以聚硫堇为电化学探针的非标记型核酸适配体传感器

采用电聚合法制备了聚硫堇氧化还原电化学探针, 以金纳米粒子为固定核酸适配体的载体构建了非标记型核酸适配体传感器. 用电化学阻抗谱对传感器的组装过程进行了监测, 用循环伏安法和差分脉冲伏安法考察了传感器的电化学行为. 结果表明, 该传感器对凝血酶的检测在1.0 pg/mL～500 ng/mL范围内呈良好的线性关系, 相关系数为0.998, 检出限为0.38 pg/mL. 该传感器制备简单、 灵敏度高且抗干扰能力强.     

Influence of ionic strength, pH and aptamer configuration for binding affinity to thrombin

We used the methods of electrochemical indicators and the quartz crystal microbalance (QCM) for detection of thrombin-aptamer interactions. We analyzed how the method of immobilization of aptamer to a solid support, the aptamer configuration as well as variation in ionic strength and pH will affect the binding of thrombin to the aptamer. The immobilization of aptamer by means of avidin-biotin technology revealed best results in sensitivity in comparison with immobilization utilizing dendrimers of first generation and in comparison with chemisorption of aptamer to a gold surface. Linear and molecular beacon aptamers of similar structure of binding site revealed similar binding properties to thrombin. Increased concentration of NaCl resulted in weakening of the binding of thrombin to the aptamers, probably due to shielding effect of Na(+) ions. The binding of the thrombin to the aptamer depends on electrolyte pH, which is presumably connected with maintaining the three dimensional aptamer configuration, optimal for binding the protein.     

Scanning electrochemical microscope observation of defects in a hexadecanethiol monolayer on gold with shear force-based tip-substrate positioning

Scanning electrochemical microscopy (SECM) was used for imaging of n-hexadecanethiol-modified Au surfaces. In these studies, small defects were observed in the monolayer when a submicrometer electrode was used as an SECM tip, although a cyclic voltammogram of a Au disk electrode showed that the surface of the Au was completely covered with n-hexadecanethiol. The dependence of the SECM images on the potential of the Au electrode was also examined. A comparison of the current at the Au electrode and the tip current in the SECM images showed that direct electron transfer through the monolayer was dominant, rather than electron transfer at the defects. The size of the defects was estimated from the tip current to be 1-100 nm, under the assumption that the defects were small compared to the SECM probe.     

Optimized preparation and scanning electrochemical microscopy analysis in feedback mode of glucose oxidase layers grafted onto conducting carbon surfaces

An optimized immobilization procedure based on the electroreduction of aryldiazonium salt followed by covalent attachment of a cross-linked hydrogel was used to graft glucose oxidase on a carbon surface. Scanning electrochemical microscopy (SECM) and cyclic voltammetry were used to follow the construction steps of the modified electrode. By adjusting the compactness of the layer through the electrografting reaction, the penetration of the mediator through the layer can be controlled to allow the monitoring of the enzymatic activity by both cyclic voltammetry and SECM in feedback mode. The enzymatic activity of the film is finally characterized by SECM.     

Fabrication of Prussian Blue modified ultramicroelectrode for GOD imaging using scanning electrochemical microscopy

A Prussian Blue (PB) film modified disk ultramicroelectrode (UME) was fabricated by electrochemical deposition technique on a Pt-disk UME. The electrocatalytical reductions of hydrogen peroxide derived from glucose oxidase (GOD) on this modified UME were investigated. The enzymatic biochemical reactivity was imaged by scanning electrochemical microscopy (SECM) utilizing the PB film modified UME. It is evident that sensitivity and spatial resolution for hydrogen peroxide measurement were improved obviously. SECM images obtained clearly revealed the concentration profile of the reaction products around the enzymes. The PB film modified microelectrode is in the nature of simple preparation, high catalytic activity on hydrogen peroxide and substrate selectivity for SECM etc.     

A Simple Method to Tune Up Screen‐Printed Carbon Electrodes Applicable to the Design of Disposable Electrochemical Sensors

We report an easy method to tune up screen‐printed carbon electrodes (SPCEs) for application in fabricating disposable electrochemical sensors. Simply by ultrasonic polishing a bare SPCE in a γ‐Al₂O₃ slurry, the surface roughness was drastically smoothed coupled with a large increase in hydrophilicity. The as‐generated micromorphology on the surface of the SPCE was found to be ideal for the immobilization of catechol to minimize the overpotential in the sensitive detection of nicotinamide adenine dinucleotide (NADH) and hydrazine. Physical characterization by both XPS and AFM studies specify that the adsorption behavior is related to the carbon surface functionalities and the trapping of γ‐Al₂O₃ on the polished‐SPCE.     

