CN104458710B - 肿瘤早期高通量电化学发光检测方法的构建 - Google Patents
肿瘤早期高通量电化学发光检测方法的构建 Download PDFInfo
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Abstract
本发明基于铱配合物分子高的电化学发光效率以及柔性阵列微芯片电极的开发，构建了高灵敏、高通量电化学发光肿瘤标志物检测新方法。将大量的具有高的电化学发光效率的铱配合物分子装入介孔硅作为电化学发光信号探针，而金纳米颗粒在这里被用来作为肿瘤标志物抗体的载体并且改善介质的电子传递从而提高电化学发光信号，通过研制高通量微芯片电极并在多阵列微芯片电极表面构建仿生传感界面，利用抗原与抗体之间的特异性识别，结合纳米粒子的信号放大技术以及优良的电子传递性质，通过表面修饰二抗的铱配合物填充的介孔硅/纳米金复合物信号探针优良的电化学发光性质，实现肿瘤标志物的高灵敏、高通量、准确性检测，并凭借其方法简便快速、价廉、无创伤等优势用于广泛人群的早期肿瘤疾病的全面筛查。
Description
技术领域
本发明涉及一种可对多种肿瘤标志物或多种样品同时检测的电化学发光纳米探针及柔性阵列微芯片电极的制备方法。
背景技术
肿瘤是目前世界上威胁人类健康及生命的重大疾病之一。我国对肿瘤疾病的防治方针是“以预防为主，防治结合，重在三早”，早期筛查及诊断是肿瘤治疗的关键。目前临床医学中对肿瘤疾病的筛查主要依靠临床表现，影像学检查、血清学检查、穿刺活检等多种手段。肿瘤起病隐匿，症状不明显，症状表现及影像学检查对早期肿瘤诊断不敏感，仍无法及时对早期肿瘤进行诊断。目前临床中检测人体血清中肿瘤标志物的方法主要有传统的酶联免疫检测法 (ELISA)、化学发光免疫分析法(CL IA )及电化学发光免疫分析法(ECLIA)，其中电化学发光检测方法灵敏度高，受环境效应影响较小，对抗原漏检率低，且自动化程度高，检测时间短，备受研究者的关注。在电化学发光技术中，发光材料的性能是影响漏检率及灵敏度的关键因素，故而开发新型无毒、稳定且发光强度高的电化学发光材料是提高电化学发光技术对血清中抗原检测能力的关键点，对实现肿瘤早期筛查和诊断具有十分重要的经济和社会意义。
为了满足临床中多种肿瘤标志物高灵敏检测需要以及大批待测样品的快速分析，该专利制备的纳米探针为铱配合物填充的介孔硅/纳米金复合物，铱配合物的电化学发光强度为传统电化学发光试剂钌配合物的24倍，极大地提高了检测的灵敏度，结合研制的高通量微芯片电极并在多阵列微芯片电极表面构建仿生传感界面，利用抗原与抗体之间的特异性识别，结合纳米探针的信号放大技术，可实现多种肿瘤标志物的高灵敏、高通量检测。
发明内容
本发明研究了以铱配合物填充的介孔硅/纳米金复合物为电化学发光信号探针的肿瘤标志物高通量、高灵敏检测方法，由于铱配合物具有很高的电化学发光效率，其电化学发光强度为传统电化学发光试剂钌配合物的24倍，将大量的具有高的电化学发光效率的铱配合物分子装入介孔硅（MSN）作为电化学发光信号探针，而介孔硅表面的金纳米颗粒在这里被用来作为肿瘤标志物抗体的载体并且改善介质的电子传递从而提高电化学发光信号。通过将聚二甲基硅氧烷与适量的固化剂混合，固化后形成透明的电极基底材料，我们可以在其表面镀上一层质地均匀的纳米金薄膜形成柔性微芯片表面，纳米金层具有优良的导电性，同时借助基底聚二甲基硅氧烷材料的柔韧性，得到的柔性微芯片电极在制备、储存、携带及使用等各方面都具有独特优势，最后利用界面修饰技术构建仿生传感界面。芯片表面形成的纳米金薄膜具有优良的导电性，在其表面可以修饰不同的肿瘤标志物第一抗体，与待测样本以及纳米信号探针反应，利用抗原与抗体的特异性结合，形成三明治的构型，然后通过探针上携带的大量铱配合物发光材料，实现肿瘤标志物高灵敏、高通量检测。本发明制备的64位点高通量柔性微芯片电极可对64个血液样本进行分析，促进了电化学发光技术在临床肿瘤疾病筛查、诊断中应用和发展。
