基于二茂铁标记的电化学DNA生物传感器研究

将二茂铁和巯基修饰的脱氧核糖核酸(DNA)探针通过自组装的方式固定到金电极表面,以二茂铁作为电子介体构建电化学DNA传感器,利用杂化前后DNA传感器所展现出峰电流的差异,实现对目标DNA的定量检测。通过研究DNA杂化前后方波伏安法的扫描频率与二茂铁电子介体传输速率关系,优化扫描频率。结果表明:当扫描频率200 Hz时,目标DNA浓度在5. 0×10~(-9)～1. 0×10~(-7)mol/L范围内,峰电流的变化与目标DNA浓度呈线性关系,线性拟合方程式为ΔI_P/I_0(%)=0. 760 95+0. 610 65 c,检测限为1. 7×10~(-9)mol/L(S/N=3)。     

基于卟啉-联吡啶的铜离子荧光化学传感器及其应用研究

通过将卟啉衍生物(H2Pbpy)固定在PVC基质中制成Cu2+光纤传感器,该传感器在其它离子共存下对Cu2+有很高的选择性.在pH(6.0～8.0)时,Cu2+浓度在2.0×10-8～1.0×10-5mol/L时相对荧光强度的对数与Cu2+度的对数有线性关系,检测限为5×10-9mol/L.当[Cu2+]＜5×10-6mol/L时,响应时间小于5 min.用0.3mol/L的EDTA(pH9)和缓冲溶液连续清洗光极膜使之再生.采用了该传感器测定碳酸饮料中Cu2+.     

p53抑癌基因电化学阻抗传感器研究

基于电化学阻抗技术构建了一种检测p53抑癌基因的电化学传感器.首先利用自组装作用将末端带巯基的探针p53抑癌基因(p53-DNA)固定于金电极表面,根据传感器结合前后电子转移阻抗值的变化,对目标p53-DNA进行了测定.以pH =7.4的5mmol/L K3[Fe(CN)6]-5mmol/L K4[Fe(CN)6]平衡电对溶液作为检测液,检测目标p53-DNA的线性浓度范围为1.0×10-8～ 1.0×10-6 mol/L,其检出限为3.0×10-9 mol/L(S/N =3).对5.0×10-8 mol/L的目标p53-DNA进行11次平行测定,其RSD为3.4％.该传感器制作简单、灵敏高、选择性好,且无需标记,易于操作.     

基于电化学还原氧化石墨烯的电化学DNA生物传感器

使用还原氧化石墨烯(rGO)制备一种简单、快速和可重复方法构建DNA生物传感器。将带负电的氧化石墨烯(GO)与半胱氨酸上带正电的氨基基团通过静电作用相互吸附,用线性扫描伏安法(LSV)电化学还原电极表面吸附的GO。将二茂铁标记的DNA(Fc-DNA)探针固定到r GO表面,成功构建DNA传感器。传感器的制备过程使用循环伏安法和拉曼光谱表征。通过杂化前后DNA传感器所展现出方波信号峰电流的差异,实现对目标DNA的定量检测。实验结果表明:目标DNA浓度在1. 0×10~(-13)～1. 0×10~(-6)mol/L范围内,峰电流变化与目标DNA浓度呈线性关系,线性相关系数为0. 981,检测限是2. 0×10~(-13)mol/L (S/N=3)。     

基于DNA-Hb修饰的聚合二氧化锆/金电极过氧化氢生物传感器的研究

以聚合二氧化锆(ZrO2)薄膜修饰的金电极为基底,通过在二氧化锆修饰电极表面滴涂DNA和血红蛋白(Hb)溶液制备了性能优良的DNA-Hb/ZrO2/Au过氧化氢传感器.该传感器对H2O2的还原显示出较好的电催化响应,固定在电极表面的Hb在0.1 mol/L(pH5.0)PBS中对过氧化氢响应灵敏度高,检测范围宽(1.7×10-7～3.0×10-3～mol/L),检测下限低(8.0×10-8～tool/L),并且表现出良好的热稳定性和高选择性.     

以丙二酰肼席夫碱为中性载体的钴离子选择性电极的研究

以双水杨醛缩丙二酰肼为中性载体制备了PVC膜Co2+离子选择性电极.该电极对Co2+呈现出良好的选择性和近Nemst电位响应性能.电极斜率为31.1 mV/dec,线性范围为1.0×10-1～3.2×10-5mol/L,检测下限1.0×10-5 mol/L.采用交流阻抗技术研究了电极响应机理,并将电极作为指示电极初步应用于EDTA的电位滴定及Co2+的回收率实验,结果令人满意.     