Localized Deposition of Chitosan as Matrix for Enzyme Immobilization

A local electrodeposition method was developed for chitosan by exploiting a pH gradient between a macroscopic electrode (the support) and a much smaller counter electrode. The deposition was confined either by using the direct mode of scanning electrochemical microscopy (SECM) or by performing the deposition in channels of a microfluidic network. The roughness was characterized by noncontact scanning force microscopy. The availability of amino groups at the surface of the microstructures was visualized after labeling by confocal laser scanning microscopy. The enzyme glucose oxidase could be entrapped during the electrochemical deposition and showed activity as seen by SECM images.     

Imaging of enzyme activity by scanning electrochemical microscope equipped with a feedback control for substrate-probe distance

The enzymatic activity of diaphorase (Dp) immobilized on a solid substrate was characterized using a scanning electrochemical microscope (SECM) with shear force feedback to control the substrate-probe distance. The shear force between the substrate and the probe was monitored with a tuning fork-type quartz crystal and used as the feedback control to set the microelectrode probe close to the substrate surface. The sensitivity and the contrast of the SECM image were improved in the constant distance mode (distance, 50 nm) with the shear force feedback compared to the image in the constant height mode without the feedback. By using this system, the SECM and topographic images of the immobilized diaphorase were simultaneously measured. The microelectrode tip used in this study was ground aslant like a syringe needle in order to obtain the shaper topographic images. This shape was also effective for avoiding the interference during the diffusion of the enzyme substrates.     

Effect of pre-treatment on the surface and electrochemical properties of screen-printed carbon paste electrodes

The effect of various electrochemical pre-treatment methods on the surface and electrochemical properties of screen-printed carbon paste electrodes (SPCE) prepared with three different commercial products was examined. It was observed that a positively charged redox couple, e.g., hexaammineruthenium(III), exhibited quasi-reversible behavior at the untreated SPCE. However, the cyclic voltammograms (CVs) of the SPCE prepared with general-purpose carbon inks did not exhibit clear redox peaks to other representative redox couples [e.g., hexacyanoferrate(III), hexachloroiridate(IV), dopamine, and hydroquinone] without activation. Electrochemical pre-treatment methods were sought in four different aqueous solutions, i.e., sulfuric acid, potassium chloride, sodium hydrogencarbonate, and sodium carbonate, applying various activation potentials. It was found that the pre-treatment procedure in saturated Na2CO3 solution at 1.2 V provides a mild and effective condition for activating the SPCE. By measuring the water contact angles at the SPCE surfaces and recording their SEM images, it was confirmed that the electrochemical pre-treatment effectively removes the organic binders from the surface carbon particles. A prolonged period of activation (> 5 min) or the use of high potentials (> 1.2 V) increased the capacitance of the electrode over 20 microF cm(-2). The pre-treated SPCE behaved like a random array microelectrode, exhibiting a sigmoidal-shaped CV at a slow scan rate. The short pre-anodization method in Na2CO3 solution was generally applicable to most SPCE prepared with general-purpose carbon inks.     

Long-Term Stability of Redox Mediators in Carbonate Solvents

Scanning electrochemical microscopy (SECM) used in the feedback mode is one of the most powerful versatile analytical tools used in the field of battery research. However, the application of SECM in the field of lithium-ion batteries (LIBs) faces challenges associated with the selection of a suitable redox mediator due to its high reactivity at low potentials at lithium metal or lithiated graphite electrodes. In this regard, the electrochemical/chemical stability of 2,5-di-tert-butyl-1,4-dimethoxybenzene (DBDMB) is evaluated and benchmarked with ferrocene. This investigation is systematically carried out in both linear and cyclic carbonates of the electrolyte recipe. Measurements of the bulk current with a microelectrode prove that while DBDMB decomposes in ethyl methyl carbonate (EMC)-containing electrolyte, bulk current remains stable in cyclic carbonates, ethylene carbonate (EC) and propylene carbonate (PC). Ferrocene was studied as an alternative redox mediator, showing superior electrochemical performance in ethyl methyl carbonate-containing electrolytes in terms of degradation. The resulting robustness of ferrocene with SECM is essential for a quantitative analysis of battery materials over extended periods. SECM approach curves depict practical problems when using the decomposing DBDMB for data acquisition and interpretation. This study sheds light towards the use of SECM as a probing tool enabled by redox mediators.     

