DNA-modified electrodes; part 4: optimization of covalent immobilization of DNA on self-assembled monolayers

The covalent immobilization of DNA onto self-assembled monolayer (SAM) modified gold electrodes (SAM/Au) was studied by X-ray photoelectron spectrometry and electrochemical method so as to optimize its covalent immobilization on SAMs. Three types of SAMs with hydroxyl, amino, and carboxyl terminal groups, respectively, were examined. Results obtained by both X-ray photoelectron spectrometry and cyclic voltammetry show that the largest covalent immobilization amount of dsDNA could be gained on hydroxyl-terminated SAM/Au. The ratio of amount of dsDNA immobilized on hydroxyl-terminated SAMs to that on carboxyl-terminated SAMs and to that on amino-terminated SAMs is (3-3.5): (1-1.5): 1. The dsDNA immobilized covalently on hydroxyl-terminated SAMs accounts for 82.8-87.6% of its total surface amount (including small amount of dsDNA adsorbed). So the hydroxyl-terminated SAM is a good substrate for the covalent immobilization of dsDNA on gold surfaces.     

用于DNA杂交检测的丝网印刷型生物传感器的研究

将单链DNA(ssDNA)固定到丝网印刷碳电极上构成电化学DNA传感器,采用电化学指示剂,建立DNA杂交的检测方法.Co(phen)33+电化学指示剂通过钴盐与配体邻菲罗啉络合制备,采用等离子发射光谱法(ICP-AES)和核磁共振法(NMR)表征功能基团,采用循环伏安法(CV)分析指示剂的电化学特性,并以此为基础研究ssDNA在电极表面的固定及DNA杂交过程.本研究探讨了直接吸附、静电吸附与键合等3种ssD-NA在电极表面的固定方法,结果表明,静电吸附法和键合法具有较高的ssDNA固定量,采用静电吸附法固定探针的电极杂交目标DNA后,Co(phen)33+易于嵌入双链DNA (dsDNA)中,CV峰电流(ip)信号随目标DNA浓度增加.本研究采用静电吸附ssDNA的电极检测DNA杂交,实验表明,当探针固定液中ssDNA浓度为5 mg/L时,目标DNA浓度在6.65×10- 8～4.26× 10-6mol/L范围内,Co(phen)33+在dsDNA修饰电极上ip值与DNA浓度呈良好的线性关系,R2为0.9819.本研究为建立新的微生物分子分型手段提供了初步依据.     

Hydrogen Evolution: Electrochemical Pretreatment for Voltammetric Analysis with Gold Electrodes

A pretreatment for gold electrodes is presented in this work. The improvement of the voltammetric responses for gold surfaces is achieved applying an adequate cathodic potential in acidic media which results on vigorous evolution of gas hydrogen at the interface between electrode and electrolyte. The effectiveness of this procedure is evidenced for gold wire electrodes by cyclic voltammetry in several redox systems. The pretreatment is also compared with other activation methods for gold electrodes in electroanalytical and electrochemical terms.     

Comparative immobilization of antibodies on modified screen-printed graphite electrodes

Immobilization of polyclonal antibodies was studied on native screen-printed graphite electrodes (SPEs) and variously modified electrodes. SPEs coated with didodecylammonium bromide (DDAB, a synthetic membranelike substance) films with gold nanoparticles gave the maximum electrochemical response. DDAB and gold nanoparticle films strongly changed the surface morphology, and the electrochemical signal became more intense and stable. This immobilization method increased the concentration of immobilized antibodies while their activity was retained. The detection limit of the enzymatic label (horseradish peroxidase) was 0.02 ng/L of sample.     

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。     

Multi-walled Carbon Nanotubes and Gold Nanorod Decorated Biosensor for Detection of microRNA-126

In this article, we introduced a novel electrochemical biosensor for the detection of microRNA-126. The biosensor utilizes a hybridization assay combined with multi-walled carbon nanotubes and gold nanorod-decorated screen-printed carbon electrodes. For electrode preparation, gold nanorods were first immobilized onto the surface of bare and multi-walled carbon nanotube-modified screen-printed carbon electrodes, and the thiol tagged-capture probe was immobilized on the electrode surface through gold and thiol group interaction. After the immobilization, thiol tagged-capture probe hybridized with the target sequence. Under optimum conditions, we determined limit of detection (LOD) and limit of quantification (LOQ) as high as 11 nM and 36 nM, respectively.     

