微流控技术筛选酶抑制剂的研究进展

随着近年新合成或提取的化合物大量涌现,药物筛选朝着快速、高效、高通量方向发展。微流控分析技术具有的分析微型化、高通量化、可集成化和良好的生物相容性等特点,为药物的筛选提供了新的方法和技术平台。本文简要介绍了酶抑制剂筛选,重点评述基于微流控技术筛选酶抑制剂的研究进展。     

基于固定化酶的乙酰胆碱酯酶抑制剂体外筛选模型的建立

以可重复使用的固定化酶代替游离态酶, 建立一种基于比色分析的乙酰胆碱酯酶(AChE)抑制剂体外筛选新模型. 采用以氨基化硅胶为载体固定的AChE优化了实验条件, 用AChE抑制剂阳性对照物他克林和毒扁豆碱对该模型进行验证, 还对模型技术参数进行评价, 并将新模型用于单体化合物及天然产物粗提物AChE抑制活性评价. 结果表明, 最佳实验条件为: 固定化酶用量55 μL, 底物浓度5 mmol/L, 甲醇、 乙醇及体积分数不高于6%的二甲基亚砜水溶液均可作为样品溶剂; 模型验证及模型技术参数评价结果良好, 该模型对AChE抑制剂筛选有较好的特异性和灵敏度, 可用于筛选AChE抑制剂. 该模型具有适用性强、 固定化酶可重复使用及结果可靠等优点, 是单体化合物及天然产物粗提物AChE抑制剂活性评价的有效方法.     

基于毛细管电泳的天然产物中酶抑制剂筛选研究进展

人类疾病的发生往往与体内各种酶的功能失调密切相关,因此酶一直是目前新药研发的重要靶标。天然产物是发现新药的宝贵资源,但是由于成分复杂,活性筛选一直受制于耗时费力的分离纯化过程。毛细管电泳(CE)技术由于具有样品和试剂消耗少、灵活多样的分离模式且不受样品基质干扰的特点,可以直接从粗提物开始筛选活性成分,在复杂样品活性筛选中显示出独特的优势。该文综述了近十年来CE在天然产物中酶抑制剂筛选的研究进展。其中重点介绍了CE应用于重要药物靶标,包括转移酶(激酶)、水解酶以及氧化还原酶等方面的应用,总结了用于酶抑制剂筛选的电泳分离模式和酶动力学研究,并展望了CE用于天然产物中活性成分筛选的应用前景。     

抗坏血酸在2-氨基吡啶修饰电极上的电催化氧化及其应用

研究了 2 -氨基吡啶聚合膜修饰玻碳电极的制备及其电化学性质 ,修饰电极对抗坏血酸 ( AA)的氧化有明显的催化作用 ,其氧化电位负移 2 72 m V。在 p H5 .7的B.R.缓冲溶液中 ,以 1 5 0 m V为工作电位 ,AA在修饰电极上的响应电流与 AA的浓度在 4× 1 0 - 6～ 1 0 - 3 mol/ L范围内呈良好的线性关系 ,检出限为 1 .3× 1 0 - 6mol/ L。在此条件下 ,多巴胺 ( DA)对 AA无干扰 ,电极重现性良好 ,可用于实际样品中 AA的测定     

基于拉伸分子动力学模拟的黄嘌呤氧化酶抑制剂解离途径的理论研究

采用拉伸分子动力学模拟的方法研究了黄嘌呤氧化酶(Xanthine oxidase, XO)抑制剂别嘌呤醇(Allopurinol)和葛根素(Puerarin)从XO离去通道解离的动态过程. 分子对接结果表明, 别嘌呤醇和葛根素均结合在XO的钼蝶呤中心(Molybdopterin, Mo-pt); 丙氨酸扫描的结果显示, Val789, Arg880, Phe911, Phe914和Val1081在XO与抑制剂的结合中起到非常重要的作用. 拉伸分子动力学模拟结果显示, 相比于葛根素, 别嘌呤醇需要更大的外力和更长的时间才能从XO中解离, 拉伸过程中Arg880, Phe1009, Thr1010, Val1011和Ala1079均对维持2种复合物的结构稳定起到重要作用, Phe649和Phe1013在抑制剂解离过程中起到门控的作用, His875起到阻碍抑制剂解离的作用.     

