CN109115734A - 一种利用氮化硼量子点-金纳米粒子纳米复合物检测人血清中乙酰胆碱酯酶含量的方法 - Google Patents
一种利用氮化硼量子点-金纳米粒子纳米复合物检测人血清中乙酰胆碱酯酶含量的方法 Download PDFInfo
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Abstract
本发明公开了一种利用氮化硼量子点‑金纳米粒子纳米复合物检测人血清中乙酰胆碱酯酶含量的方法。该方法包括以下步骤:1)建立荧光强度与乙酰胆碱酯酶浓度的标准曲线:①先制备碘化乙酰胆碱‑氮化硼量子点溶液;②向碘化乙酰胆碱‑氮化硼量子点溶液中分别加入相同体积不同浓度的乙酰胆碱酯酶溶液,随后加入HAuCl4,混合均匀;将一系列混合溶液反应后,分别测定溶液的荧光强度,绘制荧光强度与乙酰胆碱酯酶浓度的标准曲线,得线性回归方程;2)将待测血清样品按照步骤②的方法制成混合溶液,测定荧光强度;将荧光强度代入线性回归方程中,得到乙酰胆碱酯酶浓度。该方法可以应用到人血清样本的实际检测,具有步骤简单、快速和灵敏的特点。
Description
技术领域
本发明属于分析化学和纳米技术领域,具体涉及一种利用氮化硼量子点-金纳米粒子纳米复合物检测人血清中乙酰胆碱酯酶含量的方法。
背景技术
乙酰胆碱酯酶(AChE)是生物神经信号传输过程中一种非常关键的酶,这种酶通过将碘化乙酰胆碱(ATCh)分解为硫代胆碱(TCh)来减弱神经递质对突触后膜的兴奋作用,从而确保体内神经信号的正常传递。AChE还参与细胞的发育和成熟,从而促进神经元发育和神经再生。 值得注意的是,神经毒气,阿尔茨海默病(AD)药物和有机磷农药(OPs)可以抑制AChE的活性,并导致ATCh在突触间隙积累,阻碍神经递质的传输。调节AChE的水平对于维持正常的生理活动十分重要。因此,从理论和实践两个方面,简单,快速和准确的测定AChE的含量并筛选潜在抑制剂具有重要意义。
发明内容
本发明的目的在于针对目前大多数检测方法具有繁琐的操作程序,耗时,标记和低灵敏度的缺点,提供了一种利用氮化硼量子点-金纳米粒子纳米复合物检测人血清中乙酰胆碱酯酶含量的方法。本发明的检测方法步骤简单,快速,无标记且灵敏;该传感器基于AChE调节合成的AuNPs能猝灭BNQDs荧光特点,以实现对血清样本中AChE含量的检测,其方法高效、快速、免标记、灵敏,适用于临床诊断。
为实现上述目的,本发明采用如下技术方案:
一种利用氮化硼量子点-金纳米粒子纳米复合物检测人血清中乙酰胆碱酯酶含量的方法,包括以下步骤:
1)建立荧光强度与乙酰胆碱酯酶浓度的标准曲线:
①配制0.1mg/ml碘化乙酰胆碱-氮化硼量子点溶液;
②向100μL 0.1mg/ml碘化乙酰胆碱-氮化硼量子点溶液中分别加入50μL不同浓度的乙酰胆碱酯酶溶液,随后加入50μL 0.8mM HAuCl4溶液,混合均匀;将一系列混合溶液在37℃下反应32 min后,分别测定溶液的荧光强度,绘制荧光强度与乙酰胆碱酯酶浓度的标准曲线,得线性回归方程;
2)向100μL 0.1mg/ml 碘化乙酰胆碱-氮化硼量子点溶液中加入50μL稀释后的待测血清,随后加入50μL 0.8mM HAuCl4,混合均匀;将混合溶液在37℃下反应32 min后,测定溶液的荧光强度,将荧光强度代入步骤②的线性回归方程中,得到乙酰胆碱酯酶浓度,经过换算得到未经稀释的原待测血清中的乙酰胆碱酯酶含量。
步骤①具体为:向氮化硼量子点溶液中加入碘化乙酰胆碱,氮化硼量子点与碘化乙酰胆碱的摩尔比为1:1.5,混合振荡1h,溶液通过透析袋去除未键合的碘化乙酰胆碱,得到0.1mg/ml碘化乙酰胆碱-氮化硼量子点溶液。
步骤②中乙酰胆碱酯酶溶液的浓度范围为0.05~10 mU/mL。
步骤②所述的线性回归方程为:FL=406.11-49.52C,R2=0.997。
该方法对乙酰胆碱酯酶的响应范围为0.05-6 mU/mL,检测限LOD为0.0212 mU/mL。
该方法的作用机理为:
将ATCh与氮化硼量子点(BNQDs)通过静电作用结合形成ATCh-BNQDs,AChE水解ATCh产生巯基,巯基还原氯金酸(HAuCl4)产生金纳米颗粒(AuNPs),同时,金纳米颗粒与未参与反应的巯基通过金巯键结合形成氮化硼量子点-金纳米粒子纳米复合物(TCh-BNQDs/AuNPs);基于荧光共振能量转移,由于AuNPs与BNQDs分之间的距离较近,存在FRET效应,AuNPs猝灭BNQDs的荧光,通过BNQDs荧光强度的变化评估AChE的浓度。
一种利用氮化硼量子点-金纳米粒子纳米复合物检测有机磷农药双氧磷对乙酰胆碱酯酶抑制浓度的方法,包括以下步骤:
建立荧光强度与双氧磷浓度的标准曲线:
①向氮化硼量子点溶液中加入碘化乙酰胆碱,混合振荡1h,溶液通过透析袋去除未键合的碘化乙酰胆碱,得到碘化乙酰胆碱-氮化硼量子点溶液;
②将不同量的有机磷农药双氧磷分别和50μL 5mU/mL乙酰胆碱酯酶预先混匀30min,然后加入100μL 0.1mg/ml 碘化乙酰胆碱-氮化硼量子点溶液和50μL 0.8mM HAuCl4,混合溶液在37℃下反应32 min后,测定溶液的荧光强度,绘制荧光强度与双氧磷浓度的标准曲线,得到线性回归方程为:FL=159.86+31.81C,R2=0.997;并计算双氧磷抑制AChE的半抑制浓度(IC50)。其中(F -F0)/F0叫变化率,(F-F0)/F0=50%时对应的浓度即为半抑制浓度。
该法的作用机理为:
有机磷农药双氧磷抑制会降低AChE的活性,从而阻碍AChE水解ATCh的速率,导致产生的AuNPs的浓度显著降低,猝灭BNQDs的效率降低,BNQDs的荧光强度随着双氧磷浓度的增大而增强。因此,可以绘制BNQDs荧光强度与双氧磷浓度的标准曲线。可计算双氧磷抑制AChE的半抑制浓度。
本发明的有益效果在于:
本发明基于AChE调节合成的AuNPs能猝灭BNQDs荧光特点的原料,建立了一种检测血清样本中AChE含量的方法,该法高效、快速、免标记、灵敏,适用于临床诊断。
附图说明
图1为本发明所得BNQDs的表征图谱;
图2为本发明所得ATCh-BNQDs的表征图谱;
图3为AChE调节原位生长AuNPs的可行性;
图4为BNQDs响应不同浓度AChE调节AuNP诱导荧光强度变化图(A)及其绘制的标准曲线(B);
图5为本BNQDs响应不同浓度AChE抑制剂双氧磷调节AuNP诱导荧光强度变化图(A)及其绘制的标准曲线(B)。
具体实施方式
为了使本发明所述的内容更加便于理解,下面结合具体实施方式对本发明所述的技术方案做进一步的说明,但是本发明不仅限于此。
实施例1
一种利用氮化硼量子点-金纳米粒子纳米复合物检测人血清中乙酰胆碱酯酶含量的方法,具体步骤为:
1)ATCh-BNQDs合成
向20 mL 0.2mg/mL BNQDs(表征见图1)溶液中加入70mg ATCh,在37℃下混合振荡1h,混合溶液通过1000Da的透析袋除去未键合的ATCh,得到ATCh-BNQDs,通过紫外可见吸光光谱、Zeta电势和红外光谱表征产物是否合成成功,见图2,最终通过紫外可见吸收光谱定量ATCh-BNQDs的浓度;
2)AChE调节原位生长AuNPs的可行性
向100μL 0.1mg/ml ATCh-BNQDs溶液中分别加入50μL不同浓度的AChE,随后加入50μL0.8mM HAuCl4溶液,混合溶液在37℃下反应32 min后,溶液变红,测定溶液的紫外可见吸收光谱的最大吸收峰位于531nm处,粒径分布为20nm,并且通过透射电镜TEM直观观察到原位生长的AuNPs,见图3,混合反应中的pH值为7.0;
3)BNQDs响应不同浓度AChE调节AuNP诱导荧光强度变化图及其标准曲线。
使用PBS(pH=7.0)缓冲液配制不同浓度的AChE溶液(AChE的浓度依次为0.05、0.1、0.2、0.5、1、2、3、4、5、6、8、10 mU/mL),向100μL 0.1mg/ml ATCh-BNQDs溶液中分别加入50μL不同浓度的AChE溶液,随后加入50μL 0.8mM HAuCl4溶液,混合溶液在37℃下反应32 min后,测定溶液的荧光强度,绘制荧光强度与AChE浓度的标准曲线;(如图4,其中A为BNQDs响应不同浓度AChE调节AuNP诱导荧光强度变化图;B为相应的标准曲线,其响应范围为0.05-6 mU/mL,检测限LOD为0.0212 mU/mL);