A Novel DNA Probe Based on Molecularly Imprinted Polymer Modified Electrode for the Electrochemical Monitoring of DNA

A DNA probe that was based on methylene blue (MB) imprinted polyvinyl pyridine polymer (MIP) modified carbon paste electrodes were developed for the first time for electrochemical monitoring of DNA. Probes were built up by adsorbing MB onto modified electrodes prior to DNA immobilization. It was shown that DNA strongly immobilizes on MIP modified electrodes when MB was adsorbed in advance of DNA immobilization. The performance of the MB imprinted polymer modified carbon paste electrodes (MIP‐CPE) to rebind the template molecule (MB) were compared to those of control polymer modified (non‐imprinted polymer NIP‐CPE) and bare (CPE) electrodes. Electrochemical signal resulting from the oxidation of guanine moiety of the immobilized probe DNA was high enough on the constructed platform, implicating that probes of this kind could be favorably used for DNA analysis. These probes exhibited high selectivity for its complementary DNA sequences (target). HBV‐DNA hybridization was studied to evaluate the selectivity of the probes for complementary, non‐complementary and mismatch sequences. The detection limit of the probe for the target DNA was 8.72 µg/mL (1.38 µM), which was better than those attained by some earlier DNA sensor studies.     

Study of the immobilization of alcohol dehydrogenase on Au-colloid modified gold electrode by piezoelectric quartz crystal sensor,cyclic voltammetry,and electrochemical impedance techniques

The immobilization of alcohol dehydrogenase (ADH) on Au-colloid modified gold electrodes has been investigated. Colloidal Au was first self-assembled onto gold electrodes through the thiol groups of an 1,6-hexanedithiol monolayer. Piezoelectric quartz crystal sensor, cyclic voltammetry, and electrochemical impedance techniques were used to investigate the immobilization of ADH on Au colloids. The cyclic voltammogram tends to be more irreversible with increased ADH concentration. In the impedance spectroscopic study, an obvious difference of the electron transfer resistance between the Au-colloid modified electrode and the bare gold electrode was observed. Using the piezoelectric quartz crystal sensor, the Michaelis constant, K(m), and the maximum initial rate, V(max), of the immobilized ADH were estimated as 6.03 x 10(-4) M and 0.63 Hzs (-1), respectively. The binding constant of ADH with nicotinamide adenine dinucleotide (NAD) was also determined as 1.87 x 10(4) M(-1). Experimental results showed that colloidal Au can be used as a biocompatible matrix for enzyme immobilization.     

Oriented immobilization of a membrane-bound hydrogenase onto an electrode for direct electron transfer

The interaction of redox enzymes with electrodes is of great interest for studying the catalytic mechanisms of redox enzymes and for bioelectronic applications. Efficient electron transport between the biocatalysts and the electrodes has achieved more success with soluble enzymes than with membrane enzymes because of the higher structural complexity and instability of the latter proteins. In this work, we report a strategy for immobilizing a membrane-bound enzyme onto gold electrodes with a controlled orientation in its fully active conformation. The immobilized redox enzyme is the Ni-Fe-Se hydrogenase from Desulfovibrio vulgaris Hildenborough, which catalyzes H(2)-oxidation reversibly and is associated with the cytoplasmic membrane by a lipidic tail. Gold surfaces modified with this enzyme and phospholipids have been studied by atomic force microscopy (AFM) and electrochemical methods. The combined study indicates that by a two-step immobilization procedure the hydrogenase can be inserted via its lipidic tail onto a phospholipidic bilayer formed over the gold surface, allowing only mediated electron transfer between the enzyme and electrode. However, a one-step immobilization procedure favors the formation of a hydrogenase monolayer over the gold surface with its lipidic tail inserted into a phospholipid bilayer formed on top of the hydrogenase molecules. This latter method has allowed for the first time efficient electron transfer between a membrane-bound enzyme in its native conformation and an electrode.     

生物功能电极 III. 葡萄糖氧化酶的电化学固定化研究

利用磷酸盐缓冲溶液中吡咯的电聚合, 将葡萄糖氧化酶(GOD)包埋在聚吡咯(PPy)基质中以构成生物功能电极。讨论了溶液pH和聚合电位对酶固定化的影响, 并用IR和交流阻抗谱对酶膜进行表征。GOD的固定化只有当pH>5.5时才能实现, 由此推测酶是以带负电的粒子嵌入PPy的。交流阻抗谱表明这一电极具有有界多孔电极的特征。探索了酶与电子传递体Fe(CN)_6~(3-)同时固定化的可行性。电化学固定化的GOD保持其生物催化活性, 酶反应表观上遵循Michealis-Menten动力学。     

辣根过氧化物酶的电化学固定化及其对H2O2的生物电催化还原作用

利用电化学固定化方法制备了聚吡咯/辣根过氧化物酶(PP/HRP)膜电极,并研究了其电化学行为。在除氧的磷酸盐缓冲液介质中,PP/HRP电极加速H₂O₂的还原,归因于酶加成物的直接电子传递。探索HRP与电子传递体K₄Fe(CN)₆在聚吡咯(PP)膜中的同时固定化条件及其膜电极的电化学行为,实验证实,K₄Fe(CN)₆在酶膜中的存在使得H₂O₂的还原电位强烈正移,在-0.05V的工作电位下能对H₂O₂进行检测,相应的电极过程可用间接氧化还原催化机理解释。     