Electrochemical detection of thrombin by sandwich approach using antibody and aptamer

The goal of this work was to introduce a modified electrochemical sandwich model for target protein detection, exploiting antibody as the capturing probe, aptamer as the detection probe and methylene blue as the electrochemical active marker intercalating in the probing aptamer without previous labeling. With appropriate design of the sequence of the aptamer, the aptamer was successfully utilized instead of antibody for obtaining the electrochemical detection. A special immobilization interface consisting of nanogold-chitosan composite film was used to improve the conductivity and performance characteristics of the electrode. The capturing antibody was linked to the glassy carbon electrodes modified with composite film via a linker of glutaraldehyde. Differential pulse voltammetry was performed to produce the response signal. Thrombin was taken as the model target analyte to demonstrate the feasibility of proposed methodology. The sensor shows the linear response for thrombin in the range 1-60 nM with a detection limit of 0.5 nM. The proposed approach provides an alternative approach for sandwich protein assay using aptamers.     

基于铁氰化镍的非标记型核酸适配体电化学传感器检测凝血酶

在玻碳电极（GCE）表面首先用增敏作用的多壁碳纳米管(MWCNTs)夹心于两层电沉积的铁氰化镍（NiHCF）氧化还原电化学探针之间,然后以金纳米粒子为固定核酸适配体的载体,构建了检测凝血酶的非标记型核酸适配体生物传感器。 利用扫描电子显微镜(SEM)对MWCNTs和NiHCF的形貌进行了表征。 利用电化学阻抗谱对传感器的组装过程进行了监测,用循环伏安法（CV）和差分脉冲伏安法（DPV）对传感器的电化学行为进行了研究。 以铁氰化镍为探针的传感器对凝血酶的检测在1.0 ng/L~1.0 mg/L范围内呈良好的线性关系,相关系数为0.998,检测限为0.2 ng/L(S/N=3)。     

Filming a live cell by scanning electrochemical microscopy: label-free imaging of the dynamic morphology in real time

ABSTRACT: The morphology of a live cell reflects the organization of the cytoskeleton and the healthy status of the cell. We established a label-free platform for monitoring the changing morphology of live cells in real time based on scanning electrochemical microscopy (SECM). The dynamic morphology of a live human bladder cancer cell (T24) was revealed by time-lapse SECM with dissolved oxygen in the medium solution as the redox mediator. Detailed local movements of cell membrane were presented by time-lapse cross section lines extracted from time-lapse SECM. Vivid dynamic morphology is presented by a movie made of time-lapse SECM images. The morphological change of the T24 cell by non-physiological temperature is in consistence with the morphological feature of early apoptosis. To obtain dynamic cellular morphology with other methods is difficult. The non-invasive nature of SECM combined with high resolution realized filming the movements of live cells.     

Scanning electrochemical microscopy: Visualization of local electrocatalytic activity of transition metals hexacyanoferrates

The redox competition mode of scanning electrochemical microscopy (SECM) was used to visualize differences in local electrocatalytic activity of Fe and Ni hexacyanoferrates (HCFs) in hydrogen peroxide reduction. The uniform round-shaped spots of electrocatalysts for the SECM measurements were electrochemically deposited using a scanning droplet cell. A negligible activity of NiHCF towards H₂O₂ reduction compared to Prussian Blue (PB) was observed. The dependence of local Prussian Blue activity on the applied potential was investigated. The proposed strategy explores the potential application of SECM as a rapid screening tool for HCF film activity within a single experiment.     

Photosystem I patterning imaged by scanning electrochemical microscopy

We report the first directed adsorption of Photosystem I (PSI) on patterned surfaces containing discrete regions of methyl- and hydroxyl-terminated self-assembled monolayers (SAMs) on gold. SAM and PSI patterns are characterized by scanning electrochemical microscopy (SECM). The insulating protein complex layer blocks the electron transfer of the SECM mediator, thereby reducing the electrochemical current significantly. Uniformly and densely packed adsorbed protein layers are observed with SECM. Pattern images correlate with our previous studies where we showed that low-energy surfaces (e.g., CH3-terminated) inhibit PSI adsorption in the presence of Triton X-100, whereas high-energy surfaces (e.g., OH-terminated) enable adsorption. Therefore, a SAM pattern with alternating methyl and hydroxyl surface regions allows PSI adsorption only on the hydroxyl surface, and this is demonstrated in the resulting SECM images.     

