Development of a Temperature-pulse Enhanced Electrochemical Glucose Biosensor and Characterization of its Stability via Scanning Electrochemical Microscopy

Glucose oxidase (GOx) is an enzyme frequently used in glucose biosensors. As increased temperatures can enhance the performance of electrochemical sensors, we investigated the impact of temperature pulses on GOx that was drop-coated on flattened Pt microwires. The wires were heated by an alternating current. The sensitivity towards glucose and the temperature stability of GOx was investigated by amperometry. An up to 22-fold increase of sensitivity was observed. Spatially resolved enzyme activity changes were investigated via scanning electrochemical microscopy. The application of short (<100 ms) heat pulses was associated with less thermal inactivation of the immobilized GOx than long-term heating.     

扫描电化学显微镜技术近期进展

本文较详细地介绍了扫描电化学显微镜技术的近期进展,引用参考文献77篇。     

扫描电化学显微镜及其最新进展

对扫描电化学显微镜技术的基本原理,仪器设备,最新的研究进展和发展展望进行了评述。     

Fabrication of Active Horseradish Peroxidase Micropatterns with a High Resolution by Scanning Electrochemical Microscopy

We used a new reactive species OH^? to fabricate active horseradish peroxidase (HRP) micropatterns with a high resolution by scanning electrochemical microscopy (SECM) coupled with a carbon fiber disk electrode as the SECM tip. In this method, except for active HRP micropatterns predesigned other regions on a HRP‐immobilized substrate were deactivated by OH^? generated at the tip held at ?1.7 V in 1.0 mol/L KCl containing 2.0×10^?3 mol/L benzoquinone (BQ) (pH 8.0). The feedback mode of SECM with a tip potential of ?0.2 V was used to characterize the active HRP micropatterns in 1.0 mol/L KCl containing 2.0×10^?3 mol/L BQ and 2.0×10^?3 mol/L H₂O₂.     

扫描电化学显微镜用于研究生物膜微环境的电子传递

生物电化学系统(BESs)的核心是生物膜在电极/溶液界面的电子传递反应,研究生物膜微区环境中的电子传递有助于阐明微生物的胞外电子传递(EET)机制,从而有针对性地提高BESs中的电子转移效率。微生物的EET机制包括直接电子传递和间接电子传递,由于生物膜组成复杂,含有多种分泌物、胞外聚合物等,常规电化学方法只能从生物膜宏观层面研究EET机制,无法有效区分这两种电子传递途径的贡献。本文采用电化学循环伏安方法研究了电子穿梭体二茂铁甲醇(FcMeOH)与希瓦氏菌(Shewanella)相互作用的界面过程;基于扫描电化学显微技术构建了穿透模式,通过微电极介导FcMeOH与Shewanella反应,收集仅来自间接电子传递途径产生的电流,同时测定了Shewanella在电极/溶液界面的氧化还原性质和空间分布。本论文将电化学扫描探针显微技术应用于EET的研究,从物理化学角度揭示微生物在代谢过程中与外界的电子传输机制。     

Non‐destructive Patterning of Carbon Electrodes by Using the Direct Mode of Scanning Electrochemical Microscopy

Patterning of glassy carbon surfaces grafted with a layer of nitrophenyl moieties was achieved by using the direct mode of scanning electrochemical microscopy (SECM) to locally reduce the nitro groups to hydroxylamine and amino functionalities. SECM and atomic force microscopy (AFM) revealed that potentiostatic pulses applied to the working electrode lead to local destruction of the glassy carbon surface, most likely caused by etchants generated at the positioned SECM tip used as the counter electrode. By applying galvanostatic pulses, and thus, limiting the current during structuring, corrosion of the carbon surface was substantially suppressed. After galvanostatic patterning, unambiguous proof of the formation of the anticipated amino moieties was possible by modulation of the pH value during the feedback mode of SECM imaging. This patterning strategy is suitable for the further bio‐modification of microstructured surfaces. Alkaline phosphatase, as a model enzyme, was locally bound to the modified areas, thus showing that the technique can be used for the development of protein microarrays.     

Determination of Paracetamol in Presence of Ascorbic Acid in Pharmaceutical Products by Scanning Electrochemical Microscopy

The selective amperometric determination of paracetamol in pharmaceutical formulations containing ascorbate was achieved by removing the interfering species in the diffusion layer created between a platinum substrate and a disc microelectrode in a Scanning Electrochemical Microscopy (SECM) configuration, while the target analyte was kept unconsumed. After complete depletion of ascorbate, paracetamol was detected at the SECM tip in a free‐interference solution zone. The influence of the substrate potential and the gap distance on the efficiency of ascorbate removal was systematically examined. The effectiveness of the device towards the determination of paracetamol in pharmaceutical samples was evaluated and under optimal conditions the results obtained agreed well with the labeled value.     