生物功能电极 Ⅲ.葡萄糖氧化酶的电化学固定化研究

利用磷酸盐缓冲溶液中吡咯的电聚合,将葡萄糖氧化酶(GOD)包埋在聚吡咯(PPy)基质中以构成生物功能电极。讨论了溶液pH和聚合电位对酶固定化的影响,并用IR和交流阻抗谱对酶膜进行表征。GOD的固定化只有当pH>5.5时才能实现,由此推测酶是以带负电的粒子嵌入PPy的。交流阻抗谱表明这一电极具有有界多孔电极的特征。探索了酶与电子传递体Fe(CN)_6~(3-)同时固定化的可行性。电化学固定化的GOD保持其生物催化活性,酶反应表观上遵循Michealis-Menten动力学。     

Comparison of Different Strategies on DNA Chip Fabrication and DNA‐Sensing: Optical and Electrochemical Approaches

New strategies for the construction of DNA chips and the detection of DNA hybridization will be discussed in this review. The focus will be on the use of polypyrrole as a linker between a substrate and oligonucleotide probes. The modification step is based on the electrochemical copolymerization of pyrrole and oligonucleotides bearing a pyrrole group on its 5′ end. This strategy was employed for the immobilization of oligonucleotides on millimeter‐sized electrodes, microelectrode arrays, as well as for the local structuring of homogeneous gold surfaces. Our approaches for the localized patterning of gold surfaces will be also discussed. Localized immobilization was achieved by using an electrospotting technique, where a micropipette served as an electrochemical cell where spot sizes with 800 μm diameters were fabricated. The use of a microcell using a Teflon covered metal needle with a cavity of 100 μm resulted in immobilized probe spots of 300 μm. Scanning electrochemical microscopy (SECM) was also used, and surface modifications of 100 μm were obtained depending on the experimental conditions. Different detection methods were employed for the reading of the hybridization event: fluorescence imaging, surface plasmon resonance imaging (SPRI), photocurrent measurements, and voltamperometric measurements using intercalators. Their advantages concerning the various immobilization strategies will also be discussed.     

Reduction of hexavalent chromium at solid electrodes in acidic media: reaction mechanism and analytical applications

The electrochemical detection of hexavalent chromium species was investigated. It was found that Cr(VI) can undergo chemically irreversible reduction in acidic solutions at gold, glassy carbon and boron-doped diamond electrodes. The process was found to be diffusionally controlled at all three electrodes studied. The response obtained at a gold electrode towards the reduction of chromium(VI) produced an electrochemically reversible wave in contrast to those recorded at glassy carbon and boron-doped diamond electrodes. The analytical response of the hexavalent species was studied at gold electrodes in the presence of common environmental interferences: Ni²⁺, Cu²⁺, Fe³⁺, Cr³⁺ and Triton X-100 (surfactant), with an LoD of 4.3 μM obtained in the presence of 5 mM Cr(III).     

Gold Nanoparticle-based Layer-by-Layer Enhancement of DNA Hybridization Electrochemical Signal at Carbon Nanotube Modified Carbon Paste Electrode

Nanoporous materials have been widely applied to biosensor investigation. Recently, Guo et al. have investigated the mesoporous materials modified carbon paste electrode for rapid cTnI (cardiac troponin I) detection with enhanced sensitivity1-3. However, …     

Electorchemical Studies of Cytochrome c on Electodes Modified by Single—Wall Carbon Naotubes

Single-wall carbon nanotubes(SWNTs) modified gold electrodes were prepared by using two different methods.The electrochemical behavior of cytochrome c on the modified gold electrodes was investigated.The first kind of SWNT-modified electrode (noted as SWNT/Au electrode)was prepared by the adsorption of carboxylterminated SWNTs from DMF dispersion on the gold electrode.The oxidatively processed SWNT tips were covalently modified by coupling with amines (AET) to form amide linkage.Via Au-S chemical bonding,the self-assembled monolayer of thiol-unctionalized nanotubes on gold surface was fabricated so as to prepare the others SWNT-modified electrode (noted as SWNT/AET/Au electrode).It was shown from cyclic voltammetry cxperiments that cytochrome c exhibited direct electrochemical responses on the both electrodes, but only the current of controlled diffusion existed on the SWNT/Au electrode while both the currents of controlled diffusion and adsorption of cytochrome c occurred on the SWNT/AET/Au electrode.Photoelastic Modulation Infared Reflection Absorpthion Spectroscopy (PEM-IRRAS) and Quartz Crystal Microbalance (QCM) were employed to verify the adsorption of SWNTs on the gold electrodes.The results proved that SWNTs could enhance the direct electron transfer proecss between the electrodes and redox proteins.     