还原聚苯胺插层氧化石墨纳米材料修饰电极用于抗坏血酸测定的研究

本文研制成还原聚苯胺插层氧化石墨纳米材料(R-(PAI/GO))修饰电极,并用于测定抗坏血酸(AA)。在pH5.02的Brinton-Robinson(B-R)缓冲溶液中,AA可在R-(PAI/GO)修饰电极上产生一对氧化还原峰,该峰的峰电流与AA浓度的对数在1.0×10-9~4.24×10-2mol/L范围呈良好的线性关系,相关系数为0.9910。多巴胺(DA)等物质对AA的测定无干扰。该电极制作简单,有良好的稳定性和重现性,用于实际试样中AA的测定,结果满意。     

壳聚糖/氮掺杂还原氧化石墨烯修饰电极对黄嘌呤的检测及尿酸抑制的研究

用碳热还原法制备氮掺杂还原氧化石墨烯(N-RGO),用壳聚糖(CS)的乙酸溶液作为氮掺杂还原氧化石墨烯(N-RGO)的分散剂,将其修饰在玻碳电极表面,用于检测黄嘌呤(xanthine).该传感器对黄嘌呤展现出优异的电化学响应,线性范围为2.99×10^-8～1.07×10^-4mol/L,检测限为9.96×10^-9mol/L(S/N=3).此外,利用循环伏安法(CV)对黄嘌呤电化学行为进行了研究.最后,用电化学的方法研究了非布索坦(Febuxostat)和别嘌呤醇(Allopurinol)两种药物对尿酸生成的抑制.本工作为痛风的诊断和治疗提供了重要的信息.     

基于镍基纳米材料电化学传感器的α-糖苷酶抑制剂筛选方法研究

采用多周期的电化学循环伏安(CV)法在泡沫镍(NF)上一步制备了镍基纳米材料修饰电极(Ni(OH)₂/NF)用于α-糖苷酶抑制剂的酶抑制活性评价,并基于此建立了一种简便的中药糖苷酶抑制剂筛选方法。采用X射线粉末衍射仪和扫描电镜表征修饰电极表面的结构和形貌;采用CV法和计时电流法测试电极的电化学性能。结果表明,Ni(OH)₂/NF传感器检测复杂酶体系中的葡萄糖具有良好的电化学响应,灵敏度高达3222μA·mmol/(L·cm²),线性范围为3.0～6000μmol/L,检出限低至0.9μmol/L (S/N=3)。采用临床降糖药物阿卡波糖验证了此传感器用于α-糖苷酶活性检测的可行性;并将传感器应用于莲须提取液的酶抑制效果评价,发现莲须具有一定的α-糖苷酶抑制活性,其半数抑制浓度(IC₅₀)为3.31 g/L。本研究结果表明,研制的传感器适用于α-糖苷酶抑制活性分析,为天然降糖药物筛选提供了一种新方法。     

新型原卟啉原氧化酶抑制剂的固相合成及生物活性研究

采用固相有机合成技术,合成了全新结构的新型原卟啉原氧化酶抑制剂化合物.初步建立了针对特殊靶酶PPO的酶生物活性测定方法,对所合成的化合物进行了生物活性测定,初步结果表明预期有活性的化合物显示出一定的PPO酶抑制活性.     

