基于金纳米簇与纳米金的荧光内滤效应检测脂肪酶活性

Detection of lipase activity based on fluorescence internal filtration effect of gold nanoclusters and gold nanoparticles

目的建立一种基于吐温80自氧化作用催化纳米金形成与荧光内滤机制,相对简单、快速、适用于蛋白浓度较高的游离酶的脂肪酶活性检测方法。方法使用金纳米簇作为荧光基团,纳米金作为吸收剂,基于荧光内滤机制测定游离脂肪酶活力。设置对照试验对吐温80浓度、氯金酸浓度、温度、pH等反应条件进行优化,探索最适条件下脂肪酶水解反应时间及吐温80还原纳米金的时间并对反应体系进行抗干扰检测,验证该方法的可行性。结果当脂肪酶酶活力在13.5~15092.0 U/L时,荧光强度的差值随脂肪酶活力的升高而增加,相关系数为0.9977,脂肪酶活力的检出限为2.34 U/L(信噪比为3)。结论该方法操作相对简单、快速、检测范围较大、灵敏度高,具有实用性,可适用于蛋白浓度较高的游离酶的脂肪酶活力测定。

Objective To establish a relatively simple,rapid and suitable method for the detection of lipase activity of free lipase with high protein concentration based on the formation of gold nanoparticles catalyzed by Tween 80 self-oxidation and the fluorescence internal filtration effect.Methods Using gold nanoclusters as fluorophores and gold nanoparticles as absorbent,the free lipase activity was determined based on the fluorescence internal filtration effect.A control experiment was set up to optimize the reaction conditions such as Tween 80 concentration,chloroauric acid concentration,temperature,and pH.The time of lipase hydrolysis reaction under optimal conditions and the time of Tween 80 reducing gold nanoparticles were explored.The anti-interference detection of the reaction system was performed to verify the feasibility of the method.Results When the lipase activities were 13.5-15092.0 U/L,the difference of fluorescence intensity increased with the increase of lipase activity,and the correlation coefficient was 0.9977.The limit of detection of lipase activity was 2.34 U/L(signal-to-noise ratio was 3).Conclusion The method is relatively simple,rapid,wide detection range,sensitive and practical,can be applied to the detection of lipase activity of free lipase with high protein concentration.