电聚合二氧化锆固载辣根过氧化物酶的第三代生物传感器的研制

利用生物相容性良好的无机材料二氧化锆(ZrO2)对辣根过氧化物酶(HRP)进行直接固定,用交流阻抗法对修饰电极进行了表征,并用循环伏安法和计时电流法对过氧化氢(H2O2)生物传感器的性能进行了研究.结果表明,该法成功的制备了以电聚合ZrO2为载体的无介体第三代电流型生物传感器.其线性范围为1.00×10-6～1.87×10-3mol/L,线性回归方程为:ip(μA)=0.002 3 c(μmol/L)+2.9324,相关系数r为0.996 8,检测限为2.86×10-7 mol/L.     

盐酸异丙嗪荧光化学传感器研究

盐酸异丙嗪(PMH)水溶液对共价固定在玻片上的四碘荧光素衍生物有可逆荧光猝灭作用,基于此研制了一种可以测定盐酸异丙嗪的荧光化学传感器.该传感器具有良好的重现性,可逆性,选择性和较长的使用寿命,响应时间短.检测范围为5.0×10-5～1.0×10-3mol/L,检测限为6.2×10-7mol/L.     

基于纳米MnO2的免标记型的电化学适体传感器用于腺苷的测定

以纳米MnO2作为适体固定的构建平台,制备了一种用于腺苷灵敏测定的电化学交流阻抗型生物传感器.[Fe(CN)6]3-/4-作为氧化还原探针监测传感器表面电子传递电阻的变化,表面电子传递电阻的变化值与腺苷的浓度在1.0×10-9～1.0×10-7mol/L范围内有很好的线性关系,最低检测限为8.0×10-10 mol/L.传感器显示出高的灵敏度、好的选择性和稳定性.     

离子液体修饰碳糊电极方波伏安法测定氨氯地平

研究了氨氯地平(Amlodipine besylate,Aml)在离子液体(1-Benzyl-3-Methylimidazole,[BnMIM]PF6)修饰碳糊电极([BnMIM]PF6/CPE)上的电化学行为和电化学动力学性质,并用循环伏安法(CV)及计时电流法(CA)测得Aml在[BnMIM]PF6/CPE上的电极反应动力学参数。实验结果表明,Aml在[BnMIM]PF6/CPE上发生了受吸附控制的不可逆电化学氧化过程。用方波伏安法(SWV)考察了Aml氧化峰电流与其浓度在1.2×10-6~1.0×10-3mol/L范围内呈良好的线性关系,检出限(3S/N)3.3×10-7 mol/L,RSD1.3%~3.4%之间,加样回收率98.2%~103%。     

基于纳米银/石墨烯/酪氨酸酶/聚酰胺-胺的双酚A传感器

采用石墨烯纳米材料,并结合酪氨酸酶、聚酰胺-胺(PAMAM)和纳米银修饰玻碳电极研制了新型BPA生物传感器。运用循环伏安法和电化学交流阻抗考察了修饰电极的电化学行为。由于石墨烯独特的物理化学性质,结合聚酰胺-胺和纳米银的协同作用,该修饰电极对于BPA有较好的电流响应。在最佳条件下,该传感器对双酚A的线性检测范围为1.0×10-7～3.3×10-5mol/L,检测限为3.0×10-8 mol/L(信噪比为3),相关系数为0.998。     

Binding free energy calculations using MMPB/GBSA approaches for PAMAM-G4-drug complexes at neutral,basic and acid pH conditions

