| Quantum dots (QDs), also known as semiconductor nanocrystals, are novel outstanding fluorescent nano-materials, and have been widely used in biological analysis and biomedical diagnosis. QDs have attracted enormous interest due to their many novel properties such as unique optical, electrochemical and electrochemical luminescence properties. One of the most active trends is the application of QDs in electrochemical and biological sensing, due to their high surface-to-volume ratio, high reactivity and small size. Slight changes in the external environment will lead to significant changes in particle valence and electron transfer. Based on these significant changes, QDs can be used to construct electrochemical biosensor with biological macromolecules, which is characterized by high sensitivity, rapid response and high selectivity.This paper is based on the related researches of fungal laccase and new nano-materials in our laboratory. We further purified the laccase and synthesised mercaptoacetic acid (MPA) coated CdTe quantum dots (CdTe-MPA QDs). Then by sol-gel method we fixed CdTe-MPA QDs and laccase (Layer by Layer) on the electrode. So, we prepared a new type of QDs modified laccase biosensor, and to determine the optimal immobilization conditions.In this research, the most important and difficult things were the variety of gel-sol's selection, preparation and the exploration of electrode modification methods. By changing a variety of factors, we have been reached our goal. The main results are as follows:1. The water-phase synthesised CdTe-MPA QDs have a strong exciton absorption peak in 314nm, and the diameter was about 2.9nm, under the excitation spectra of 370nm, the emission peak was 570nm, the half-peak width was 70nm and the quantum yield was 54%. The prepared CdTe-MPA QDs in the solution could maintain good stability and the semiconductor nanocrystals were wurtzite CdTe (JCPDS 65-1082).2. The extracted Trametes gallica laccase activity was 3700U/L. The optimum temperature is 50℃, as electrochemical sensors usually work at room temperature, so in the QDs modified electrochemical biosensor construction process, we choosed room temperature 25℃as working temperature. Under this condition, laccase activity can reach 70%. The optimum pH was 4.5, when the pH above to 6.5, the laccase decreased rapidly.3. When the bare gold electrode in buffer solution, it was found that organic acids had a greater impact on the electrochemical biosensor. The directly modified laccase would led to most of the laccase inactivation and it was easy to fall off from the electrode and had only a weak reduction current.The blended modified would prevent most of the laccase inactivation, in this way, laccase significantly improved the contact with the gold electrode surface and the enzyme activity, the process was reversible redox process, and the redox current increased nearly 1.5 times. CdTe-MPA QDs had a little small impact on electrochemical biosensor background currents, which could improve the electron transfer efficiency. The redox reaction of CdTe-MPA QDs/laccase/sol-gel modified gold electrode was not reversible. With a larger redox current, oxidation and reduction current increased significantly by nearly 12 times, and was conducive to the reasonable position of laccase. The addition of CdTe-MPA QDs could improve the ability of electron transfer of laccase, and alter the catalytic properties of laccase. Redox potential increased from 0.129V to 0.570V, which could detect more target substances.4. In the optimization of the sensor, it was found that the best pH was 6.0, the pH was higher than the free enzyme (4.5), mainly through the sol-gel and CdTe-MPA QDs changed the fixed enzyme microenvironment and the charge distribution, which was the result of interaction. With the increasing of modified layer thickness, the redox potential unchanged, but the oxidation current was increased doubled and the reduction current decreased almost doubled. It was due to the increased distance between the enzyme and the electrode, and blocked the proliferation of substrate channels and products, thus extended the electron transfer time, resulted in longer sensor response time and lower sensitivity. The electrical activity of immobilized laccase on the electrode surface coverageΓwas: 3.26×10-11mol/cm2. Michaelis constant Km was 0.86mmol/L. The smaller Km means by sol-gel fixed, the substrate had a high affinity.5. When the CdTe-MPA QDs/laccase/sol-gel electrochemical biosensor used in detection of catechol. It was found that the detected concentration could range from 1.0×10-6-1.0×10-4mol/L. Beyond this concentration interval, the laccase reached its saturation state gradually. The regression equation was i(μA)=0.0286+26.107C (mmol/L), the correlation coefficient was 0.9993 and the detection limit (signal to noise ratio S/N=3) was 7.5×10-7mol/L. |
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