CN111579613B - 一种基于光电化学传感的脂肪酸结合蛋白检测方法 - Google Patents

一种基于光电化学传感的脂肪酸结合蛋白检测方法 Download PDF

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CN111579613B
CN111579613B CN202010397550.7A CN202010397550A CN111579613B CN 111579613 B CN111579613 B CN 111579613B CN 202010397550 A CN202010397550 A CN 202010397550A CN 111579613 B CN111579613 B CN 111579613B
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韩得满
陈逢灶
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Abstract

本发明属于分析化学中的光电化学传感技术领域,特别涉及一种基于脂质体信号放大策略的新型光电化学生物传感器的设计及在检测脂肪酸结合蛋白FABP中的应用技术。所述光电化学传感器由多孔二氧化钛纳米管TiO2 NTs与金纳米簇Au NCs组合而成。其制备方法简便易行、快速、成本低、所制备的光电化学传感器性能稳定。光电化学传感器可在温和条件下应用于高选择性、高灵敏度、快速检测脂肪酸结合蛋白。本发明提供了一种新型的基于光电化学传感检测脂肪酸结合蛋白的方法,具有重要的意义和很好的应用前景。

Description

一种基于光电化学传感的脂肪酸结合蛋白检测方法
技术领域
本发明属于分析化学中的光电化学传感技术领域,特别涉及一种基于脂质体信号放大策略的新型光电化学生物传感器的设计及在检测脂肪酸结合蛋白FABP中的应用技术。
背景技术
脂肪酸结合蛋白FABP是一种小分子量的细胞溶质蛋白,广泛分布于人体内的各个组织中并发挥重要的作用如:作为脂肪酸的运载体参与调节细胞脂肪酸隔室化分布和代谢;作为协同因子增强以脂肪酸代谢为基础的细胞合成或氧化;参与调节细胞的增殖与生长、胰岛素信息传递及胆红素、甾醇、前列腺素等的代谢活动。研究表明,在发生心肌梗死或急性心梗病后30分钟到3小时内,血浆中的FABP将开始升高并与12-24小时内恢复正常,其灵敏度可达78%,明显高于其他常规指标如肌酸激酶、肌酸激酶同工酶等,因此FABP可作为急性心梗疾病早期诊断的一种重要指标。常见的检测FABP的方法主要有放射免疫法、ELISA酶联免疫法、荧光分析法等。虽然这些方法取得一定的成功,但仍存在不足如灵敏度低、硬件设备要求高、成本高等问题。因此,开发一种新型的简便的高灵敏度检测FABP的传感器具有重要的实用价值和应用前景。
随着材料化学和生物技术的快速发展,在光电化学过程和电化学生物传感技术的基础上已开发出新一代光电化学(Photoelectrochemical,PEC)生物传感器技术,为生物分析提供了一种新的检测手段。光电化学过程指具有光电活性的材料在合适的光照作用下所发生的经由电子激发和电荷转移形成的光电转换过程。光电活性物质在受光激发后产生电子空穴对,随后发生复合或电荷转移,一定条件下,导带上的电子可以向电极或电解池溶液中电子受体转移而产生相应的阳极或阴极光电流响应。光电化学生物传感技术的定量分析基础是基于光照作用下,目标物与光电活性材料之间直接或间接的相互作用或在目标物进行高特异性识别反应后(改变电极表面或周围状态,如产生位阻效应等)工作站所监测到的电信号变化与目标物浓度之间的关系。检测过程中,光被用作激发信号来激发光电活性材料而电信号则作为检测信号,二者分属于不同形式的能量,因此该技术可进一步降低背景信号,实现更高灵敏的检测。
