番茄蛋白酶类劣变因子荧光传感器阵列检测技术研究

Detection of Protease Deterioration Factor in Tomato by Fluorescence Sensor Array

基于荧光光谱法原理,结合免疫荧光法和荧光探针法,建立了番茄蛋白酶类劣变因子荧光传感器阵列检测技术。该研究合成了能被果胶酶、过氧化氢酶和超氧化物歧化酶抗体识别的量子点荧光探针,基于抗原抗体特异性识别原理建立了果胶酶、过氧化氢酶和超氧化物歧化酶的免疫荧光光谱分析法,并对其反应参数进行了优化,在最优条件下,通过研究其相应的荧光光谱变化规律,发现果胶酶、过氧化氢酶和超氧化物歧化酶的活性检测范围分别为0.05~500, 0.02~800和0.5~900U·mL^-1,且其免疫荧光强度与果胶酶、过氧化氢酶和超氧化物歧化酶的活性呈线性关系,相关系数分别为0.989 4, 0.993 8和0.981 9,最低检测限分别为5.0×10^-3, 2.0×10^-3和5.0×10^-2 U·mL^-1,与现有的分析法相比,该方法操作简单,线性范围宽,检测限低。针对多酚氧化酶和羧酸酯酶的活性检测,合成了能被羧酸酯酶和多酚氧化酶特异性识别的水溶性荧光探针,并对其响应机制和反应参数进行探究。在37℃pH 7.4条件下通过研究其相应的荧光光谱变化规律,发现羧酸酯酶和多酚氧化酶的存在可催化荧光团与探针中识别部分的价键裂解,释放出近红外荧光团,且荧光基团的释放量与羧酸酯酶(0.01~0.3U·mL^-1)及多酚氧化酶(10~70U·mL^-1)的活性呈线性相关,其相关系数分别为0.991 0和0.997 2,最低检测限分别为3.4×10^-3和1.1×10^-2 U·mL^-1。与现有的近红外荧光探针法相比,合成的近红外荧光探针具有较好的水溶性,且特异性较高。基于荧光光谱法原理,利用荧光免疫法和荧光探针法相结合的荧光分析方法,通过荧光酶标仪可针对不同荧光物质设置相应的激发波长和发射波长,实现多个样品中多种酶活性的同时可视化阵列检测。通过样品加标回收实验,结果表明回收率在90.0%~102.3%范围内,变异系数<15%,且具有较好的特异性,说明所建立方法准确度好,可应用于实际样品的检测。

Based on fluorescence spectroscopy,combined with immunofluorescence and fluorescence probe,a fluorescence sensor array detection technology of tomato protease deterioration factor was established.In this study,a quantum dot fluorescent probe capable of being recognized by pectinase,catalase and superoxide dismutase antibodies was synthesized by carbodiimide method.Immunofluorescence analysis of pectinase,catalase and superoxide dismutase was established based on the principle of antigen-antibody specific recognition,and the influence of its reaction parameters was investigated.Under the optimized reaction conditions(60-min reaction time at 37℃),the immunofluorescence intensity presented excellent linearity with the activities of pectinase,catalase and superoxide dismutase by studying the corresponding fluorescence spectrum changes.The detection range of activity was 0.05~500,0.02~800 and 0.5~900 U·mL-1,respectively.The correlation coefficients were 0.9894,0.9938,0.9819,and the detection limits were 5.0×10^-3,2.0×10^-3,5.0×10^-2 U·mL^-1,respectively.Compared with the existing analysis method,the method is simple in operation,low detection line and linear range.Based on the near-infrared fluorescent probe method,a novel hydrosoluble near-Infrared fluorescence off-on probe has been developed for detecting carboxylesterase and polyphenol oxidase activities.The probe was designed by introducing(4-acetoxybenzyl)oxy and 3-hydroxybenzyloxy respectively as quenching and recognizing moiety to the decomposed product of IR-783,which exhibits excellent near-infrared fluorescence feature and good water solubility.The responding mechanism of the novel probe to carboxylesterase and polyphenol oxidase was investigated.By studying the corresponding fluorescence spectrum changes at 37℃pH 7.4,it was found that the presence of carboxylesterase and polyphenol oxidase will cut off the bonds that connect the fluorophore with a recognition moiety in the probe,resulting in the release of the fluorophore,which achieves the purpose of detecting carboxylesterase and polyphenol oxidase.Moreover,the release amount of the fluorophore is linearly related to the activity of carboxylesterase(0.01~0.3 U·mL^-1)and polyphenol oxidase(10~70 U·mL^-1).This behaviour leads to the development of a simple and sensitive fluorescent method for assaying carboxylesterase and polyphenol oxidase activity,with detection limits of 3.4×10^-3 and 1.1×10^-2 U·mL^-1,respectively.The correlation coefficients were 0.9972 and 0.9910,respectively.Compared with the existing near-infrared fluorescent probe method,the near-infrared fluorescent probe synthesized in this study has better water solubility and higher specificity.The research realized the one-time and visual array detection of various enzyme activities in the sample,which is based on the multi-function fluorescent microplate reader to set the corresponding excitation wavelength and emission wavelength for different fluorescent substances.Through the sample spike recovery experiment,it is found that the recovery rate of the method is within the range of 90.0%~102.3%,the coefficient of variation is<15%,and it has good specificity,which indicated that the method established in this study has good accuracy and is expected to be applied.