Dendrimers are synthetic macromolecules with a highly-branched structure and high concentration of surface groups. Among dendrimers, Poly(amidoamine) (PAMAM) has received substantial attention as a novel drug carrier and delivery system. Depending on the generation and type of terminal groups, dendrimer toxicity could change and include cytotoxicity. Although PAMAM is water soluble, molecular modeling of the dendrimer-drug complex is considered challenging for exploring the conformational mobility of dendrimers and atomic specific interactions during the dendrimer-drug association. However, conventional protocols for predicting binding affinities have been designed for small protein molecules or protein-protein complexes that can be applied to study the dendrimer-drug association. In this work, we performed docking calculations for a set of 94 previously reported compounds on PAMAM of fourth generation (G4-PAMAM) to select six compounds, cromoglicic acid (CRO) − a mast cell stabilizer, Fusidic acid (FUS) − a bacteriostatic antibiotic, and Methotrexate (MTX) − a chemotherapy agent and immune system suppressant, which have the highest affinities for G4-PAMAM, and Lidocaine (LDC) − used to numb tissue in a specific area and for ventricular tachycardia treatment, Metoprolol (MET) − a β₁ receptor blocker, and Pindolol (PIN) − a β blocker, which have the lowest affinities for the G4-PAMAM dendrimer, to perform MD simulations combined with the molecular mechanics generalized/Poisson-Boltzmann surface area MMGBSA/MMPBSA approach to investigate the interactions of generating 4 charge-neutral, charge-basic and charge-acid G4-PAMAM dendrimers. In addition, to validate these theoretical G4-PAMAM-drug complexes, the complexes were experimentally conjugated to determine their stability in aqueous solubility studies immediately and over one year. Our results show that among the different commercial drugs, both charged and neutral PAMAM have the most favorable binding free energies for CRO, MTX, and FUS, which appears to be due to a complex counterbalance of electrostatics and van der Waals interactions. These theoretical and aqueous solubility studies supported the high affinity of methotrexate for the G4-PAMAM-drug due to its carboxyl and aryl moieties that favor its accommodation by noncovalent interactions.     

基于NaOH刻蚀玻碳电极的电化学传感器在检测膀胱癌DNA中的应用

制备了一种基于活化的玻碳电极的新型电化学DNA生物传感器,可用于膀胱癌DNA的检测.通过循环伏安法(CV)实现玻碳电极在NaOH溶液中的刻蚀,使电极表面负载大量官能团,为DNA提供连接位点,由Laviron方程计算得到玻碳电极表面的羧基浓度为 1.022×10-6 mol/cm2.亚甲基蓝(MB)作为电化学检测的杂交指示剂.采用原子力显微镜(AFM)对刻蚀后的电极进行了形貌表征.在最优杂交条件下,通过差分脉冲法(DPV)计算出最佳检测限为5.677×10-13 mol/L(n=5),适用目标 DNA浓度范围1×10-8 mol/L~1×10-12 mol/L.该传感器有望用于实际样品中膀胱癌DNA的快速检测.     

Locked nucleic acids biosensor for detection of BCR/ABL fusion gene using benzoate binuclear copper (II) complex as hybridization indicator

Benzoate binuclear copper (II) complex, [Cu₂(C₇H₅O₂)₄(C₂H₆O)₂] (abbreviated as CuR₂) was prepared and its interaction with double-stranded salmon sperm DNA (dsDNA) in pH 7.4 phosphate buffer solution was studied by electrochemical experiments at the Au electrode (AuE). It was revealed that CuR₂ presented an excellent electrochemical activity on AuE and could bind with dsDNA by intercalation mode. The CuR₂ was further utilized as a new indicator in the fabrication of an electrochemical DNA biosensor for detection of BCR/ABL fusion gene. The biosensor based on nanogold (NG) modified AuE was developed by using thiolated-hairpin locked nucleic acids (LNA) as the capture probe for hybridization with BCR/ABL fusion gene. The results indicated this new method has excellent specificity for single-base mismatch and complementary after hybridization. The constructed electrochemical DNA biosensor achieved a detection limit of 1.0 × 10⁻¹⁰ M for complementary target DNA with a good stability.     

基于氧化石墨烯修饰的DNA生物传感器用于苯酚的检测

作为一种典型的优先控制污染物,苯酚一直是环境监测和污染控制的重要对象。基于氧化石墨烯大的比表面积、优良的电子传导性等特性,以其为桥梁,为DNA在玻碳电极上的固定提供了可能,并加大了DNA在电极上的电化学响应信号,由此而构建了一种性能优良的DNA生物传感器。将该传感器浸在含有苯酚的溶液中,由于苯酚对DNA的损伤作用,降低了DNA在电极上的电化学响应。实验发现,响应信号与苯酚的浓度对数呈现良好的线性关系,响应范围为1.0×10-8～1.0×10-4mol/L,此外,该生物传感器表现出良好的稳定性和重现性。     

Electrochemical performance of a DNA-based sensor device for detecting toxic algae

This work illustrates the electrochemical performance of a DNA-based sensor device for detecting toxic algae. This biosensor uses an electrochemical detection of the species in a sandwich hybridisation. A thiol (biotin) labelled capture probe was immobilized onto gold (carbon) electrodes. Synthetic positive control DNA was applied to the sensor and allowed to hybridize to the capture probe. A signal probe with a horseradish peroxidase (HRP) label was then applied, followed by an antibody to the HRP and a substrate. The electrical signal obtained from the redox reaction was proportional to the amount of DNA applied to the biosensor, which in turn would be proportional to the number of cells harvested when applied to real samples. Optimization of the hybridization process was already achieved in a previous work. Elucidation of the different steps of the fabrication process from the electrochemical point of view, proof of concept with different algal species and evaluation of the influence of the transducer platform geometry and material in the biosensor analytical performance are the main achievements reported here.     