发明内容
本发明的目的在于解决现有技术问题的不足,提供一种基于脂质体信号放大策略的新型光电化学生物传感器的设计及在检测脂肪酸结合蛋白FABP中的应用技术。所述技术通过改变电极表面Au NCs对TiO2NTs敏化作用来实现对FABP的灵敏检测。
为了实现上述发明目的,本发明提供以下技术方案:
一种基于光电化学传感的脂肪酸结合蛋白检测方法,包括以下步骤:
(1)光电化学传感器的制备
a.金纳米簇Au NCs的制备:
将23mg谷胱甘肽溶于23mL水中,滴加2mL质量分数1%的HAuCl4溶液,然后加热至70℃并搅拌一定时间,待反应结束,冷却至室温得到金纳米簇Au NCs溶液;
b.多孔二氧化钛纳米管TiO2NTs电极的制备:
将金属钛片连接于电源正极,石墨棒做负极,以质量分数0.3%的NH4F乙二醇溶液为电解液,设定直流电压为50V通电2h进行电氧化,用超声仪清洗钛片并吹干,设定直流电压为50V通电30min,水洗并吹干得到TiO2NTs,将得到的TiO2NTs置于450℃的马弗炉中煅烧60min,得到多孔二氧化钛纳米管TiO2NTs电极;
c.Au NCs@TiO2NTs电极的制备:
将步骤b中得到的TiO2NTs电极置于步骤a中得到的Au NCs溶液中浸泡一段时间,使得Au NCs吸附在TiO2NTs表面并对TiO2NTs进行敏化,得到Au NCs@TiO2NTs电极;
(2)脂肪酸结合蛋白的检测方法
d.脂质体Ab2-ALPL的制备:
将二棕榈酰基磷脂酰乙醇胺DPPE、1,2-棕榈酰磷脂酰甘油DPPG、二棕榈酰磷脂酰胆碱DPPC和胆固醇CHOL溶于二氯甲烷/甲醇混合溶液中,减压脱溶得到脂质体膜后加入碱性磷酸酶ALP溶液水化过夜,超声并过滤得到装载有ALP的脂质体ALPL,将鼠抗人脂肪酸结合蛋白单克隆抗体2(Ab2)通过戊二醛修饰到ALPL表面得到Ab2-ALPL;
e.光电化学脂肪酸结合蛋白检测体系的构建:
1)取60uL 0.2mg/mL的鼠抗人脂肪酸结合蛋白单克隆抗体1(Ab1)加入96孔板中4℃下孵育12h,将Ab1修饰在96孔板中,然后用PBS缓冲液小心地洗去未结合在96孔板上的Ab1,然后加入牛血清蛋白BSA 4℃下孵育2h对多余位点进行封闭;
2)加入待检测的目标抗原Ag(FABP)在4℃下孵育1h,Ag通过免疫反应结合到Ab1上形成Ab1-Ag复合物,PBS缓冲液洗去未结合的Ag;
3)加入40uL步骤d得到的Ab2-ALPL在37℃下孵育1h,通过三明治免疫反应结合到Ab1-Ag复合物形成Ab1-Ag-Ab2-ALPL免疫复合物,PBS缓冲液洗去未结合的Ab2-ALPL,然后加入曲拉通Triton X-100进行脂质体裂解,得到含有ALP的脂质体裂解液;
4)将3)得到的脂质体裂解液滴加到步骤c得到的Au NCs@TiO2NTs电极上,然后再加入磷酸抗坏血酸酯AAP和HAuCl4溶液37℃孵育1h,使得ALP可以水解AAP产生具有还原性的抗坏血酸AA,以Au NCs作为晶种,AA将还原HAuCl4溶液中的Au3+在Au NCs上原位生长,使得Au NCs原位长大成金纳米颗粒Au NPs,改变了原先Au NCs的敏化效果,抑制电极的光电流信号;
