基于聚硫堇/亚甲基蓝和纳米金放大的免疫传感器检测贝类毒素大田软海绵酸

采用聚硫堇(PTH)修饰电极为传感界面提供一个生物修饰功能基质膜,借助纳米金(GNPs)的导电性、生物相容性与高比表面积特性实现抗体的有效固定,并以亚甲基蓝(MB)为电子媒介加速电极表面电化学反应的电子传递,构建了一种高灵敏的非标记电化学免疫传感器,用于贝类毒素大田软海绵酸(OA)的检测。当分子结构中含有羧基和酚基的OA与其抗体特异性结合后,生成以阴离子形式存在的抗原-抗体复合物,阻碍了传感器表面电子的传递,导致峰电流下降。利用免疫反应前后峰电流的变化,可对OA进行特异性识别和准确定量。在优化实验条件下,OA浓度的对数在0.2~100 μg/L范围内与其峰电流的变化值(ΔI)呈线性相关,线性方程为ΔI=1.721 7+1.083 6lgρ,相关系数为0.992 0,检出限为0.1 μg/L。该免疫传感器重现性好、特异性强,用于实际贝类样品的测定,回收率为85.3%~112%。     

A thionine-based reversible redox sensor in a sequential injection system

According to the current demands of Green Analytical Chemistry and regarding the need for lower reagent consumption with improved analytical performance, an automatic methodology with a flow-through optosensor incorporating solid-phase spectrophotometric detection was developed. The sensor used in this work was based on the redox state of thionine whose oxidized form is blue and reduced form is colorless with monitoring carried out at 621 nm. This redox indicator was immobilized on gel beads and subsequently packed into a flow-through cell. It was then assembled into a sequential injection system and was shown to be an excellent alternative to monitor enzymatic redox reactions automatically as the redox catalysis is performed by glucose dehydrogenase. This enzyme is a representative dehydrogenase enzyme and uses NAD⁺ as cofactor, promoting the oxidation of glucose to glucono-lactone and reduction of NAD⁺ to NADH. The produced NADH promotes color depletion on the surface of the sensor. The calibration graph for glucose was linear between 5.74 × 10⁻⁴ and 2.00 × 10⁻³ mol L⁻¹ and detection limit was 1.72 × 10⁻⁴ mol L⁻¹. Glucose concentration in different samples including sera, salines, perfusion solutions, powder for preparing oral solutions and solutions for hemodialysis was determined. The method proved to be reproducible with a RSD < 5% for glucose determinations.     

Complexation of heterocyclic ligands with DNA in aqueous solution

We have used spectrophotometry to study self-association and complexation with DNA by organic heterocyclic compounds in the acridine and phenothiazine series: proflavin, thionine, and methylene blue. Based on the experimental concentration dependences of the molar absorption coefficient of the molecules in an aqueous buffer solution (0.01 M NaCl, 0.01 M Na₂EDTA, 0.01 M Tris, pH 7.4, T = 298 K), we have determined the equilibrium dimerization constants for the dyes and the DNA complexation parameters using the Scatchard and McGhee-von Hippel models. The observed increase in the cooperativity parameters as the dimerization constants of the ligands increase allowed us to hypothesize that the same interactions occur between dye molecules adsorbed on DNA as in their self-association. The equilibrium DNA-binding constants for the ligands, obtained using the McGhee-von Hippel cooperative model, are (20.9 ± 2.7)·10³ M⁻¹ for proflavin and (33.8 ± 4.1)·10³ M⁻¹ for thionine. Using the Scatchard model, taking into account intercalation and “external” binding of ligands with DNA, we determined the DNA complexation constants for methylene blue: (26.4 ± 4.6)·10³ and (96 ± 17)·10³ M⁻¹ respectively. Based on analysis of the data obtained, we hypothesized that the predominant type of binding with DNA is intercalation binding in the case of proflavin and thionine, and “external” binding with the DNA surface in the case of methylene blue. __________ Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 75, No. 2, pp. 242–249, March–April, 2008.     

A Novel Amperometric Nitric Oxide Sensor Based on Polythionine / Nation Modified Glassy Carbon Electrode

A novel amperometric sensor for the determination of nitric oxide was developed by coating polythionine / nafion on a glassy carbon electrode. This sensor exhibited a great enhancement to the oxidation of nitric oxide. The oxidation peak currents were linear to the concentration of nitric oxide over the wide range from 3.6×10-7 to 6.8×10-5 mol.L-1, and the detection limit was 7.2×10-8 mol.L-1. Experimental results showed that this nitric oxide sensor possessed excellent selectivity and longer stability. NO releasing from rat kidney was monitored by this sensor.     

