| Electrochemical sensors have become a hot research topic due to their good selectivity, high sensitivity and easy miniaturization. In the field of the proteins (enzymes)-based electrochemical biosensors, researchers are looking for new strategies to maintain the enzymatic activity and enhance the direct electron transfer signal of the redox proteins (enzymes). In the field of the nanomaterials-based nonenzymatic electrochemical sensors, they devote themselves to investigating the evolution mechanism of the nanomaterials and preparing the nanomaterials with high electrocatalytic activity, which are the key problems for constructing high-performace electrochemical sensors.As novel sensing materials, the "green" ionic liquids and nanomaterials have attracted great attention recently. The ionic liquids have played a great role in the electrochemical biosensing systems. As binders, ionic liquids are used to prepare carbon ionic liquid electrodes (CILEs), which have many advantages. However, one drawback is their high background current. For the enzyme-containing CILE, decreasing its background current while maintaining the enzymatic activity is the key problem. As supporting electrolytes, ionic liquids have different levels of effect on the direct electron transfer process of the immobilized and free proteins (enzymes), but the effect mechanisms are not clear and deserve to be investigated. The understanding of the mechanisms will help to develop high-performance biosensors. Moreover, their correlation with the Hofmeister effects of ionic liquids will also favor for establishing a bioelectrochemical method to characterize the Hofmeister effect. The nano/micro-sized CuO and nanoporous gold (NPG) have played a great role in the nonenzymatic electrochemical sensing systems. The performance of the electrochemical sensors is correlated closely with the size, morphology of the nanomaterials. Thus exploring the formation mechanism of the nanomaterials and establishing facile, low-cost and green methods to fabricate the nanomaterials with high electrocatalytic activity are crucial for the high-performance electrochemical sensors. To this end, some new strategies are tried and the results obtained are presented in the following sections:1. An ionic liquid-based new strategy to enhance the performance of the enzyme-containing CILECompared with the traditional carbon paste electrode (CPE), the CILE has many advantages and the CILE-based electrochemical biosensors have been tried in electroanalysis. However, one drawback is their high background current, which limits their further application. Currently, it is shown that heating can decrease the background current of the CILE prepared with the high-melting-point ionic liquid as the binder, but this measure also decreases the activity of the enzyme entrapped in the CILE. From the perspective of biocompatibility, an ionic liquid with proper melting point should be chosen so that the background current would decrease but the enzymatic activity be kept, and the signal to noise ratio thus increase. In the present study, a new CILE has been constructed using a low melting point (39℃) hydrophobic ionic liquid1-propyl-3-methylimidazolium hexafluorophosphate ([Pmim][PF6]) as the binder. Both cyclic voltammograms and electrochemical impedance spectroscopy demonstrate that, in addition to the composition optimization of the ionic liquid/graphite composite, heating the composite at a temperature a little higher than the melting point of [Pmim][PF6] can also lower the background current and enhance the mechanical strength of the CILE. The heated CILE is more sensitive than the traditional CPE for the detection of H2O2. Glucose oxidase (GOx) can be easily entrapped in the bulk composite. Heating the GOx-modified CILE (GOx-CILE) at the melting point of [Pmim][PF6] does not lower the catalytic activity of GOx. As compared with n-octylpyridinium hexafluorophosphate (melting point65℃) as the binder,[Pmim][PF6]-based CILE is much better in signal to noise ratio. Under the optimum conditions, the [Pmim][PF6]-based GOx-CILE has a linear amperometric response to glucose over a concentration range of2.0-26mM with the detection limit as low as0.39mM. It follows that choosing an ionic liquid with a melting point a little higher than the room temperature as a binder to fabricate enzyme-entrapped CILEs is a good strategy for the enhancement of the performance of the electrode.2. Effects of ionic liquids on the direct electron transfer of the immobilized enzyme and the construction of an anti-fluoride biosensorThe direct electron transfer of redox proteins (enzymes) is the prerequisite for the fabrication of the third generation electrochemical biosensor. Ionic liquids play an important role in the construction and application of these biosensors. However, the effects as well as their mechanism of the ionic liquids as the supporting electrolytes on the direct electron transfer of the immobilized proteins (enzymes) are not clear and the use of various electron promoters or mediators and inappropriate films also makes the study of the effect of the single cation or anion difficult. In the present study, the direct electrochemistry and bioelectrocatalysis of horseradish peroxidase (HRP) in Nafion film at glassy carbon electrode (GCE) were investigated in three [BF4]--type ionic liquids to understand the structural effect of imidazolium cations. A small amount of water in the three ionic liquids is indispensable for maintaining the electrochemical activity of HRP in Nafion film, and the optimum water contents decrease with the increase of alkyl chain length on imidazole ring. Analysis shows that the optimum water contents are primarily