5)孵育后的Au NCs@TiO2NTs电极用PBS缓冲液冲洗,然后在实验室自行搭建的光电化学测试系统上测量该电极的光电流强度,测试条件为:5W的LED灯作为激发光源;光电测试采用三电极体系:Au NCs@TiO2NTs电极为工作电极,Ag/AgCl为参比电极,Pt丝为对电极;光电流由上海辰华仪器公司的CHI660C工作站测定,设置偏压为0V;电解液为pH值7.4的含20mM三乙醇胺的10mM的PBS缓冲液。
优选的,所述步骤a中搅拌时间为10-48h,更优选的搅拌时间为24h。
优选的,所述步骤c中浸泡时间为1-48h,更优选的浸泡时间为8h。
优选的,所述步骤d中二棕榈酰基磷脂酰乙醇胺DPPE、二棕榈酰磷脂酰胆碱DPPC、1,2-棕榈酰磷脂酰甘油DPPG、胆固醇CHOL的质量比为5:15:15:7。
优选的,所述步骤d中二氯甲烷/甲醇混合溶液的体积比为4:1。
优选的,所述步骤e 3)中使用40uL 0.05%的曲拉通Triton X-100进行脂质体裂解。
优选的,所述步骤e中PBS缓冲液为10mM pH 7.4的磷酸盐缓冲液。
优选的,Au NCs@TiO2NTs电极实现对脂肪酸结合蛋白的检测在于其光电流的变化程度与脂肪酸结合蛋白浓度有关。
本发明与现有技术相比,其有益效果主要体现在:
本发明所述的基于脂质体信号放大策略的新型光电化学生物传感器在检测脂肪酸结合蛋白FABP中的应用技术,具有制备方法简单易行、成本低、无污染的优点,并且所制备的光电化学生物传感器稳定性好、输出信号稳定;同时,在温和条件下,该传感器的灵敏度高,可实现对低浓度FABP的灵敏检测,并且选择性好,对FABP具有非常好的专一性。因此,该光电化学生物传感器具有重要的意义和很好的应用前景。
附图说明
图1为所述的光电化学生物传感器稳定性测试的光电流图;
图2为所述的光电化学生物传感器检测FABP的光电流图;
图3为所述的光电化学生物传感器的选择性实验光电流图。
具体实施方式
下面通过具体实施例,并结合附图对本发明的技术方案作进一步的具体说明。
实施例1:
(1)光电化学传感器的制备
a.金纳米簇Au NCs的制备:
将23mg的谷胱甘肽溶于23mL水中,然后滴加入质量分数为1%的2mL的HAuCl4溶液,然后加热至70℃并搅拌24h,待反应结束,将其冷却至室温并存储备用;
b.多孔二氧化钛纳米管TiO2NTs电极的制备:
将金属钛片连接于电源正极,石墨棒做负极,以质量分数为0.3%的NH4F乙二醇溶液为电解液,设定直流电压为50V并通电2h进行电氧化,之后用超声仪清洗钛片,洗净并吹干,然后设定直流电压为50V并通电30min,之后水洗并吹干得到TiO2NTs,将得到的TiO2NTs置于450℃的马弗炉中煅烧60min,得到TiO2NTs电极;
c.Au NCs@TiO2NTs电极的制备:
将步骤b中得到的TiO2NTs电极置于步骤a中得到的Au NCs溶液中浸泡8h,使得AuNCs吸附在TiO2NTs表面并对TiO2NTs进行敏化,得到Au NCs@TiO2NTs电极;
(2)脂肪酸结合蛋白的检测方法
d.脂质体Ab2-ALPL的制备:
将5mg的二棕榈酰基磷脂酰乙醇胺DPPE、15mg的1,2-棕榈酰磷脂酰甘油DPPG、15mg的二棕榈酰磷脂酰胆碱DPPC、7mg的胆固醇CHOL溶于12.8mL的二氯甲烷和3.2mL的甲醇混合液中,之后用真空旋转蒸发仪除去溶液得到脂质体膜,然后加入10mL的6U/mL的碱性磷酸酶ALP溶液水化过夜,然后超声并用过滤得到装载有ALP的脂质体ALPL,将鼠抗人脂肪酸结合蛋白单克隆抗体2(Ab2)通过戊二醛修饰到ALPL表面得到Ab2-ALPL;