黄嘌呤和尿酸在修饰电极上的电化学行为及测定研究

用循环伏安法制备了银掺杂聚L-赖氨酸修饰玻碳电极,并对修饰电极表面进行了表征,研究了黄嘌呤和尿酸在该修饰电极上的电化学行为,建立了循环伏安法和示差脉冲伏安法同时测定黄嘌呤和尿酸的新方法。研究表明,修饰电极对尿酸和黄嘌呤的氧化具有较好的电催化活性,在pH 3.0磷酸盐缓冲溶液中,黄嘌呤和尿酸在银掺杂聚L-赖氨酸修饰电极上的氧化峰电位分别为0.980和0.600 V,两者的峰电位差达0.380 V。在最优实验条件下,用差分脉冲伏安法同时测定黄嘌呤和尿酸的线性范围为1.0×10-6~2.5×10-4mol/L,检出限为5.0×10-7mol/L。用于人尿样中黄嘌呤和尿酸的测定,获得满意结果。     

多壁碳纳米管及氧化锌纳米复合物修饰丝印电极的制备及在去甲肾上腺素测定中的应用

建立了多壁碳纳米管及氧化锌纳米复合物修饰的丝印电极(MWNTs-ZnO SPE)测定去甲肾上腺素(NE)的方法。利用丝网印刷技术制备即抛型MWNTs-ZnO丝印电极,去甲肾上腺素在此电极上0.28 V处可产生灵敏的响应电流。应用方波伏安法测定NE的线性范围为0.75～50μmol/L,r2=0.999 3,检出限(S/N=3)为0.3μmol/L。该法简单、快速、灵敏,且电极成本低、可批量制作,适用于大批量样品中NE的测定。     

基于对氨基苯硼酸修饰磁性纳米粒子及丝网印刷技术测定糖化血红蛋白

制备了对氨基苯硼酸修饰的磁性纳米粒子,其与血液中的糖化血红蛋白(Hb A1c)结合,通过网状玻璃态碳(RVC)电极的磁性区域后,可实现与未糖基化的血红蛋白分离。通过壳聚糖(CS)、正硅酸乙酯(TEOS)和碳纳米管构成的溶胶-凝胶膜修饰丝网印刷电极对血红蛋白进行电化学检测,从而建立了一种用于检测糖化血红蛋白的新方法。在0.10 V电位下,丝网印刷电极的电量与Hb A1c和Hb的浓度有较好的线性关系,检出限分别为6 mg/m L和0.05 mg/m L。该方法电极制作简单,有较好的重现性和稳定性,且能有效排除抗坏血酸(AA)、尿酸(UA)等的干扰,已成功应用于实际样品中Hb A1c的检测。     

用聚苯胺膜从牛乳中直接分离黄嘌呤氧化酶

酶电极;新鲜牛乳;固定分离;用聚苯胺膜从牛乳中直接分离黄嘌呤氧化酶     

Fabrication of Silver Nanoparticles Decorated on Activated Screen Printed Carbon Electrode and Its Application for Ultrasensitive Detection of Dopamine

In the present study, we report the fabrication of silver nanoparticles (AgNPs) decorated on activated screen printed carbon electrode (ASPCE). The AgNPs were prepared by using Justicia glauca leaf extract as a reducing and stabilizing agent and the ASPCE was prepared by a simple electrochemical activation of screen printed carbon electrode (SPCE). The ASPCE/AgNPs shows a reversible electrochemical behaviour with enhanced response for DA than that of other modified SPCEs. Under optimum conditions, the electrochemical oxidation current response of DA is linear over the concentration range from 0.05 to 45.35 µM. The limit of detection is found as 0.017 µM with a high sensitivity of 7.85 µA µM^?1 cm^?2.     

基于丝网印刷电极的阻抗型免疫传感器用于检测烯效唑

采用循环伏安法在丝网印刷碳电极表面电聚合聚苯胺/壳聚糖复合膜,利用静电吸附作用固定烯效唑抗体,制备了检测烯效唑的阻抗型免疫传感器.采用循环伏安法和电化学阻抗谱对电极修饰过程进行了表征,并考察了缓冲溶液pH值、温育温度及时间对免疫反应的影响.在优化的条件下研究了免疫传感器的性能.此免疫传感器具有灵敏度高、特异性强、重现性和稳定性好的特点,对烯效唑的线性检测范围为0.01 ～100 mg/L,相关系数为0.999,检出限为8μg/L(3σ).白菜样品中添加0.05,0.10和1.0 mg/kg烯效唑,平均回收率为98.5％ ～ 104.6％,相对标准偏差为3.3％ ～4.3％,检测结果与气相色谱法的检测结果相符.     

