CN111624338A - 一种检测前列腺特异性抗原的光电化学免疫传感器的制备方法 - Google Patents
一种检测前列腺特异性抗原的光电化学免疫传感器的制备方法 Download PDFInfo
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Abstract
本发明公开了一种用于检测前列腺特异性抗原的环金属铱配合物敏化NiO的阴极光电免疫传感器的制备方法。首先采用水热合成法在ITO电极上制备纳米多孔NiO薄膜，然后将光敏剂[Ir(C6)2(dcbpy)]+PF6 ‑(其中C6为香豆素6，dcbpy为2,2'‑联吡啶‑4,4'‑二羧酸)通过缩合作用固定在纳米NiO膜表面，进而借助壳聚糖和戊二醛将前列腺特异性抗原捕获抗体通过共价修饰在电极上制得传感工作电极。通过前列腺特异性抗原和其抗体之间的特异性免疫反应，实现对前列腺特异性抗原浓度的检测，具有高灵敏度和高特异性。
Description
技术领域
本发明属于光电功能材料及生物分析领域，具体涉及一种基于环金属铱配合物敏化NiO的光电化学免疫传感器用于检测前列腺特异性抗原的制备方法。
背景技术
前列腺特异性抗原(PSA)是由前列腺上皮细胞分泌的一种丝氨酸蛋白酶，是目前应用最为广泛的前列腺癌生物标志物。前列腺癌是严重危害男性健康的恶性肿瘤之一，近年来我国发病率呈现明显的上升趋势。其早期检测、诊断及治疗成为提高患者生存率及预后效果的关键。前列腺特异性抗原越来越广泛地应用于前列腺癌的早期筛查，以便可以发现早期、无症状的前列腺癌。因此开发高灵敏、高特异性检测PSA含量的检测方法具有重要意义。目前用于PSA检测的方法主要包括酶联免疫测定、放射免疫测定、荧光法、表面增强拉曼、化学发光等，但这些方法均需要昂贵的仪器，操作复杂、耗时长。
近年来光电化学免疫传感因具有灵敏度高、仪器简单等特点而备受关注，被广泛应用于肿瘤标志物的检测。光电材料根据电荷载体形式的不同，可以分为n型和p型。n型材料，如TiO2，CdS，Bi2S3等，基于其导带上电子的注入，而p型材料则通过在其价带中注入空穴来实现。目前，基于n型材料的光阳极分析系统由于具有较高的检测灵敏度，在光电生物分析领域占有主导地位。而基于p型材料的光电阴极体系研究较少，与光阳极分析体系相比，光阴极体系因能避免光阳极界面固有氧化反应干扰而具有高的特异性。目前光阴极体系主要集中于染料敏化的NiO半导体，而NiO中的空穴与光敏剂之间快速的电荷复合降低了光电转换效率从而使得体系灵敏度低。解决这一问题的关键在于设计高效的敏化剂减少电子-空穴之间的复合，使其产生长寿命的分离状态。本发明采用具有长寿命三线态的环金属铱配合物敏化NiO作为光阴极，组建光电化学免疫传感器用于前列腺特异性抗原的检测。
发明内容：
鉴于现有技术的不足，本发明旨在提供一种用于检测前列腺特异性抗原的环金属铱配合物敏化NiO的阴极光电免疫传感器的制备方法，具有高灵敏度、高特异性的特点。
基于上述目的，本发明所涉及的技术方案如下：
采用水热合成法在ITO电极上制备纳米多孔NiO薄膜，然后将光敏剂[Ir(C6)2(dcbpy)]+PF6 -(其中C6为香豆素6，dcbpy为2,2'-联吡啶-4,4'-二羧酸)通过化学作用固定在纳米NiO膜表面(ITO/NiO/[Ir(C6)2(dcbpy)]+PF6 -)。然后借助壳聚糖和戊二醛将PSA捕获抗体(Ab)共价键合在ITO/NiO/[Ir(C6)2(dcbpy)]+PF6 -上。通过PSA和其Ab之间的特异性免疫反应，实现对前列腺特异性抗原的检测。包括以下步骤：
(1)NiO修饰ITO电极的制备：将面积固定的ITO电极(0.5cm×4.0cm)依次放入盛有10mL超纯水、10mL无水乙醇、10mL丙酮、10mL无水乙醇、10mL超纯水中，超声清洗10分钟，以除去ITO电极表面的杂质，自然干燥。采用水热法在ITO电极上修饰NiO纳米膜，将清洗干净的ITO玻璃浸入400μL含0.1～0.5M Ni(NO3)2·6H2O和0.1～0.5M六亚甲基四胺的水溶液的离心管中，80～100℃加热150～300分钟，冷却至室温后，用超纯水洗涤冲洗三次，在氮气氛围下干燥。然后将ITO电极放置到马弗炉中300～400℃并保持30～50分钟，升温速率为5℃/分钟，自然降至室温，制得NiO修饰ITO电极(ITO/NiO).