A novel bienzyme glucose biosensor based on three-layer Au-Fe3O4@SiO2 magnetic nanocomposite

The magnetic core-shell Au-Fe₃O₄@SiO₂ nanocomposite was prepared by layer-by-layer assembly technique and was used to fabricate a novel bienzyme glucose biosensor. Glucose oxidase (GOD) and horseradish peroxidase (HRP) were simply mixed with Au-Fe₃O₄@SiO₂ nanocomposite and cross-linked on the ITO magnetism-electrode with nafion (Nf) and glutaraldehyde (GA). The modified electrode was designated as Nf-GOD-HRP/Au-Fe₃O₄@SiO₂/ITO. The effects of some experimental variables such as the pH of supporting electrolyte, enzyme loading, the concentration of the mediator methylene blue (MB) and the applied potential were investigated. The electrochemical behavior of the biosensor was studied using electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV) and chronoamperometry. Under the optimized conditions, the biosensor showed a wide dynamic range for the detection of glucose with linear ranges of 0.05-1.0 mM and 1.0-8.0 mM, and the detection limit was estimated as 0.01 mM at a signal-to-noise ratio of 3. The biosensor exhibited a rapid response, good stability and anti-interference ability. Furthermore, the biosensor was successfully applied to detect glucose in human serum samples, showing acceptable accuracy with the clinical method.     

Preparation of an electrochemical PNA biosensor for detection of target DNA sequence and single nucleotide mutation on p53 tumor suppressor gene corresponding oligonucleotide

Development of an electrochemical biosensor based on peptide nucleic acid (PNA) probe for detection of target DNA sequence and single nucleotide mutation in p53 tumor suppressor gene corresponding oligonucleotide using methylene blue (MB) as an electrochemical indicator is described. The interaction between MB and short sequence of p53, one of the most important tumor suppressor genes due to its dysfunction in the majority of human cancers, was studied by differential pulse voltammety (DPV). Probe modified electrode was prepared by self-assembled monolayer (SAM) formation of thiolated PNA molecules on the surface of gold electrode (GE). The hybridization of PNA probe with target DNA was performed in solution to form PNA-DNA hybrid on the surface of the GE. A significant increase in the reduction signal of MB was observed upon hybridization of the probe with the complementary DNA. The selectivity of the biosensor was studied using noncomplementary oligonucleotides. Furthermore, our results confirmed the ability of the sensor to detect single base mismatch in the sample oligonucleotide. The influence of probe concentration on the effective discrimination against noncomplementary sequence and point mutation was also investigated. Diagnostic performance of the biosensor is described and the detection limit is found 6.82 × 10⁻¹⁰ M. The electrochemical impedance spectroscopy was also employed to further investigate the sensor function.     

New amperometric immunosensor with response enhanced by PAMAM-dendrimers linked via self assembled monolayers for determination of alpha-fetoprotein in human serum

A new amperometric immunosensor for alpha-fetoprotein (AFP), based on nanobiocomposite substrate and with response enhanced by polyamidoaminic (PAMAM) dendrimers was developed and characterized. The nanostructurated substrate obtained by electrochemical deposition of 100 nm-sized gold nanoparticles on glassy carbon electrodes (GCE) was functionalized by deposition of a SAM of 2-aminoethanethiol (AET), used as linker for the subsequent immobilization of polyamidoaminic dendrimers (PAMAM G.1.5). Two different modes were investigated for the reading of the assay: cyclic voltammetry (CV) or Double Step ChronoAmperometry (DSCA). Satisfying results in terms of response range and precision were reached with both methods. Immunosensors were tested and validated for AFP determination in human serum, showing a limit of detection of 3 ng/mL and a limit of quantitation of 15 ng/mL. The enhanced immunosensor has proved an attractive diagnostic tool able to match the needs of clinical monitoring purposes for AFP quantification in human serum at levels useful both for prognosis of pregnancy progression, and for the identification of the occurrence of neoplastic diseases.