Alternating Current Measurements in Scanning Electrochemical Microscopy,Part 2: Detection of Adsorbates

A scanning electrochemical microscope (SECM) in ac mode is used for the characterisation of the adsorption process during the hydrogen evolution reaction (HER) in sulfuric acid solution. It is shown that this technique allows quantitative analysis of the adsorption process, and measurements of the differential capacitance with the frequency as parameter are obtained. The time constant for relaxation of adsorbed hydrogen (H_ads) is approximately 2 Hz, and analysis of the Nyquist plot allows direct evaluation of the charge involved. In addition, the direct comparison of the usual electrochemical impedance data and ac‐SECM results obtained simultaneously permits characterisation of processes occurring at the surface and in solution.     

ITO基底上ZnO薄膜的制备以及刻蚀图形的扫描电化学显微镜表征

在氧化铟锡(ITO)导电玻璃的衬底上,利用直接电沉积方法制备了ZnO纳米线或ZnO薄膜.然后利用存储有HCI刻蚀剂的琼脂糖微图案印章对其进行了化学刻蚀以形成不同的图形.利用扫描电子显微镜(SEM)、X射线衍射(XRD)和扫描电化学显微镜(SECM)分别对ITO衬底上的ZnO薄膜的结构、形貌和电化学性质进行表征.     

扫描探针显微镜在纳米加工中的应用

扫描探针显微镜不仅能对材料表面形貌进行原子级观测，还能够对单个的分子、原子及纳米粒子进行操纵。本文综述了扫描探针显微镜在纳米加工中对自组装单层膜的扫描探针刻蚀以及“蘸写笔”两方面的应用情况。     

Detection and Quantification of Sulfonamide Antibiotic Residues in Milk Using Scanning Electrochemical Microscopy

The use of scanning electrochemical microscopy (SECM) for the qualitative and quantitative determination of sulfapyridine (SPY) in milk is described. A direct competitive immunoassay was performed involving an antibiotic horseradish peroxidase (HRP)‐labeled analog and using selective capture antibodies immobilized on the surface of Protein G‐modified glassy carbon plates. SECM detection was accomplished by means of the sample generator/tip collector (GC) mode involving the reduction of benzoquinone (BQ) generated upon the HRP‐catalyzed oxidation of hydroquinone (HQ) at the modified substrate surface in the presence of H₂O₂. The detection limit for SPY in milk samples was as low as 0.13 ng mL^?1.     

Evaluation of Enzymatic Kinetics of GOx‐based Electrodes by Scanning Electrochemical Microscopy at Redox Competition Mode

Glucose oxidase (GOx) is an enzyme, which is used for the development of enzymatic biofuel cells. Therefore in this research redox competition mode of scanning electrochemical microscopy (RC‐SECM) was applied for the investigation of glucose oxidase (GOx) catalyzed reaction kinetics. The GOx was immobilized by glutaraldehyde on substrates of different electrical conductivity: (i) gold covered glass was used as conducting substrate and (ii) plastic poly(methyl methacrylate) was used as non‐conducting substrate. Current vs distance dependencies were registered by SECM at different concentrations of glucose in the absence of redox mediator. The potential of −750 mV vs Ag/AgCl(3 M KCl) was applied to the microelectrode (ME), which was used as a probe in SECM, in order to register oxygen reduction current. Consumption of oxygen by the GOx based layer was evaluated according to principles determined by Michaelis‐Menten kinetics. Apparent Michaelis constants K _{M (app.)} were calculated from the dependencies of current vs glucose concentration. In both these cases the K _{M (app.)} value increased when the distance between ME and enzyme modified surface was increasing from 10 to 30 μm, while the K _{M (app.)} value decreased by increasing the distance from 30 to 60 μm.     

Alternating‐Current Measurements in Scanning Electrochemical Microscopy,Part 1: Principle and Theory

The development of the scanning electrochemical microscope in ac mode is presented from both experimental and theoretical point of views. The experiments are performed with the ferri/ferrocyanide redox mediator as model system. Based on analysis of the frequency‐dependent collection efficiency, diffusion between the probe and the substrate is investigated, and analysis of time constants allows evaluation of the size of the sensing area under investigation. The experimental results are in good agreement with numerical simulations.     