Spectroelectrochemical detection of phenothiazine and phenoxazine derivatives covalently bound to self-assembled cystamine monolayers

The phenothiazine derivatives, Toluidine Blue O and Azur A, and the phenoxazine derivative Nile Blue were bound covalently to self-assembled cystamine monolayers chemisorbed on gold electrodes by derivatization of the surface amino groups with two different bifunctional spacers: terephthaloyl chloride and 1,6-hexamethylene-diisocyanate.The formation of the amido- and urea-derivatives of the parent compounds after covalent immobilization induces a shift of their redox potentials towards more positive values which can easily be detected by cyclic voltammetry.UV-Vis difference spectroelectrochemistry has been used to characterize the electroactive species immobilized onto transparent gold electrode surfaces in both oxidation states. In every case, the oxidized-minus-reduced (and reduced-minus-oxidized) difference spectra of the immobilized redox species show a shift of the UV maxima towards longer wavelengths and a shift of the Vis maximum towards shorter wavelengths when compared with their parent compounds. Each redox species showed different optical characteristics depending on the spacer used for immobilization.For phenothiazine derivatives immobilized with 1,6-hexamethylene di-isocyanate the total surface coverages obtained by optical methods were close to those obtained by cyclic voltammetry. However, for the same derivatives immobilized with terephthaloyl chloride, and for Nile Blue, independently of the spacer used, higher surface coverages were found by optical methods than by cyclic voltammetry.     

Electrically Addressed Fabrication of Aptamer‐Based Array Electrodes

An aptamer immobilization method based electrically addressed fabrication has been developed for the preparation of aptamer‐modified arrayed electrodes, by which the human IgE aptamer was oriented and immobilized on the gold electrode surface. The optimization of the experimental conditions including the applied potential, time and scan rate of potential was investigated. The method was successfully used to immobilize the aptamer onto the desired electrodes, pixel by pixel, based on the electrically addressed approach. Compared to the control electrodes, the resulting aptamer‐modified electrodes showed their specific recognition for human IgE. The present method owns several advantages such as rapid and simple immobilization as well as its automatic addressed capability by the electric approach.     

基于纳米铂黑修饰的快速检测用乳酸生物传感器研究

制备了一种可用于运动员血清样品乳酸快速检测的L-乳酸传感器.这种便携式平面电化学生物传感器采用金薄膜两电极系统;先后修饰纳米铂黑粒子层和铁氰化钾媒介体.铂黑纳米粒子沉积于金电极表面以提高传感器的灵敏度和稳定性,然后将乳酸氧化酶(LOD, E.C.1.1.3.2)和相关试剂固定在工作电极表面,铁氰化钾作为媒介体用以提高电极表面电子传递能力,并将工作电压降低为0.2 V.通过优化铂黑颗粒的沉积、乳酸氧化酶的浓度、铁氰化钾的浓度、添加剂的成分和浓度等条件,将传感器的检测范围扩展至1～20 mmol/L乳酸,检测灵敏度提高到1.43 μA·L/mmol,检测时间为50 s.生物传感器的批间r为0.0549;生物传感器经室温储存1年后仍可保持90%的活性.这种传感器成功地用于无稀释乳酸血清样品的快速检测,结合便携式检测仪(YT 2005-1 乳酸测试仪)将在快速诊断领域具有很好的应用前景.     

Immobilization of Magnetic Nanoparticles onto Conductive Surfaces Modified by Diazonium Chemistry

Core-shell γ-Fe(2)O(3)@SiO(2) nanoparticles (NPs) substituted by PEG and NH(2) groups may be immobilized on metal surfaces (glassy carbon or gold) substituted by 4-carboxyphenyl groups through electrostatic interactions. Such immobilization is evidenced by (i) IRRAS owing to the Si-O band, (ii) SEM images, which show that the surface coverage by the NPs is nearly 100%, and (iii) the NPs film thickness measured by ellipsometry or AFM, which corresponds to about one NPs monolayer. Such NPs film is permeable to redox probes, which allows us to propose electrochemical methods based on direct or local measurements as a way to inspect the NPs assembly steps through their ability to alter mass and charge transfer. This process also applies to patterned polystyrene surfaces, and selective immobilization of NPs substituted by amino groups was carried out onto submillimeter patterns obtained by local oxidation. Biological applications are then expected for hyperthermia activation of the NPs to trigger cellular death. Finally, some tests were performed to further derivatize the immobilized NPs onto surfaces through either a covalent bond or electrostatic interactions. Future work will be dedicated to the recovery of such Janus NPs from the substrate surface.