(2)将步骤(1)制得的ITO/NiO电极浸入浓度为1×10-5～10-4M的[Ir(C6)2(dcbpy)]+PF6 -DMF溶液中反应12～18小时，分别用DMF和CH3CN洗涤，以降低非特异性吸附。然后放入恒温干燥箱37℃下干燥，除去溶剂，制得ITO/NiO/[Ir(C6)2(dcbpy)]+PF6 -电极。
(3)将步骤(2)制得的ITO/NiO/[Ir(C6)2(dcbpy)]+PF6 -电极固定有效的传感面积为0.5cm×0.5cm，滴涂0.1％的壳聚糖乙酸溶液，干燥后均匀滴涂20μL 5～10％(体积分数)的戊二醛(GLD)溶液，通过戊二醛的醛基与前列腺特异性抗原抗体中胺基之间的反应将其固定在电极表面，用PBS缓冲溶液进行清洗。滴加牛血清蛋白溶液进行封板，制得用于检测前列腺特异性抗原的传感工作电极。
(4)将步骤(3)制得的工作电极浸入不同浓度的前列腺特异性抗原溶液中，在37℃下静置孵育1小时，冲洗后进行光电流检测。光电流检测采用三电极系统：传感工作电极，铂丝电极作为辅助电极，Ag/AgCl电极作为参比电极。在激发光475nm下，0.1M的PBS缓冲溶液(pH＝7.4)作为检测液，偏置电压为-0.3～0V，光源开启和关闭时间均为20s。
本发明的有益效果：
(1)本发明公开了一种环金属铱配合物敏化纳米NiO产生阴极光电流用于光电化学免疫传感器的制备，避免了阳极型传感器造成的干扰，具有选择性高的特点。
(2)采用三线态寿命长的环金属铱配合物作为光敏剂，降低了NiO中的空穴与光敏剂之间的电荷复合，增加了光电流强度，提高了检测灵敏度。
附图说明：
图1光电化学生物传感器制备示意图；
图2传感器对前列腺特异性抗原浓度的光电流响应示意图；
图3传感器对前列腺特异性抗原浓度的线性关系图。
具体实施方式：
结合实施例对本发明作进一步的说明，应该说明的是，下述说明仅仅是为了解释本发明，并不对其内容进行限定。
实施例1
光敏剂[Ir(C6)2(dcbpy)]+PF6 -的合成与表征：
称取IrCl3·3H2O固体(0.34g,1mmol)溶于水(5mL)，搅拌使其充分溶解，加入香豆素-6(0.77g,2.2mmol)和2-乙氧基乙醇(15mL)到50mL的圆底烧瓶中，在氮气保护下120℃下回流24小时，将反应产物自然冷至室温，过滤收集滤饼固体，然后依次用水、乙醇洗涤，得到铱二氯桥联二聚体产物。将2,2'-联吡啶-4,4'-二羧酸(0.047g,0.25mmol)和2-乙氧基乙醇(25mL)以及得到的二聚体(0.175g,0.1mmol)加入到50mL圆底烧瓶中。搅拌溶解后，将过量的Na2CO3(5mmol)加入到上述溶液中，搅拌加热，氮气保护下回流20小时。反应结束，利用减压蒸馏除去溶剂，加入1M盐酸(10mL)，中和过量的Na2CO3，搅拌20分钟。反应产物经过过滤，得到固体，然后用超纯水(2×15mL)洗涤，得到的固体干燥后，用二氯甲烷和甲醇混合溶液溶解。加入含饱和六氟磷酸铵的甲醇(5mL)溶液，搅拌20分钟。通过减压蒸馏除去甲醇和二氯甲烷，通过柱层析色谱对粗产物分离纯化，洗脱剂选择二氯甲烷：甲醇＝5：1，得到橘红色粉末(0.087g,39％)。1H NMR(500MHz,DMSO)δ:8.71(d,J＝10.0,2H)；8.59(s,2H)；8.08(d,J＝5.0,2H)；7.95(d,J＝10.0,2H)；7.24(t,J＝7.5,2H)；7.00(t,J＝7.5,2H)；6.46(s,2H)；6.03(s,4H)；5.88(d,J＝10.0,2H)；3.40(q,J＝6.5,8H)；0.96(t,J＝6.5,12H)。
实施例2
传感工作电极的制备：
(1)NiO修饰ITO电极的制备：将面积固定的ITO电极(0.5cm×4.0cm)依次放入盛有10mL超纯水、10mL无水乙醇、10mL丙酮、10mL无水乙醇、10mL超纯水中，超声清洗10分钟，以除去ITO电极表面的杂质，自然干燥。采用水热法在ITO电极上修饰NiO纳米膜。将清洗干净的ITO玻璃浸入配置含400μL含0.25M Ni(NO3)2·6H2O和0.25M六亚甲基四胺的水溶液的离心管中，90℃加热200分钟，冷却至室温后，用超纯水洗涤冲洗三次，在氮气氛围下干燥。然后将ITO电极放置到马弗炉中350℃并保持30分钟，升温速率为5℃/分钟，自然降温，制得NiO修饰ITO电极(ITO/NiO).