Electron‐Transfer Mediator Microbiosensor Fabrication Based on Immobilizing HRP‐Labeled Au Colloids on Gold Electrode Surface by 11‐Mercaptoundecanoic Acid Monolayer

In this paper, a new strategy for constructing a mediator‐type amperometric hydrogen peroxide (H₂O₂) microbiosensor was described. An electropolymerized thionine film (PTH) was deposited directly onto a gold electrode surface. The resulting redox film was extremely thin, adhered well onto a substrate (electrode), and had a highly cross‐linked network structure. Consequently, horseradish peroxidase (HRP) was successfully immobilized on nanometer‐sized Au colloids, which were supported by thiol‐tailed groups of 11‐mercaptoundecanoic acid (11‐MUA) monolayer covalently bound onto PTH film. With the aid of the PTH mediator, HRP‐labeled Au colloids microbiosensor displayed excellent electrocatalytical response to the reduction of H₂O₂. This matrix showed a biocompatible microenvironment for retaining the native activity of the covalent HRP and a very low mass transport barrier to the substrate, which provided a fast amperometric response to H₂O₂. The proposed H₂O₂ microbiosensor exhibited linear range of 3.5 μM–0.7 mM with a detection limit of 0.05 μM (S/N=3). The response showed a Michaelis‐Menten behavior at larger H₂O₂ concentrations. The K_M^app value for the biosensors based on 24 nm Au colloids was found to be 47 μM, which demonstrated that HRP immobilized on Au colloids exhibited a high affinity to H₂O₂ with no loss of enzymatic activity. This microbiosensor possessed good analytical performance and storage stability.     

Soluble polymers with pendant thionine groups: Synthesis and evaluation as singlet oxygen photosensitizers

Thionine ( 1 ) has been covalently bound to linear copoly(styrene-p-vinylbenzyl chloride) and to linear copoly(acrylic acid-2-ethylhexyl acrylate). The resulting polymeric dyes, purified by ultrafiltration, present quantum yields of singlet oxygen generation in dimethylformamide solution lower than their corresponding low-molecular-weight models thionine hydrochloride ( 1.HCl ) and N(3)-acetylthionine ( Ac-1 ), the decrease being more pronounced in the case of the polystyrene-dye photosensitizer. High chromophore concentrations within the volume encompassed by each macromolecule in the solution can explain this fall in efficiency. The free dyes thionine hydrochloride and its hydrolysis products thionoline ( 2 ) and thionol ( 3 ) behave as good singlet oxygen generators, with quantum yields of 0.71, 0.62, and 0.63, respectively. © 1993 John Wiley & Sons, Inc.     

基于纳米金和硫堇固定酶的过氧化氢生物传感器

在铂电极上自组装一层纳米金(GNs), 构建负电荷的界面, 然后通过金-硫、金-氮共价键合作用和静电吸附作用自组装一层阳离子电子媒介体硫堇(Thio). 再以同样的作用自组装一层GNs和辣根过氧化酶(HRP)的混合物, 最后在电极最外层滴加一层疏水性聚合物壳聚糖(Chit), 由此制备了一种新型的过氧化氢生物传感器. 研究了工作电位、检测底液pH、温度对响应电流的影响, 以及GNs和HRP之间的相互作用, 探讨了传感器的表面形态、交流阻抗、重现性和稳定性. 该传感器的酶催化反应活化能为12.4 kJ/mol, 表观米氏常数为6.5×10^－4 mo/L, 在优化的实验条件下, 所研制的传感器对H₂O₂的线性范围为5.6×10^－5～2.6×10^－3 mol/L, 检出限为1.5×10^－5 mol/L. 应用此方法制备了HRP和葡萄糖氧化酶(GOD)双酶体系葡萄糖生物传感器, 并应用于实验样品葡萄糖含量的测定.     

基于壳聚糖膜固定双酶的胆碱传感器的研究

提出了一种基于壳聚糖膜固定辣根过氧化物酶 胆碱氧化酶的胆碱传感器的制备方法。该传感器以电聚合于玻碳电极的硫堇作为电子传递介体,在pH6. 8,外加电压-0. 2V(vs.SCE)条件下,其峰电流与浓度范围 5. 0×10-5 ~3. 0×10-3 mol/L的胆碱呈良好的线性响应;检出限为 1. 0×10-5 mol/L。传感器有良好的选择性和稳定性,使用一月后,仍能保持其初始活性的 80%。     

三氧化二铝溶胶-凝胶固定过氧化氢生物传感器

三氧化二铝溶胶凝胶将辣根过氧化物酶和硫堇同时固定在玻碳电极上,紫外-可见光谱结果表明酶在固定过程中生物活性保持得较好.传感器在pH 7.0外加电压为-0.22 V条件下,对过氧化氢浓度为1.76×10-3～5.5×10-2 mol/L的范围内呈线性响应,检出限为1.1×10-4 mol/L.传感器的选择性和稳定性较好,使用3个星期后,仍能保持其初始活性的80%.     

基于硫堇衍生化自组装膜的丙肝电化学免疫传感器

报道了一种制备电流型免疫传感器的新方法。在金电极表面形成巯基自组装单分子层膜,活化后以共价方式竞争键合硫堇分子和丙肝辣根过氧化酶酶标抗体,制得基于硫堇衍生化自组装膜的丙肝电化学免疫传感器。采用循环伏安法和线性扫描伏安法考察了传感器的组装过程,响应电流的性质,以及传感器对丙肝病毒的响应特性,采用线性扫描伏安法对丙肝抗原进行定量分析,线性范围为3．2—16mg／L;检出限为1．2mg／L;线性相关系数r为0．995。取临床血清进行检测,将结果与临床常用的ELISA法比较,探索了该传感器应用于临床检验的可行性。