determined by the hydrophilicity of ionic liquids used. In contrast to aqueous medium, ionic liquids media facilitate the direct electron transfer of HRP, and the electrochemical parameters obtained in different ionic liquids are obviously related to the nature of ionic liquids. The direct electron transfer between HRP and GCE is a surface-confined quasi-reversible single electron transfer process. The apparent heterogeneous electron transfer rate constant decreases gradually with the increase of alkyl chain length on imidazole ring, but the changing extent is relatively small. The electrocatalytic reduction current of H2O2at the present electrode decreases obviously with the increase of alkyl chain length, and the mass transfer of H2O2via diffusion in ionic liquids should be responsible for the change. In addition, the modified electrode has good stability and reproducibility; the ability to tolerate high levels of F-has been greatly enhanced due to the use of the Nafion film. Thus, an HRP-based biosensor with high anti-fluoride performance was constructed.3. Effects of ionic liquids on the direct electron transfer of the free enzyme and bioelectrochemical characterization of the Hofmeister effects of ionic liquidsIn order to understand the mechanism of the effects of ionic liquids on the direct electron transfer of HRP more intuitively, the effects of the cations and anions of different ionic liquids in aqueous media on the direct electron transfer and structural stability of HRP were systematically investigated using electrochemical methods. It is found that without ionic liquids no direct electron transfer current signals of HRP appear at bare GCE in phosphate buffer (pH7.0) even after incubation and accumulation at a negative potential. In the presence of ionic liquids, however, a current signal occurs at GCE, depending on the structure of the ionic liquid and its concentration. A linear relationship between the peak currents and the scan rates demonstrates that the direct electron transfer is a surface-confined thin-layer electrochemical process. The redox signal at GCE is from the heme of HRP. An ionic liquid has a perturbing effect on the HRP structure. The anodic peak current of HRP at GCE, the catalytic activity of HRP, and the secondary structure of HRP are well correlated. Different cations or anions at varied concentrations have different effects on the structural stability of HRP, resulting in different current signals at GCE. Studies on the structure of proteins and enzymatic activity are effective routes to investigate the Hofmeister effect of ionic liquids. Thus, the anodic peak current of HRP at GCE can be used as an indicator to quantitatively characterize the effect of ionic liquids on the structural stability of HRP, and a bioelectrochemical method is established to investigate the Hofmeister effect of ionic liquids. The present study not only favors for understanding the effects of ionic liquids on the direct electron transfer of free HRP, providing theoretical support for high-performance electrochemical sensors, but also offers a practical guidance to designing "green" and biocompatible ionic liquids for protein (enzyme) separation, purification, and enzymatic catalysis and conversion.4. New strategies to fabricate nanomaterials and studies on their electrocatalytic activityThe stability of the nonenzymatic electrochemical sensors is good due to the absence of biologically active substances. For the construction of nonenzymatic electrochemical sensors, the prerequisite is to obtain novel nanomaterials with high electrocatalytic activity using facile, low-cost and green methods. In the present study, a facile electrochemical method is established for the synthesis of CuO flower-like microspheres (CuO FMs) by anodic dissolution of bulk Cu in sodium hydroxide solution at room temperature and without any additives. Scanning electron microscopy (SEM) and X-ray diffraction reveal that the CuO FMs are phase-pure monoclinic crystallites and comprised of CuO nanoflakes. The infrared spectrum shows that the CuO FMs are very pure and don’t contain Cu(OH)2impurity. The evolution process of the material at different stages was investigated by controlling the electrolysis time and a proper evolution mechanism was presented. Compared with the bare GCE, the onset potential of the H2O2oxidation is more negative and the current is bigger at the CuO FMs modified GCE, indicating that the CuO FMs have good electrocatalytic activity towards H2O2oxidation. The response current increases linearly with the increase of the H2O2concentration, indicating that the CuO FMs modified GCE has good sensing ability. Self-supporting nanoporous gold electrodes (NPGEs) were fabricated by in-situ electrochemical alloying/dealloying method without any protection using a water/air-stable ionic liquid ([Choline]C1·2ZnCl2) as the electrolyte. The effect as well as its mechanism of the temperature on the alloying/dealloying process of the gold surface was investigated. The cyclic voltammograms and SEM images show that the amount of the Au-Zn alloy and the porosity of NPG increase with the increase of the temperature. Compared with the smooth gold electrode, the electroactive area and the electrooxidation ability toword glucose in neutral or alkaline solution at the NPGE prepared at120℃increase drastically. In the neutral solution, the NPGE can detect glucose effectively. This study not only favors for understanding the mechanism of the electrochemical alloying/dealloying process, but also provides a self-supporting substrate electrode for constructing higher-perpormance nonenzymatic electrochemical sensors. |
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