然后将其浸入浓度为5×10-5M的[Ir(C6)2(dcbpy)]+PF6 -DMF溶液中反应12小时，分别用DMF和CH3CN洗涤，以降低非特异性吸附。然后放入恒温干燥箱37℃下干燥，除去溶剂，制得ITO/NiO/[Ir(C6)2(dcbpy)]+PF6 -电极。将制得的ITO/NiO/[Ir(C6)2(dcbpy)]+PF6 -电极固定有效的传感面积为0.5cm×0.5cm，滴涂0.1％的壳聚糖乙酸溶液，干燥后均匀滴涂20μL 5％(体积分数)的戊二醛(GLD)溶液，静置反应30分钟后用去离子水冲洗电极。取20μL浓度为100μg/mL的前列腺特异性抗原抗体滴涂在修饰电极表面，并在4℃下孵育12小时后，用pH＝7.4、10nM的PBS缓冲溶液清洗电极。然后滴涂20μL牛血清蛋白封闭液(1％(w/v))，在37℃下静置孵育30分钟，随后用pH＝7.4、10mM的PBS缓冲溶液洗涤修饰电极三次，制得用于检测前列腺特异性抗原的传感工作电极。
实施例3
样品的测定：
将制备好的传感工作电极浸入不同浓度的前列腺特异性抗原溶液中，在37℃下静置孵育1小时，冲洗后进行光电流检测，由光电化学分析仪记录光电流强度。光电流检测采用三电极系统：制备的传感工作电极，铂丝电极作为辅助电极，Ag/AgCl电极作为参比电极在激发光475nm下，0.1M的PBS缓冲溶液(pH＝7.4)作为检测液，偏置电压为0V，光源开启和关闭时间均为20s。当前列腺特异性抗原浓度在1pg/mL到10ng/mL范围内，光电流信号和其浓度的对数具有很好的线性关系，线性回归方程为ΔI＝-720.151+53.800lg CPSA(pg/mL)，线性相关系数R2为0.9966，检测限为240fg/mL(S/N＝3)。
Claims (2)
1.一种基于环金属铱配合物敏化NiO的光电化学免疫传感器用于检测前列腺特异性抗原的制备方法；采用水热合成法在ITO电极上制备纳米NiO薄膜，然后将光敏剂环金属铱配合物[Ir(C6)2(dcbpy)]+PF6 -(其中C6为香豆素6，dcbpy为2,2'-联吡啶-4,4'-二羧酸)通过缩合作用固定在纳米NiO膜表面；进而借助壳聚糖和戊二醛将前列腺特异性抗原捕获抗体共价键合在修饰电极上制得传感工作电极。通过前列腺特异性抗原和抗体之间的特异性免疫反应，实现对前列腺特异性抗原的检测，包括以下步骤：
(1)纳米NiO修饰ITO电极的制备：将面积固定的ITO电极(0.5cm×4.0cm)依次放入盛有10mL无水乙醇、丙酮及超纯水中，超声清洗10分钟，自然干燥。将清洗干净的ITO玻璃浸入400μL含0.1～0.5M Ni(NO3)2·6H2O和0.1～0.5M六亚甲基四胺的水溶液的离心管中，80～100℃加热150～300分钟，冷却至室温后，用超纯水洗涤冲洗，在氮气氛围下干燥。然后将ITO电极放置到马弗炉中300～400℃并保持30～50分钟，升温速率为5℃/分钟，自然降温，制得NiO修饰ITO电极(ITO/NiO)。
(2)传感工作电极的制备：将制备的ITO/NiO电极浸入浓度为1×10-5～10-4M的[Ir(C6)2(dcbpy)]+PF6 -DMF溶液中反应12～18小时，分别用DMF和CH3CN洗涤，放入恒温干燥箱37℃下干燥。干燥的电极固定有效的传感面积为0.5cm×0.5cm后，滴涂0.1％壳聚糖的乙酸溶液，干燥后滴涂20μL 5～10％(体积分数)的戊二醛(GLD)溶液，用PBS缓冲溶液进行清洗。将20μL浓度为100μg/mL的前列腺特异性抗原抗体滴涂在电极表面，并在4℃下孵育12小时，进而滴加20μL牛血清蛋白溶液(1％(w/v))进行封板，制得用于检测前列腺特异性抗原的传感工作电极。
(3)光电信号检测：制得的传感工作电极浸入不同浓度的前列腺特异性抗原溶液中，37℃孵育1小时，冲洗后进行光电流检测。光电流检测采用三电极系统：传感工作电极，铂丝电极作为辅助电极，Ag/AgCl电极作为参比电极。在激发光475nm下，0.1M的PBS缓冲溶液(pH＝7.4)作为检测液，偏置电压为-0.3～0V，光源开启和关闭时间均为20s。
2.根据权利要求1所述方法制备得到的光电化学传感器在检测前列腺特异性抗原中的应用。
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