrochemical Performance Based on a Graphene‐Ionic Liquid Doped Chitosan Film Modified Glassy Carbon Electrode

This paper describes a highly sensitive and label‐free electrochemical immunosensor for the detection of 1‐pyrenebutyric acid (PBA) which is based on a graphene (GS), chitosan (CS), and ionic liquid (IL) composite modified glassy carbon electrode (GS‐CS‐IL/GCE). The modification process was monitored by transmission electron microscopy (TEM) and cyclic voltammetry (CV). Due to the synergistic effects of GS, CS, and IL, the biosensor exhibits excellent selectivity to PBA. The current response of the proposed immunosensor decreases linearly at two concentration ranges from 0.01 to 5 and from 5 to 150 ng mL^?1 with a detection limit of 0.01 ng mL^?1.     

Quantitative determinations of SiC and SiO2 in new ceramic materials by Fourier transform infrared spectroscopy

In this paper, we have critically evaluated the electrochemical behavior of the products of seven substrates of the enzyme label, alkaline phosphate, commonly used in electrochemical immunosensors. These products (and the corresponding substrates) include indigo carmine (3-indoyl phosphate), hydroquinone (hydroquinone diphosphate), 4-nitrophenol (4-nitrophenol phosphate), 4-aminophenol (p-aminophenyl phosphate), 1-naphthol (1-naphthyl phosphate), phenol (phenyl phosphate), and L-ascorbic acid (2-phospho-L-ascorbic acid). Cyclic voltammetry and amperometry of these products were carried out at glassy carbon (GC), screen-printed carbon (SPC) and gold (Au) electrodes, respectively. Among the products, L-ascorbic acid showed the most sensitive (24.8 microA cm(-2), 12.0 microA cm(-2), and 48.0 microA cm(-2) of 100 microM ascorbic acid at GC, SPC, and Au electrodes, respectively) and well-defined amperometric response at all electrodes used, making 2-phospho-l-ascorbic acid the best substrate in electrochemical detection involving an alkaline phosphatase (ALP) enzyme label. The 2-phospho-L-ascorbic acid is also commercially available and inexpensive. Therefore, it was the best choice for electrochemical detection using ALP as label. Using mouse IgG as a model, an ALP enzyme-amplified sandwich-type amperometric immunosensor was constructed. The immunosensor was designed by electropolymerization of o-aminobenzoic acid (o-ABA) conductive polymer on the surface of GC, SPC, and Au electrodes. The anti-mouse IgG was subsequently attached on the electrode surface through covalent bonding between IgG antibody and the carboxyl groups from poly(o-ABA). Using 2-phospho-L-ascorbic acid as a substrate, the poly(o-ABA)/Au immunosensor produced the best signal (about 297 times of current density response ratio between 1000 ng mL(-1) and 0 ng mL(-1) of mouse IgG), demonstrating that amperometric immunosensors based on a conducting polymer electrode system were sensitive to concentrations of the mouse IgG down to 1 ng mL(-1), with a linear range of 3-200 ng mL(-1) (S.D.<2; n=3), and very low non-specific adsorption.     

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.     

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.     

Amperometric immunosensors based on layer-by-layer assembly of gold nanoparticles and methylene blue on thiourea modified glassy carbon electrode for determination of human chorionic gonadotrophin

Nano-Au and methylene blue (MB) were assembled layer-by-layer (LBL) into films on the glassy carbon electrode modified by thiourea for detection of human chorionic gonadotrophin (HCG). The electrode modification process was characterized by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and scanning electron micrograph (SEM). The factors influencing the performance of the amperometric immunosensor were studied in detail. Tests performed with this immunosensor showed good linearity, the working range for the system was 1.0–100.0 mIU/mL with a detection limit of 0.3 mIU/mL at 3σ. Moreover, the studied immunosensor exhibited high sensitivity and long-term stability. The present work supplied a promising test method for clinical immunoassay.     

Improved electrochemical immunosensor for myeloperoxidase in human serum based on nanogold/cerium dioxide-BMIMPF6/L-Cysteine composite film

An electrochemical immunosensing assay for myeloperoxidase (MPO) determination in human serum has been developed. Firstly, L-Cysteine was initially electropolymerized on an Au electrode to form L-Cysteine film. After that cerium dioxide (CeO2) dispersed in 1-butyl-3-methylimidazolium hexafluorophosphate (BMIMPF6) were immobilized on the L-Cysteine film. Then the negatively charged nanogold particles were adsorbed onto the membrane via the positive charge of CeO2, which aimed at assembling more antibody of MPO (anti-MPO). The resulting immunosensor showed a high sensitivity, broad linear response to the MPO concentration comprised between 10 ng/mL and 400 ng/mL with a detection limit of 0.06 ng/mL. Moreover, the surface morphology of the electrode was studied by means of a scanning electron microscope and the electrochemical properties of the fabricated immunosensor were further characterized by cyclic voltammetry. Also, factors influencing the performance of the resulting immunosensors were studied in detail.     

