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Fe3O4@Au磁性纳米粒子修饰丝网印刷电极的微囊藻毒素免疫传感器研究

将核壳型Fe3O4@Au磁性纳米粒子修饰在丝网印刷工作电极表面,再通过纳米金和微囊藻毒素-(亮氨酸-精氨酸)抗体(anti-MCLR)之间的吸附作用,将抗体固定于电极表面,以牛血清白蛋白(BSA)封闭非特异性吸附位点,制得了检测MCLR的电流型免疫传感器。该传感器基于直接竞争的免疫分析模式,以辣根过氧化物酶偶联的微囊藻毒素(MCLR-HRP)为标记物,用差分脉冲伏安法检测微囊藻毒素,在优化的实验条件下,此免疫传感器响应的峰电流值与微囊藻毒素浓度在0.79～12.9μg/L范围内呈良好的线性关系,检测限为0.38μg/L。对实际水样进行了微囊藻毒素的加标回收实验,回收率在95%～107%之间。此免疫传感器具有测定速度快、灵敏高、携带方便等优点。     

碳纳米管/纳米金复合膜电化学免疫传感器用于微囊藻毒素的检测研究

在玻碳电极表面修饰碳纳米管,并用多电位阶跃法在碳纳米管表面沉积纳米金制得碳纳米管/纳米金复合膜。通过纳米金和微囊藻毒素-(亮氨酸-精氨酸)抗体之间的吸附作用,将抗微囊藻单克隆抗体固定于电极表面,以牛血清白蛋白封闭非特异性吸附位点,研制了检测微囊藻毒素的电化学免疫传感器。利用微囊藻毒素与其抗体之间的特异性识别作用构建"三明治"夹心结构的免疫分析模式,以辣根过氧化物酶标记抗体为二抗,利用微分脉冲伏安法实现了对微囊藻毒素的检测。在优化条件下,此传感器的响应电流与微囊藻毒素浓度在0.50~12.0μg/L范围内呈良好的线性关系,检出限为0.30μg/L(S/N=3)。对实际水样进行了微囊藻毒素的加标回收实验,回收率在93.0%~108.5%之间,相对标准偏差为3.8%~5.0%。     

基于Nafion-氧化石墨烯复合物/硫堇/纳米金构建过氧化氢传感器的研究

利用Nafion(全氟聚苯乙烯磺酸溶液)-氧化石墨烯复合物、硫堇和纳米金构建了H2O2酶传感器。首先将氧化石墨烯分散在体积分数0.2%Nafion溶液中制得Nafion-氧化石墨烯的复合物,并将其固定在玻碳电极表面,通过静电吸附将带正电荷的硫堇吸附到Nafion-氧化石墨烯复合膜修饰的玻碳电极表面,再利用静电吸附将纳米金修饰于电极上,通过纳米金来固定辣根过氧化物酶从而制得H2O2传感器。用循环伏安法和计时电流法考察该修饰电极的电化学特性。H2O2浓度为5.5×10-6~1.0×10-3mol/L时,酶电极的响应电流值与H2O2的浓度呈良好的线性关系,检出限为1.80×10-6mol/L。     

基于中性红电聚合膜的伏安型甲胎蛋白免疫传感器

用循环伏安法在玻碳电极上电聚合一层稳定的中性红聚合物膜,再通过共价键合作用将戊二醛和甲胎蛋白抗体自组装到电极表面,最后用辣根过氧化物酶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,制得高灵敏伏安型甲胎蛋白抗原免疫传感器。实验结果表明,该传感器对甲胎蛋白抗原具有良好的电流响应,该传感器的线性范围为1.00～10.0ng/mL和10.0～200ng/mL,检出限(3σ)为0.40ng/mL。     

