农药残留有机磷水解酶生物传感器的研究
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摘要
为了开发有机磷水解酶生物传感器,本文采用重复银镜反应制备银准参比电极及导电条,采用阳极氧化法制备银/氯化银准参比电极,探讨了碳糊工作电极制作工艺;研究了固定化材料、规格、辅助蛋白浓度对酶固定化效果的影响;优化了温度、pH等传感器检测参数,确定了酶生物传感器最佳检测条件,并检验了传感器的一致性、检测限及稳定性。主要研究内容包括三个部分,第一部分:薄层基础电极制备;第二部分:一次性固定化酶片制备;第三部分:酶生物传感器性能检测。取得以下研究成果:
(1)银镜反应最佳基质为PVC绝缘胶布、反应试剂组成为2%硝酸银、5%氢氧化钠、2%氨水、10%葡萄糖;最佳反应温度为45℃;光照有利于银镜反应进行,但避免阳光直射;最佳反应时间为10min;基质是否事先于碱液中浸泡对银镜反应效果影响不显著。所有试剂应现配现用,银氨溶液在配制过程中切不可加入过量氨水。
(2)银准参比电极最佳制备工艺为:银镜反应9次、面积98mm2;底物浓度、温度、pH值等因素对银准参比电极抗极化能力影响不显著。银∕氯化银准参比电极最佳制备工艺为:稀盐酸浓度0.1mol/L、阳极氧化时间3min;电极面积对抗极化能力影响不显著。二甲基硅油作为制备碳糊电极的黏结剂,其效果要好于液体石蜡;本文确定由M高纯石墨粉:M二甲基硅油7:3制备碳糊工作电极。通过循环扫描伏安法确定以银为准参比电极的薄层电极之间的差异小于10%。
(3)酶固定化载体的孔径是影响固定化酶片效果的主要因素,最佳固定材料及试剂组成为:孔径0.8μm、厚度135μm的硝酸纤维素膜及10%牛血清白蛋白。
(4)传感器的响应信号在起始时波动较大,最佳采集时间在45s;酶解时间为3min、温度为60℃、pH为8.0、酶量为280U;酶传感器检测的差异为7.5%;酶片在4℃条件下储存,24h后活性损失既大于50%,因此本实验制备的酶片只能一次性使用。在最优检测条件下,得到传感器的最低检测限为0.001 g/L,农药残留的标准曲线呈幂函数分布,幂函数方程为:y = 3.277x-1.4486,R2= 0.9828。以还原信号为检测对象的传感器,其信号要几倍于氧化信号,但易受背景信号影响,检测限比较高,并且不能呈现相关线性关系,数据间差异较大,需要进一步研究。
In order to prepare enzyme biosensor, the technology of repetition silver mirror reaction on surface of PVC was used to generated Ag electrod and conductive-strip, the technology of dilute hydrochloric acid on Ag electrod surface was used to made Ag/AgCL reference electrod and discussed the way for madding carbon working electrod; The influence of immobilized material、material size and auxiliary proteins concerntration was studied for enzyme immobilized effect evaluating. The parameters of temperature、suffer pH etc were studied for optimizing enzyme biosensor’s working environment, then, we detected the consistency、stability and detect-limited of the biosensor. Main contents of the paper were contained three aspects, First: Prepare thin-layer electrod; Second: Prepare one-time using carrier-bound immobilized enzyme papers; Third: Detecting the performance of enzyme biosensor. The results as follows:
(1) The results showed that the optimum matrix is friction mandrel tape; The optimum reagent were composed of 2% silver nitrate、5%sodium hydroxide、2% amminia and 10% glucose; The optimum temperature was 45℃, light was helpful to silver mirror reaction, but avoided direct-sunlight; The optimum reaction time was 10 min; The results also showed that alkali steeping before had no significant effects to silver mirror reaction. All reagents must be present-distribution and present-used, ammonia solution could not be overdose-added in the process of distributed silver-ammonia solution.
(2) Found the Ag reference electrod was made by 9-times of repetition silver mirror reaction and 98mm2electrod area; Substrate concentration、temperature、pH had no significant effects to anti-polarized ability. And found the best way for madding Ag/AgCL reference electrod was: 0.1 mol/L concentration of dilute hydrochloric acid,reaction time was 3 min,and electrod area had no significant effects to anti-polarized ability. Dimethyl silicone used as bonding agent for carbon paste electrod was better than liquid paraffin; The thesis choice M Dimethyl silicone:M graphite powder(3:7)made carbon paste working-electrod.The paper used cycle sweep voltammetry to detecting variance between thin-layer electrod, the result showed that the variance between thin-layer electrod was less than 10%.
(3) Characterized enzyme paper on electrod, the result showed that membrane pore size had significant effects to carrier-bound immobilized enzyme, the optimum carrier-bound and regents were 0.8μm nitrocellulose membrane and 10% bovine serum albumin (BSA).
(4) Regarded oxidation current as detecting object, optimized enzyme sensor parameters, the results showed that the beginning sensor signal was greatly fluctuated and the optimum acquisition time was 45S; The optimum enzyme degradation reaction time、temperature and pH was 3min、60℃and 8.0, enzyme reaction needed pure enzyme solution. The variance between sensor signal was 7.5%; More than 50% activity of carrier-bound immobilized enzyme paper which was stored at 4℃was lost after 24 hours, so the enzyme paper must under single-use.Under the optimal detecting condition, its detection limit was 0.001g/L, its standard curve was power-function and power-function equation was y = 3.277x-1.4486,R2= 0.9828. Regarded reduction current as detecting object. The results showed that the signal was many times than oxidation current sensor, but had significant effects by background signal and with high detecting limit; The signal data were also existed big variance and without any linear relationships between them.