Study on Electrocatalytic Activity of Platinum for Hydrogen Oxidation in Acidic Solution by Scanning Electrochemical Microscopy

The electrocatalytic activity of platinum for hydrogen oxidation in 0.01 M H₂SO₄ + 0.1 MNa₂SO₄ solution has been investigated by scanning electrochemical microscopy (SECM) technique. The cyclic voltammogram (CV), approach curve, area scan imaging and chronoamperometric methods have been used. The results indicate that the imaging capability of the SECM feedback mode can be used more efficiently to visually identify materials' electrocatalytic activity, compared with the approach curve method for identification of the conductive or insulating nature of a surface. The SECM imaging method has demonstrated the effects of Pt substrate potential on the electrocatalytic oxidation of hydrogen under a constant tip potential. It is found that the more positive the Pt substrate potential, the lower the electrocatalytic activity of the Pt. Furthermore, the chronoamperometric results support the variation of the electrocatalytic activity with the Pt substrate potential as well.     

扫描探针显微术在巯醇自组装单分子膜纳米刻蚀中的应用

介绍了近十年来扫描探针显微术(SPM)在巯醇自组装单分子膜纳米刻蚀中的应用. 依据扫描探针的工作原理, 依次讨论了扫描隧道显微镜、原子力显微镜和导电原子力显微镜的工作特点和适用范围. 同时也讨论了自组装单分子膜纳米刻蚀术在生物分子传感器、超高密度信息存储等领域的应用前景.     

Non‐Invasive Monitoring of Osteogenic Differentiation on Microtissue Arrays under Physiological Conditions Using Scanning Electrochemical Microscopy

In this paper, we present a non‐invasive assay using scanning electrochemical microscopy (SECM) for detecting osteogenic differentiation at physiological conditions (pH 7.5) on arrays of C2C12 microtissues. Upon exposure to bone morphogenic protein 2 (BMP‐2), C2C12 microtissues differentiate and express alkaline phosphatase (ALP), which is indirectly detected through an enzymatic assay producing an electroactive species. The latter is detected using SECM by scanning at constant height over live microtissues at physiological pH (7.5) as well as more alkaline pH (8.5). As a control, expression of ALP is confirmed using a standard colorimetric assay. Detecting differentiation on live samples at physiological conditions represents a significant improvement for continuous monitoring of tissue differentiation or further use of the microtissues for, e.g., regenerative medicine.     

应用扫描电化学显微镜研究极化液/液界面上的电子转移反应

将含有氧化还原电对的水溶液滴涂在铂盘电极表面, 然后将该电极插入到1,2-二氯乙烷溶液中, 形成稳定的油/水界面. 液滴中的K3Fe(CN)6和K4Fe(CN)6氧化还原电对既可以作为水相中的参比电对参与控制液/液界面上的电势差, 同时又可以作为水相的电子授受体参与界面上的电子转移反应. 结合扫描电化学显微镜电化学系统的特点, 利用其双恒电位仪分别控制界面电势差和现场扫描的优点, 通过扫描电化学显微镜的渐进曲线得到了不同界面电势差控制的电子转移反应速率常数. 实验结果表明, 应用此方法获得的液/液界面可以被外加电位极化, 在一定的电势差范围内, 反应速率常数与界面电势差的关系遵守Butler-Volmer公式.     

Application of AC impedance techniques to Scanning Electrochemical Microscopy

Fundamental aspects of alternating current Scanning Electrochemical Microscopy (AC-SECM) have been studied. In particular, the flow of AC currents in the cell and simplified equivalent circuit diagrams are discussed. In the case of conductive substrates, the local (1^st and 2^nd order) impedance characteristic of the substrate is presented as a function of DC potential. 3-D imaging of mixed substrates (containing both conductive and non-conductive areas) was performed in the supporting electrolyte alone, without an added redox mediator.Dedicated to Professor Zbigniew Galus on the occasion of his 70^th birthday.     

应用扫描电化学显微镜研究水/1,2-二氯乙烷界面上离子诱导的电子转移反应

应用扫描电化学显微镜和微电极技术研究了水/1,2二氯乙烷界面上的反向电子转移反应.分别以K₄Fe(CN)₆和7,7,8,8四氰代二甲基苯醌(TCNQ)作为水相和有机相的电活性物质,通过选择合理的共同离子(TPAs⁺与TBA⁺)来控制界面电位差,实现了这一在热力学上通常不可能实现的反向电子转移反应.利用扫描电化学显微镜给出的正负反馈信息,研究了界面电位差驱动的液/液界面上的电子转移反应,并进一步得到了在不同的共同离子浓度比时,此异相界面反应速率常数k_f为1.3×10^-31.8×10^-2cm/s(共同离子为TBA⁺)和2.5×10^-32.8×10^-2cm/s(共同离子为TPAs⁺).验证了此反应速率常数k_f是由界面电位差所决定的.在此实验条件下,此反应速率常数kf与界面电位差的关系遵守Butler-Volmer公式.