Voltammetric Detection of Captopril on Graphite Screen Printed Electrodes

We report the electrochemical detection of captopril on commercially available screen printed electrodes (GSPE); it exploits the silver residue left behind on a GSPE during its manufacturing process and involves the catalytic formation of the silver thiol complex (Ag⁺+RSH→AgSR+H⁺) at potentials corresponding to the oxidation of silver. The oxidation of the silver thiol complex was found to vary linearly with the captopril concentration up to 0.8 mM. A sensitivity of 13.34±0.58 μA mM^?1 is reported with a limit of detection of 4.27±0.18 μM.     

Preparation of a Xanthine Sensor Based on the Immobilization of Xanthine Oxidase on a Chitosan Modified Electrode by Cross‐linking

Here in this paper, xanthine oxidase (XOD) was immobilized onto the chitosan (CHT) modified electrode by a simple way of cross‐linking with glutaraldehyde (GTD) and 3‐aminopropyltriethoxysilane (KH). The electrode displayed a sharp peak to the oxidation of xanthine at a potential about 0.67 V and the optimum of pH for determination was investigated. Under the optimum conditions, the biosensor fabricated on the KH/GTD/XOD/CHT modified electrode showed excellent response to the oxidation of xanthine within the range of 0.5 to 18 μmol/L with a low detection limit of 0.0215 µmol/L, a good stability and a high selectivity. The sensor can also be used for the determination of hypoxanthine. The electrochemical results indicated that the immobilized enzyme still retained its biological activity and this provided a new way for the construction of biosensors and determination of xanthine.     

Determination of Total PSA Using Magnetic Beads and a Re‐usable Screen Printed Carbon Electrode Array

A total PSA (tPSA) assay using magnetic beads (1 µm) and a re‐usable 8‐channel screen printed electrochemical array is here reported. The reaction scheme is based on a one step sandwich assay. The linear range obtained is comprised between 5 and 100 ng/mL of tPSA and have a limit of detection of 1.86 ng/mL. This methodology was validated, performing determination of tPSA in serum and the results were compared with a commercial ELISA kit.     

Nickel/Copper Bilayer‐modified Screen Printed Electrode for Glucose Determination in Flow Injection Analysis

This work reports about the performance of a Ni/Cu‐modified screen printed electrodes (SPE/Ni/Cu), prepared by physical vapor deposition (PVD) in an oblique angle configuration (OAD), for non‐enzymatic glucose sensing applications. SPE/Ni/Cu electrodes showed an excellent reversibility and a catalytic behavior for detection of glucose that were controlled by the diffusion of reactants up to the active sites at the electrode surface. The study with a flow injection analysis (FIA) setup of the main experimental variables affecting the detection process has shown that the developed electrode system had an excellent glucose sensitivity of 1.04 A M⁻¹cm⁻² (R²:0.999), a linear response up to 1 mM, a limit of detection of 0.33 μM and a time of analysis of ca. 30 s per sample. The selectivity of the sensor was checked against various interferences, including ascorbic acid, uric acid, acetaminophen and other sugars, in all cases with excellent results. The feasibility of using this sensor for practical applications was successfully confirmed by determining the glucose concentration in different commercial beverages.     

A Sensitive Electrochemical Approach for Melamine Detection Using a Disposable Screen Printed Carbon Electrode

We report here a simple and easy electrochemical approach for sensitive detection of non‐electroactive melamine using a disposable screen printed carbon electrode (SPCE) with uric acid as the recognition element. It is based on the competitive adsorptive behavior of melamine at the preanodized SPCE causing suppression in the oxidation current of uric acid. A linear range up to 126 ppb with a detection limit of 1.6 ppb (S/N=3) is achieved at the preanodized SPCE by differential pulse voltammetry. The electrochemical method is successfully applied to detect the melamine content in tainted milk powder and dog food.