高灵敏电位型免疫传感器对乙型肝炎表面抗原的诊断技术研究

以乙型肝炎表面抗原和乙型肝炎表面抗体为模型免疫蛋白，对传染病的诊断技术进行了研究. 利用吸附在铂电极表面Nafion膜中负电性的磺酸基与乙型肝炎表面抗体(HBsAb)分子中的氨基阳离子之间的静电作用实现抗体的结合，同时通过纳米金(Au)增加抗体的固定量，以及聚乙烯醇缩丁醛(PVB)薄膜的笼效应把乙型肝炎表面抗体和纳米金固定在铂电极上，从而制得高灵敏、高稳定电位型免疫传感器(PVB/Au/HBsAb/Nafion/Pt). 通过循环伏安法和交流阻抗技术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. 该免疫传感器具有制备简单、灵敏度高、线性范围宽、响应时间快(<3 min)、稳定性好、寿命长(>4个月)、选择性高等特点，将其用于病人的血清样品分析，其结果令人满意.     

基于双层酶信号放大及纳米功能界面的微囊藻毒素电化学免疫传感器

设计了一种基于纳米复合膜双层酶信号放大的竞争型电化学免疫传感器,用于检测痕量微囊藻毒素-LR(Microcystin-(leucine-arginine),MCLR)。在多壁碳纳米管修饰玻碳电极上电沉积金纳米粒子形成纳米复合膜,作为MCLR抗体(anti-MCLR)和辣根过氧化物酶(HRP)的固定化载体;引入HRP作为封闭剂或者通过抗体捕获HRP标记的MCLR(HRP-MCLR),起封闭活性位点及协同催化的作用。利用MCLR与HRPMCLR对纳米复合膜所固载抗体活性位点的竞争结合模式,采用微分脉冲伏安法(DPV)测定电极界面上HRP酶催化过氧化氢(H_2O_2)氧化对苯二酚(HQ)产生的还原电流,实现MCLR的定量检测。此传感器具有良好的特异性、稳定性与灵敏度,线性检测范围为0.1~100.0 μg/L,检出限为0.038 μg/L(S/N=3),对实际水样的加标回收率为72.9%~117.3%。     

基于聚(3,4-乙烯二氧噻吩)/天青Ⅰ复合物薄膜和纳米金修饰的电流型甲胎蛋白免疫传感器的研究

研制了一种基于聚(3,4-乙烯二氧噻吩)(PEDOT)与天青Ⅰ(AzureⅠ)为基体的电化学免疫传感器,可灵敏检测甲胎蛋白(AFP)。在铂盘电极表面,电化学聚合PEDOT为基体,利用静电组装技术固定AzureⅠ和纳米金颗粒(nanoAus),将甲胎蛋白抗体(anti-AFP)组装到nanoAus的表面。采用辣根过氧化物酶(HRP)封闭非特异性吸附位点,制得电流型AFP免疫传感器。采用循环伏安、扫描电镜技术研究组装过程及电极性质,探讨了影响免疫传感器性能的因素。在优化实验条件下,电极响应与AFP的浓度在0.01~120μg/L的范围内呈线性关系,检出限为0.003μg/L。取临床血清样品用本方法检测AFP含量,得到的结果与临床常用的ELISA法得到的结果无显著性差异。     

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。     

Disposable electrochemical immunosensor for myeloperoxidase based on the indium tin oxide electrode modified with an ionic liquid composite film containing gold nanoparticles, poly(o-phenylenediamine) and carbon nanotubes

A disposable electrochemical myeloperoxidase (MPO) immunosensor was fabricated based on the indium tin oxide electrode modified with a film composed of gold nanoparticles (AuNPs), poly(o-phenylenediamine), multi-walled carbon nanotubes and an ionic liquid. The composite film on the surface of the electrode was prepared by in situ electropolymerization using the ionic liquid as a supporting electrolyte. Negatively charged AuNPs were then adsorbed on the modified electrode via amine-gold affinity and to immobilize MPO antibody. Finally, bovine serum albumin was employed to block possible remaining active sites on the AuNPs. The modification of the electrode was studied by cyclic voltammetry and scanning electron microscopy. The factors affecting the performance of the immunosensor were investigated in detail using the hexacyanoferrate redox system. The sensor exhibited good response to MPO over two linear ranges (from 0.2 to 23.4 and from 23.4 to 300 ng.mL^?1), with a detection limit of 0.05 ng.mL^?1 (at an S/N of 3).