4)人血清中AChE含量的检测
计算加标回收率以及验证方法实用性:将实际的人血清样本稀释一定倍数并分成五组,分别向人血清样本中加入0.05,0.1,0.15,0.20,0.25mU/mL AChE,基于标准加入方法的原理,以步骤3)的操作步骤在同一条件下反应后测定荧光强度,根据AChE标准曲线计算加标回收率和RSD验证方法的实用性;
测定实际血清中AChE含量:向100μL 0.1mg/ml 碘化乙酰胆碱-氮化硼量子点溶液中加入50μL稀释后的待测血清,随后加入50μL 0.8mM HAuCl4溶液,混合均匀;将混合溶液在37℃下反应32 min后,测定溶液的荧光强度,将荧光强度代入步骤②的线性回归方程中,得到乙酰胆碱酯酶浓度,经过换算得到未经稀释的原待测血清中的乙酰胆碱酯酶含量,见表1。
表1
a实际血清样品稀释10000倍. b 本方法检测血清样本中AChE浓度为7380 U/L; Ellman法检测血清样本中AChE浓度为8576 U/L.
实施例2
BNQDs响应不同浓度AChE抑制剂双氧磷调节AuNP诱导荧光强度变化图及其标准曲线。
将不同量有机磷农药双氧磷和50μL AChE预先混匀30min,然后加入100μL 0.1mg/ml ATCh-BNQDs和50μL 0.8mM HAuCl4溶液;混合溶液在37℃下反应32 min后测定溶液的荧光强度,绘制荧光强度与双氧磷浓度的标准曲线,并计算双氧磷抑制AChE的半抑制浓度(IC50),见图5。
以上所述仅为本发明的较佳实施例,凡依本发明申请专利范围所做的均等变化与修饰,皆应属本发明的涵盖范围。
Claims (5)
1.一种利用氮化硼量子点-金纳米粒子纳米复合物检测人血清中乙酰胆碱酯酶含量的方法,其特征在于:包括以下步骤:
1)建立荧光强度与乙酰胆碱酯酶浓度的标准曲线:
①配制0.1mg/ml碘化乙酰胆碱-氮化硼量子点溶液;
②向100μL 0.1mg/ml碘化乙酰胆碱-氮化硼量子点溶液中分别加入50μL不同浓度的乙酰胆碱酯酶溶液,随后加入50μL 0.8mM HAuCl4溶液,混合均匀;将一系列混合溶液在37℃下反应32 min后,分别测定溶液的荧光强度,绘制荧光强度与乙酰胆碱酯酶浓度的标准曲线,得线性回归方程;
2)向100μL 0.1mg/ml 碘化乙酰胆碱-氮化硼量子点溶液中加入50μL稀释后的待测血清,随后加入50μL 0.8mM HAuCl4,混合均匀;将混合溶液在37℃下反应32 min后,测定溶液的荧光强度,将荧光强度代入步骤②的线性回归方程中,得到乙酰胆碱酯酶浓度,经过换算得到未经稀释的原待测血清中的乙酰胆碱酯酶含量。
2.根据权利要求1所述的利用氮化硼量子点-金纳米粒子纳米复合物检测人血清中乙酰胆碱酯酶含量的方法,其特征在于:步骤①具体为:向氮化硼量子点溶液中加入碘化乙酰胆碱,氮化硼量子点与碘化乙酰胆碱的摩尔比为1:1.5,混合振荡1h,溶液通过透析袋去除未键合的碘化乙酰胆碱,得到0.1mg/ml碘化乙酰胆碱-氮化硼量子点溶液。
3.根据权利要求1所述的利用氮化硼量子点-金纳米粒子纳米复合物检测人血清中乙酰胆碱酯酶含量的方法,其特征在于:步骤②中乙酰胆碱酯酶溶液的浓度范围为0.05~10mU/mL。
4.根据权利要求1所述的利用氮化硼量子点-金纳米粒子纳米复合物检测人血清中乙酰胆碱酯酶含量的方法,其特征在于:步骤②所述的线性回归方程为:FL=406.11-49.52C,R2=0.997。
5.根据权利要求1所述的利用氮化硼量子点-金纳米粒子纳米复合物检测人血清中乙酰胆碱酯酶含量的方法,其特征在于:该方法对乙酰胆碱酯酶的响应范围为0.05-6 mU/mL,检测限LOD为0.0212 mU/mL。
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