Scanning electrochemical microscopy study of laccase within a sol-gel processed silicate film

The enzyme p-diphenol:dioxygen oxidoreductase (laccase, EC 1.10.3.2) was isolated from Cerrena unicolor fungus and embedded in a sol-gel film obtained by acidic condensation of TMOS. The gel was cast to thin films on glass. The laccase-containing silicate films were inspected by confocal laser scanning microscopy (CLSM), scanning force microscopy (SFM) and scanning electrochemical microscopy (SECM). CLSM images in the reflection mode showed aggregates within the silicate films. SECM images in the substrate-generation/tip-collection mode using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) as electron donor for laccase showed that the position of aggregates coincides with increased enzymatic activity within the silicate film. The flux from individual aggregates was detected. SECM images in the redox competition mode confirmed the assignment and could exclude that topographic features observed by CLSM and SFM could be the reason for the image contrast. SFM images showed that the aggregates partially dissolve during prolonged exposure to aqueous buffer. The experimental setup allowed following one individual aggregate over time with all three microscopic techniques which enabled the collection of complementing information on morphology and catalytic activity as well as their development over time.     

Scanning electrochemical microscopy study of ion transfer process across water/2-nitrophenyloctylether interface supported by hydrophobic carbon ceramic electrode

A carbon ceramic electrode (CCE) modified with the redox probe—decamethylferrocene solution in hydrophobic organic solvent—2-nitrophenyloctyl ether and immersed into an aqueous solution was studied by scanning electrochemical microscopy (SECM). After the electrochemical oxidation of decamethylferrocene, its cations were detected near the electrode surface in the aqueous phase. This indicates that some fraction of the redox-active cations electrochemically produced in the organic phase is transferred across the liquid/liquid interface. They are reduced at the SECM tip and form a solid deposit. The amount of deposited decamethylferrocene was estimated by the anodic reaction at the tip. It is affected by the substrate–tip distance, deposition time, and electrolyte concentration. The SECM images of unmodified and modified CCEs are consistent with their heterogeneous structure.     

A disposable impedance sensor for electrochemical study and monitoring of adhesion and proliferation of K562 leukaemia cells

A polyaniline-modified screen-printed carbon electrode (PANI/SPCE) was prepared by electropolymerization for the construction of a novel disposable cell impedance sensor. The conductive polymer improved greatly the electron transfer of SPCE and was very effective for cell immobilization. The adhesion of cells increased the electron transfer resistance (R_et) of redox probe on the PANI/SPCE surface, producing an impedance sensor for K562 leukaemia cells with a semilogarithm linear range from 10⁴ to 10⁷ cells ml⁻¹ and a limit of detection of 8.32 × 10³ cells ml⁻¹ at 10σ. The proliferation of cells on the conductive polymer increased the R_et, leading to a novel way to monitor the growth process of cells on the PANI/SPCE. The electrochemical monitoring indicated K562 leukaemia cells cultured in vitro on the PANI surface were viable for 60 h, consistent with the analysis from microscopic imaging and MTT assay. This method for monitoring the surface proliferation and detecting the number of viable cells was simple, low-cost and disposable, thus providing a convenient avenue for electrochemical study of cell immobilization, adhesion, proliferation and apoptosis.     

Localized Visualization of Chemical Cross-Talk in Microsensor Arrays by Using Scanning Electrochemical Microscopy

 An investigation of an array of four Pt microband electrodes 25 μm wide and spaced 25 μm apart was performed with the scanning electrochemical microscope (SECM). Where possible the SECM measurements were confirmed with conventional electrochemical measurements. It is shown how the sensiti- vity of the SECM recycling current to the activity of the underlying surface can be used to probe the homogeneity of enzyme-modified microelectrodes. The diffusion of H₂O₂ between these micro enzyme- electrodes and unmodified electrodes was investigated and it was demonstrated how the SECM can be a powerful tool in the elucidation of the properties of these electrodes. Received June 8, 1998. Revision November 12, 1998.     

Novel coupling mechanism-based imaging approach to scanning electrochemical microscopy for probing the electric field distribution at the microchannel end

A novel coupling mechanism-based imaging approach to scanning electrochemical microscopy (SECM) was used to image the distribution of electric field at the end channel of a poly(dimethylsiloxane) (PDMS) capillary electrophoresis (CE) microchip in the absence of redox species. The coupling imaging mechanism was systematically investigated and qualitatively illustrated. It was proved that the distribution of solution potentials within the scanning plane caused a different reduction rate of water at the tip electrode, which led to the variation in tip current. Within the scanning plane, the solution potentials measured in the central area of the microchannel were usually higher than those measured outside. The SECM images showed a strong dependence on tip potential, tip-to-channel distance, and separation potential. According to the Tafel equation, SECM images were converted to parameters that directly showed the distribution of solution potential. Change in the solution potential along the central axial line of the microchannel was also continuously sensed by allowing the tip to approach the microchannel in the presence of high voltage. Using dopamine as a model compound, the effect of solution potential on electrochemical detection was estimated by detecting separation parameters.