(1)银镜反应最佳基质为PVC绝缘胶布、反应试剂组成为2%硝酸银、5%氢氧化钠、2%氨水、10%葡萄糖;最佳反应温度为45℃;光照有利于银镜反应进行,但避免阳光直射;最佳反应时间为10min;基质是否事先于碱液中浸泡对银镜反应效果影响不显著。所有试剂应现配现用,银氨溶液在配制过程中切不可加入过量氨水。
(2)银准参比电极最佳制备工艺为:银镜反应9次、面积98mm2;底物浓度、温度、pH值等因素对银准参比电极抗极化能力影响不显著。银∕氯化银准参比电极最佳制备工艺为:稀盐酸浓度0.1mol/L、阳极氧化时间3min;电极面积对抗极化能力影响不显著。二甲基硅油作为制备碳糊电极的黏结剂,其效果要好于液体石蜡;本文确定由M高纯石墨粉:M二甲基硅油7:3制备碳糊工作电极。通过循环扫描伏安法确定以银为准参比电极的薄层电极之间的差异小于10%。
(3)酶固定化载体的孔径是影响固定化酶片效果的主要因素,最佳固定材料及试剂组成为:孔径0.8μm、厚度135μm的硝酸纤维素膜及10%牛血清白蛋白。
(4)传感器的响应信号在起始时波动较大,最佳采集时间在45s;酶解时间为3min、温度为60℃、pH为8.0、酶量为280U;酶传感器检测的差异为7.5%;酶片在4℃条件下储存,24h后活性损失既大于50%,因此本实验制备的酶片只能一次性使用。在最优检测条件下,得到传感器的最低检测限为0.001 g/L,农药残留的标准曲线呈幂函数分布,幂函数方程为:y = 3.277x-1.4486,R2= 0.9828。以还原信号为检测对象的传感器,其信号要几倍于氧化信号,但易受背景信号影响,检测限比较高,并且不能呈现相关线性关系,数据间差异较大,需要进一步研究。
In order to prepare enzyme biosensor, the technology of repetition silver mirror reaction on surface of PVC was used to generated Ag electrod and conductive-strip, the technology of dilute hydrochloric acid on Ag electrod surface was used to made Ag/AgCL reference electrod and discussed the way for madding carbon working electrod; The influence of immobilized material、material size and auxiliary proteins concerntration was studied for enzyme immobilized effect evaluating. The parameters of temperature、suffer pH etc were studied for optimizing enzyme biosensor’s working environment, then, we detected the consistency、stability and detect-limited of the biosensor. Main contents of the paper were contained three aspects, First: Prepare thin-layer electrod; Second: Prepare one-time using carrier-bound immobilized enzyme papers; Third: Detecting the performance of enzyme biosensor. The results as follows:
(1) The results showed that the optimum matrix is friction mandrel tape; The optimum reagent were composed of 2% silver nitrate、5%sodium hydroxide、2% amminia and 10% glucose; The optimum temperature was 45℃, light was helpful to silver mirror reaction, but avoided direct-sunlight; The optimum reaction time was 10 min; The results also showed that alkali steeping before had no significant effects to silver mirror reaction. All reagents must be present-distribution and present-used, ammonia solution could not be overdose-added in the process of distributed silver-ammonia solution.
(2) Found the Ag reference electrod was made by 9-times of repetition silver mirror reaction and 98mm2electrod area; Substrate concentration、temperature、pH had no significant effects to anti-polarized ability. And found the best way for madding Ag/AgCL reference electrod was: 0.1 mol/L concentration of dilute hydrochloric acid,reaction time was 3 min,and electrod area had no significant effects to anti-polarized ability. Dimethyl silicone used as bonding agent for carbon paste electrod was better than liquid paraffin; The thesis choice M Dimethyl silicone:M graphite powder(3:7)made carbon paste working-electrod.The paper used cycle sweep voltammetry to detecting variance between thin-layer electrod, the result showed that the variance between thin-layer electrod was less than 10%.
(3) Characterized enzyme paper on electrod, the result showed that membrane pore size had significant effects to carrier-bound immobilized enzyme, the optimum carrier-bound and regents were 0.8μm nitrocellulose membrane and 10% bovine serum albumin (BSA).
(4) Regarded oxidation current as detecting object, optimized enzyme sensor parameters, the results showed that the beginning sensor signal was greatly fluctuated and the optimum acquisition time was 45S; The optimum enzyme degradation reaction time、temperature and pH was 3min、60℃and 8.0, enzyme reaction needed pure enzyme solution. The variance between sensor signal was 7.5%; More than 50% activity of carrier-bound immobilized enzyme paper which was stored at 4℃was lost after 24 hours, so the enzyme paper must under single-use.Under the optimal detecting condition, its detection limit was 0.001g/L, its standard curve was power-function and power-function equation was y = 3.277x-1.4486,R2= 0.9828. Regarded reduction current as detecting object. The results showed that the signal was many times than oxidation current sensor, but had significant effects by background signal and with high detecting limit; The signal data were also existed big variance and without any linear relationships between them.
引文
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