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.     

Development and validation of a screening procedure for the assessment of inhibition using a recombinant enzyme

Myeloperoxidase (MPO, E.C. 1.1.11.7) is a heme-containing enzyme that catalyses the synthesis of hypochlorous acid (HOCl) in the presence of hydrogen peroxide (H2O2) and chlorine anions. The production of HOCl is kept under strict control of neutrophils. However, in several pathological conditions, MPO is leaked in the extracellular fluid, which involves an over-production of reactive oxygen species like HOCl and promotes the damages caused by neutrophils. As a consequence, the inhibition of MPO by various agents has been investigated and a variety of molecules have been evaluated for this activity in different models. The present study aims to describe and validate a rapid screening method based on the taurine assay and using a recombinant MPO. After validation of the stock solutions used during the experiments, the amount of MPO for the completion of the reaction was measured and fixed to an optimal value. The inhibiting concentration at 50% of flufenamic acid (taken as a reference molecule) was then assessed in both a simple tube test and a microplate test and delivered similar results (1.3+/-0.2 microM vs 1.4+/-0.2 microM, P=0.2). Finally, different molecules able to inhibit MPO were evaluated in this rapid assay system providing results comparable to literature. The high throughput screening is undoubtedly a first line assessment method which affords the selection of inhibitors and permits to reduce the number of candidates for a further elucidation of the mechanism of MPO inhibition.     

Carbon nanotube wiring for signal amplification of electrochemical magneto immunosensors: application to myeloperoxidase detection

In this work, chronoamperometric myelo-peroxidase (MPO) detection was accomplished using immunofunctionalized magnetic microparticles (MPs), disposable carbon screen-printed electrodes (C-SPEs), and a ready-to-use commercially available tetramethylbenzidine (TMB)-based enzymatic substrate. In order to reach the limit of detection (LOD) needed to study real blood serum samples, assay performance was additionally improved by exploiting CNT wiring, which amplified the signal and decreased the LOD. The optimized assay can be performed in 30 min and yields LODs of 6 and 55 ng mL^?1 in PBS and undiluted human serum, respectively, making it useful for the identification of patients at risk of cardiovascular disease. These results demonstrate that electrode nanostructuring can be accomplished “post-assay,” which favors the development of enhanced magneto immunosensors based on the exploitation of cheap and simple SPE devices.     

聚邻苯二胺/镍氢氧化物修饰电极的研究

采用循环伏安法(CV)在聚邻苯二胺修饰玻碳电极表面络合Ni2+,然后将其置于NaOH溶液中CV扫描成功制备了镍氢氧化物/聚邻苯二胺/玻碳修饰电极(Ni(OH)2/PoPD/GC).通过CV探讨了聚合和负载机理,电化学交流阻抗谱(EIS)表征了电极修饰过程中界面阻抗变化,扫描电镜表征了PoPD膜负载Ni(OH)2后的形态...     

A new easy method for specific measurement of active myeloperoxidase in human biological fluids and tissue extracts

The SIEFED (“Specific Immunological Extraction Followed by Enzymatic Detection”) method already developed for the specific detection of the activity of equine myeloperoxidase (MPO) was adapted for the specific measurement of active human MPO in biological fluids or tissue extracts. The method consists of the extraction of MPO from aqueous solutions by immobilized anti-MPO antibodies followed by a washing (to eliminate the extraction medium and the biological fluid with their possible interfering molecules) and the measurement of the activity of MPO with a detection system containing a fluorogenic substrate, H₂O₂ and nitrite ions as reaction enhancer. The SIEFED was applied to study active MPO in human biological fluids (plasma, bronchoalveolar lavage fluid and supernatant from carotids extracts). The SIEFED for human MPO has a sensitivity limit of 0.080 mU/mL and showed good precision with intra- and inter-assay coefficients of variation below 10 and 20% respectively within a broad range of MPO activities establish from 0.156 to 473 mU/mL. The SIEFED for human MPO will be useful for the specific detection of active MPO in complex fluids and can be complementary to an ELISA to determine an active/total MPO ratio in healthy volunteers and patients especially in case of chronic or acute inflammatory diseases.     