e.光电化学脂肪酸结合蛋白检测体系的构建:
1)取60uL 0.2mg/mL的鼠抗人脂肪酸结合蛋白单克隆抗体1(Ab1)加入96孔板中4℃下孵育12h,将Ab1修饰在96孔板中,然后用10mM pH7.4的磷酸盐缓冲液PBS小心地洗去未结合在96孔板上的Ab1,然后加入40uL 10mg/mL牛血清蛋白BSA 4℃下孵育2h对多余位点进行封闭;
2)然后加入待检测的目标抗原Ag(FABP)在4℃下孵育1h,Ag通过免疫反应结合到Ab1上形成Ab1-Ag复合物,PBS(10mM,pH 7.4)缓冲液洗去未结合的Ag;
3)然后加入40uL步骤d所得到的Ab2-ALPL在37℃下孵育1h,通过三明治免疫反应结合到Ab1-Ag复合物形成Ab1-Ag-Ab2-ALPL免疫复合物,PBS(10mM,pH 7.4)缓冲液洗去未结合的Ab2-ALPL,然后加入40uL0.05%的曲拉通(Triton X-100)进行脂质体裂解,得到含有ALP的脂质体裂解液;
4)将3)得到的脂质体裂解液滴加到步骤c得到的Au NCs@TiO2NTs电极上,然后再加入磷酸抗坏血酸酯AAP和HAuCl4溶液37℃孵育1h,使得ALP可以水解AAP产生具有还原性的抗坏血酸AA,以Au NCs作为晶种,AA将还原HAuCl4溶液中的Au3+在Au NCs上原位生长,使得Au NCs原位长大成金纳米颗粒Au NPs,改变了原先Au NCs的敏化效果,抑制电极的光电流信号;
5)孵育后的Au NCs@TiO2NTs电极用PBS(10mM,pH 7.4)缓冲液冲洗,然后在实验室自行搭建的光电化学测试系统上测量该电极的光电流强度,测试条件为:5W的LED灯作为激发光源;光电测试采用三电极体系:Au NCs@TiO2NTs电极为工作电极,Ag/AgCl为参比电极,Pt丝为对电极;光电流由上海辰华仪器公司的CHI660C工作站测定,设置偏压为0V;电解液为pH值为7.4的含20mM三乙醇胺的10mM的PBS缓冲液。
应用实例1:如图1是光电化学生物传感器稳定性测试的光电流图。每隔10s给一次光照并持续照射10s,Au NCs@TiO2NTs电极对光响应迅速,且光电流强度在400s内输出稳定,说明该电极性能稳定,适合于传感器的构建。
应用实例2:如图2是所述的光电化学生物传感器检测FABP的光电流图。用不同浓度的FABP参与免疫反应所对应得到的脂质体裂解液处理Au NCs@TiO2NTs电极,然后分别测得其光电流强度。如图2所示所测得的光电流信号与不同浓度的FABP存在良好的相关性,R2=0.992,检测限达0.1pg/mL。说明该传感器具有高灵敏度。图中△I代表Au NCs@TiO2NTs电极在响应前后光电流的变化差值。
应用实例3:如图3是所述的光电化学生物传感器的选择性实验光电流图。在相同的测试体系和测试条件下分别加入不同的干扰物如:癌胚抗原(CEA),肌钙蛋白(cTnT),免疫球蛋白G(IgG),免疫球蛋白A(IgA),P53蛋白和脂蛋白磷脂酶A2(Lp-PLA2)。实验结果证明,所述传感器对FABP具有极高选择性,表现为只有存在FABP时Au NCs@TiO2NTs电极的光电流才会发生明显的降低。I0代表Au NCs@TiO2NTs电极响应前的光电流,I代表Au NCs@TiO2NTs电极响应后的光电流。