Immunosensor for the determination of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> using a tyrosinase–mercaptopropionic acid modified electrode as an amperometric transducer

An amperometric immunosensor for the quantification of Staphylococcus aureus based on the coimmobilization of rabbit immunoglobulin G (RbIgG) and tyrosinase on a mercaptopropionic acid self-assembled monolayer modified gold electrode is reported. A competitive mode in which protein-A-bearing S. aureus cells and antiRbIgG labeled with alkaline phosphatase (AP) compete for the binding sites of immobilized RbIgG was used. Monitoring of the affinity reaction was carried out by the amperometric detection at -0.15 V of phenol generated in the enzyme reaction with AP, at the tyrosinase-modified electrode through the electrochemical reduction of the o-quinone formed. Optimization of the working variables, such as the immunosensor composition and incubation times, the applied potential, the working pH and the concentration of phenyl phosphate used as the AP substrate, was carried out. Under the optimized conditions, both the repeatability of the measurements and the reproducibility of the responses obtained with different immunosensors yielded relative standard deviation values for the steady-state current lower than 10%. The immunosensor showed a dynamic range from 4.4x10(5) to 1.8x10(7) S. aureus cells mL(-1), with a detection limit of 1.7x10(5) cells mL(-1). The limit of detection was remarkably improved by subjecting S. aureus cells to wall lysis by heat treatment. The value obtained was 2.3x10(3) cells mL(-1), which is adequate for the monitoring of S. aureus contamination levels in some foodstuffs. As an application, milk samples spiked with bacteria at the 4.8x10(3) cells mL(-1) level were analyzed.     

基于纳米银与有机多孔材料/纳米金修饰的甲胎蛋白免疫传感器研究

在金电极表面电沉积银为氧化还原探针,利用有机多孔材料(PTC-NH2)、纳米金(nano-Au)固载甲胎蛋白抗体(anti-AFP),制备出用于检测甲胎蛋白(AFP)的安培型免疫传感器。通过交流阻抗技术、循环伏安法研究了电极的电化学特性,考察了孵育时间、测试液pH值等实验条件对传感器性能的影响,并利用扫描电子显微镜(SEM)对电极的修饰过程进行了表征。该传感器对AFP有良好的电流响应,线性范围分别为1.0～20.0ng/mL和20.0～60.0 ng/mL,检测限为0.6 ng/mL。     

One‐Step Electrochemical Immunoassay for Carcinoembryonic Antigen in Human via Back‐Filling Immobilization of Gold Nanoparticles on DNA‐Modified Gold Electrodes

A new amperometric immunobiosensor for carcinoembryonic antigen (CEA) determination in human serum was developed via encapsulation of horseradish peroxidase‐labeled carcinoembryonic antibody (HRP‐anti‐CEA) in a gold nanoparticles/DNA composite architecture. The presences of gold nanoparticles provided a congenial microenvironment for the immobilized biomolecules and decreased the electron transfer impedance, leading to a direct electrochemical behavior of the immobilized HRP. The formation of the antibody–antigen complex by a simple one‐step immunoreaction between the immobilized HRP‐anti‐CEA and CEA in sample solution introduced a barrier of direct electrical communication between the immobilized HRP and the gold electrode surface. Under optimal conditions, the current change obtained from the labeled HRP relative to H₂O₂ system was proportional to the CEA concentration in two linear ranges from 0.5 to 15 ng/mL and 15 to 300 ng/mL with a detection limit of 0.1 ng/mL (at 3δ). The precision and reproducibility are acceptable with the intraassay CV of 6.3% and 4.7% at 8 and 60 ng/mL CEA, respectively. The storage stability of the proposed immunosensor is acceptable in a pH 7.0 PBS at 4 °C for 9 days. Moreover, the proposed immunosensors were used to analyze CEA in human serum specimens. Analytical results of clinical samples show the developed immunoassay has a promising alternative approach for detecting CEA in the clinical diagnosis.     

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.