AAO-CNTs electrode on microfluidic flow injection system for rapid iodide sensing

In this work, carbon nanotubes (CNTs) nanoarrays in anodized aluminum oxide (AAO-CNTs) nanopore is integrated on a microfluidic flow injection system for in-channel electrochemical detection of iodide. The device was fabricated from PDMS (polydimethylsiloxane) microchannel bonded on glass substrates that contains three-electrode electrochemical system, including AAO-CNTs as a working electrode, silver as a reference electrode and platinum as an auxiliary electrode. Aluminum, stainless steel catalyst, silver and platinum layers were sputtered on the glass substrate through shadow masks. Aluminum layer was then anodized by two-step anodization process to form nanopore template. CNTs were then grown in AAO template by thermal chemical vapor deposition. The amperometric detection of iodide was performed in 500-μm-wide and 100-μm-deep microchannels on the microfluidic chip. The influences of flow rate, injection volume and detection potential on the current response were optimized. From experimental results, AAO-CNTs electrode on chip offers higher sensitivity and wider dynamic range than CNTs electrode with no AAO template.     

Surface-renewable cobalt(II) hexacyanoferrate-modified graphite organosilicate electrode and its electrocatalytic oxidation of thiosulfate

Cobalt(II) hexacyanoferrate (CoHCF) was deposited on graphite powder by an in situ chemical deposition procedure and then dispersed into methyltrimethoxysilane-derived gels to prepare a surface-renewable CoHCF-modified electrode. The electrochemical behavior of the modified electrode in different supporting electrolyte solutions was characterized by cyclic voltammetry. In addition, square-wave voltammetry was employed to investigate the pNa-dependent electrochemical behavior of the electrode. The CoHCF-modified electrode showed a high electrocatalytic activity toward thiosulfate oxidation and could thus be used as an amperometric thiosulfate sensor.     

Sb在Au电极上不可逆吸附的电化学过程

用电化学循环伏安法和电化学石英晶体微天平(EQCM)技术研究了Sb在Au电极上不可逆吸附的电化学过程. 研究结果表明, 在-0.25 V到0.18 V(vs SCE)范围内, Sb可在Au电极上稳定吸附, 并且在0.15 V附近出现特征氧化还原峰. 根据EQCM实验数据, 在电位0.18 V时, Sb在Au电极上的氧化产物是Sb2O3; 同时Sb的吸附阻止了电解液中阴离子和水在Au电极上的吸附. 当电极电位超过0.20 V时, Sb2O3会被进一步氧化成Sb5+化合物, 同时逐渐从Au电极表面脱附.     

Electrochemical measurement of phenothiazine-interacted DNA

The amount of DNA was measured by using thioridazine, which would be attached to the DNA, as an electrochemical indicator. An indicator (thioridazine) solution, a test solution (DNA solution), and a poly-l-lysine solution were successively placed on a glassy carbon electrode, and the electrode was allowed to dry; DNA was immobilized on an electrode surface by the electrostatic binding between DNA and poly-l-lysine. The electrode was immersed into a buffer solution for 15 min, and then differential pulse voltammetry (DPV) was carried out: the oxidation current peak of thioridazine was observed, and its magnitude depended on the amount of DNA in the solution which was used for preparing the electrode. It could be estimated between 0.2 microg DNA (corresponds to 630 pmol nucleotides) to 20 microg DNA (63 nmol nucleotides) from the oxidation peak current of DPV.