实施例2:
(1)光电化学传感器的制备
a.金纳米簇Au NCs的制备:
将23mg的谷胱甘肽溶于23mL水中,然后滴加入质量分数为1%的2mL的HAuCl4溶液,然后加热至70℃并搅拌10h,待反应结束,将其冷却至室温并存储备用;
b.多孔二氧化钛纳米管TiO2NTs电极的制备:
将金属钛片连接于电源正极,石墨棒做负极,以质量分数为0.3%的NH4F乙二醇溶液为电解液,设定直流电压为50V并通电2h进行电氧化,之后用超声仪清洗钛片,洗净并吹干,然后设定直流电压为50V并通电30min,之后水洗并吹干得到TiO2NTs,将得到的TiO2NTs置于450℃的马弗炉中煅烧60min,得到TiO2NTs电极;
c.Au NCs@TiO2NTs电极的制备:
将步骤b中得到的TiO2NTs电极置于步骤a中得到的Au NCs溶液中浸泡48h,使得AuNCs吸附在TiO2NTs表面并对TiO2NTs进行敏化,得到Au NCs@TiO2NTs电极;
步骤(2)脂肪酸结合蛋白的检测方法同实施例1。
实施例3:
(1)光电化学传感器的制备
a.金纳米簇Au NCs的制备:
将23mg的谷胱甘肽溶于23mL水中,然后滴加入质量分数为1%的2mL的HAuCl4溶液,然后加热至70℃并搅拌48h,待反应结束,将其冷却至室温并存储备用;
b.多孔二氧化钛纳米管TiO2NTs电极的制备:
将金属钛片连接于电源正极,石墨棒做负极,以质量分数为0.3%的NH4F乙二醇溶液为电解液,设定直流电压为50V并通电2h进行电氧化,之后用超声仪清洗钛片,洗净并吹干,然后设定直流电压为50V并通电30min,之后水洗并吹干得到TiO2NTs,将得到的TiO2NTs置于450℃的马弗炉中煅烧60min,得到TiO2NTs电极;
c.Au NCs@TiO2NTs电极的制备:
将步骤b中得到的TiO2NTs电极置于步骤a中得到的Au NCs溶液中浸泡1h,使得AuNCs吸附在TiO2NTs表面并对TiO2NTs进行敏化,得到Au NCs@TiO2NTs电极;
步骤(2)脂肪酸结合蛋白的检测方法同实施例1。
以上所述的实施例只是本发明的较佳方案,并非对本发明作任何形式上的限制,在不超出权利要求所记载的技术方案的前提下还有其它的变体及改型。

Claims (10)

1.一种基于光电化学传感的脂肪酸结合蛋白检测方法,其特征在于,包括以下步骤:
(1)光电化学传感器的制备
a.金纳米簇Au NCs的制备:
将23mg谷胱甘肽溶于23mL水中,滴加2mL质量分数1%的HAuCl4溶液,然后加热至70℃并搅拌一定时间,待反应结束,冷却至室温得到金纳米簇Au NCs溶液;
b.多孔二氧化钛纳米管TiO2 NTs电极的制备:
将金属钛片连接于电源正极,石墨棒做负极,以质量分数0.3%的NH4F乙二醇溶液为电解液,设定直流电压为50V通电2h进行电氧化,用超声仪清洗钛片并吹干,设定直流电压为50V通电30min,水洗并吹干得到TiO2 NTs,将得到的TiO2 NTs置于450℃的马弗炉中煅烧60min,得到多孔二氧化钛纳米管TiO2 NTs电极;
c.Au NCs@TiO2 NTs电极的制备:
将步骤b得到的TiO2 NTs电极置于步骤a得到的Au NCs溶液中浸泡一段时间,得到AuNCs@TiO2 NTs电极;
(2)脂肪酸结合蛋白的检测方法
d.脂质体Ab2-ALPL的制备:
将二棕榈酰基磷脂酰乙醇胺DPPE、1,2-棕榈酰磷脂酰甘油DPPG、二棕榈酰磷脂酰胆碱DPPC和胆固醇CHOL溶于二氯甲烷/甲醇混合溶液中,减压脱溶得到脂质体膜后加入碱性磷酸酶ALP溶液水化过夜,超声并过滤得到装载有ALP的脂质体ALPL,将鼠抗人脂肪酸结合蛋白单克隆抗体2 Ab2通过戊二醛修饰到ALPL表面得到Ab2-ALPL;
e.光电化学脂肪酸结合蛋白检测体系的构建:
1)取60uL 0.2mg/mL的鼠抗人脂肪酸结合蛋白单克隆抗体1 Ab1加入96孔板中4℃下孵育12h,用PBS缓冲液洗去未结合在96孔板上的Ab1,然后加入牛血清蛋白BSA 4℃下孵育2h;
2)加入待检测的目标FABP抗原Ag在4℃下孵育1h形成Ab1-Ag复合物,PBS缓冲液洗去未结合的Ag;
3)加入40uL步骤d得到的Ab2-ALPL在37℃下孵育1h形成Ab1-Ag- Ab2-ALPL免疫复合物,PBS缓冲液洗去未结合的Ab2-ALPL,然后加入曲拉通Triton X-100进行脂质体裂解,得到含有ALP的脂质体裂解液;
4)将3)得到的脂质体裂解液滴加到步骤c得到的Au NCs@TiO2 NTs电极上,再加入磷酸抗坏血酸酯AAP和HAuCl4溶液37℃孵育1h,改变原先Au NCs的敏化效果,抑制电极的光电流信号;
5)孵育后的Au NCs@TiO2 NTs电极用PBS缓冲液冲洗,然后在光电化学测试系统上测量该电极的光电流强度。
2.根据权利要求1所述一种基于光电化学传感的脂肪酸结合蛋白检测方法,其特征在于,所述步骤a中搅拌时间为10-48h。
3.根据权利要求1所述一种基于光电化学传感的脂肪酸结合蛋白检测方法,其特征在于,所述步骤c中浸泡时间为1-48h。
4.根据权利要求1所述一种基于光电化学传感的脂肪酸结合蛋白检测方法,其特征在于,所述步骤d中二棕榈酰基磷脂酰乙醇胺DPPE、二棕榈酰磷脂酰胆碱DPPC、1,2-棕榈酰磷脂酰甘油DPPG、胆固醇CHOL的质量比为5:15:15:7。
5.根据权利要求1所述一种基于光电化学传感的脂肪酸结合蛋白检测方法,其特征在于,所述步骤d中二氯甲烷/甲醇混合溶液的体积比为4:1。
6.根据权利要求1所述一种基于光电化学传感的脂肪酸结合蛋白检测方法,其特征在于,所述步骤e3)中使用40uL 0.05%的曲拉通Triton X-100进行脂质体裂解。
7.根据权利要求1所述一种基于光电化学传感的脂肪酸结合蛋白检测方法,其特征在于,所述步骤e中PBS缓冲液为10mM pH 7.4的磷酸盐缓冲液。
8.根据权利要求1所述一种基于光电化学传感的脂肪酸结合蛋白检测方法,其特征在于,所述步骤e5)中测试条件为:5W的LED灯作为激发光源;光电测试采用三电极体系:AuNCs@TiO2 NTs电极为工作电极,Ag/AgCl为参比电极,Pt丝为对电极;光电流由上海辰华仪器公司的CHI660C工作站测定,设置偏压为0V;电解液为pH值7.4的含20mM三乙醇胺的10mM的PBS缓冲液。
9.根据权利要求1所述一种基于光电化学传感的脂肪酸结合蛋白检测方法,其特征在于,所述步骤e4)中Au NCs敏化效果的改变是通过ALP水解AAP产生具有还原性的抗坏血酸AA,进而使得在Au3+存在下Au NCs原位生长成金纳米颗粒Au NPs实现的。
10.根据权利要求1所述一种基于光电化学传感的脂肪酸结合蛋白检测方法,其特征在于,Au NCs@TiO2 NTs电极是通过其光电流的变化程度与脂肪酸结合蛋白浓度有关来实现对脂肪酸结合蛋白的检测。
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