本发明的主要创新性和优越性为：
1、开发新型离子型铱配合物（[(ppy)2Ir(dcbpy)]+PF6−）作为电化学发光材料，与传统联吡啶钌等发光材料相比，能耗低且发光效率高，是传统电化学发光材料钌配合物的24倍，极大的提高了检测的灵敏度；
2、 铱配合物填充的介孔硅/纳米金复合物信号探针制备方法简便，生物相容性好，毒性低，其表面修饰的金纳米粒子既可作为肿瘤标志物抗体的载体又可改善介质的电子传递从而提高电化学发光信号；
3、 首次设计可与电化学发光技术联用的高通量柔性微阵列芯片电极，电极表面均匀修饰纳米金膜，导电性好且具有柔韧性，微芯片电极可设计性强、制备方法简便、表面易于修饰且价格低廉适宜批量生产，弥补了目前现有传统电极及微芯片电极的不足，可实现早期疾病筛查中快速、简便、高通量等需求。
附图说明
图1为铱配合物（[(ppy)2Ir(dcbpy)]+ PF6 -）的结构；
图2为纳米信号探针的合成示意图；
图3 铱配合物与传统联吡啶钌的电化学发光强度；
图4为柔性阵列微芯片电极的设计示意图；
图5基于信号探针和微芯片电极的高灵敏、高通量电化学发光传感方法检测
的原理图。
具体实施方式
以下为实施本发明的具体示例，其作用在于进一步阐明本发明的内容，使阅读者更容易理解，但不构成对本发明要求的保护范围的限定或限制。
本方法所用仪器装置为： MPI-E型电化学发光分析系统(西安瑞迈分析仪器有限公司)；CHI 660电化学分析仪（上海辰华仪器公司）；三电极系统：柔性微芯片电极为工作电极，Ag/AgCl为参比电极，铂丝为对电极；FA2004N电子天平（上海恒平科学仪器有限公司）；TJL-16G高速冷冻离心机（上海安亭科学仪器厂）；DF-101S集热式恒温加热磁力搅拌器(江苏省金坛市医疗仪器厂)；DZF-6051真空干燥箱（上海一恒科技有限公司）； SZ-93自动双重纯水蒸馏器(上海亚荣生化仪器厂)。
所用试剂为：IrCl3.3H2O（陕西开达化工有限公司）；苯基吡啶、2-乙氧基乙醇、2，2-联吡啶-4，4，-二甲酸(Alfa Aesar，A Johnson Mathcy Company)；NH4PF6（上海睿腾化工有限公司）；柠檬酸三钠、氯金酸（国药集团化学试剂有限公司）；正硅酸乙酯、聚二甲基硅氧烷、3-巯基丙基三甲氧基硅烷（Sigma公司）。实验所用试剂均为分析纯，水均为二次蒸馏水。
实施例一 结合图1，图2和图3，制备铱配合物填充的介孔硅/纳米金复合物信号探针
1.0mL 浓度为2.5 ×10−4 M [(ppy)2Ir(dcbpy)]+PF6 −（图1）的乙腈溶液加入到同体积的1.0 mg/mL 的介孔硅溶液中37℃恒温搅拌反应过夜，然后加入1.5 mL的3-巯基丙基三甲氧基硅烷搅拌反应2h后，加入纳米金溶液，通过金-巯基共价键的结合将纳米金组装到介孔硅的表面形成稳定的铱配合物填充的介孔硅/纳米金复合物信号探针，取1mL上述纳米复合物，加入1mL 0.02mg/mL抗体（二抗，0. 01M PBS，pH=7.4）室温孵育2 h。离心，用pH=7.4的0.0 1M PBS洗涤，然后将所得物溶于1 mL 1%BSA，孵育1 h，离心洗涤。将最终得到的二抗修饰的铱配合物填充的介孔硅/纳米金复合物信号探针分散于pH=7.4 的0.0 1M PBS中于4°C保存（总体积2 mL），并且所得铱配合物的电化学发光强度为传统电化学发光试剂钌配合物的24倍（图3）。
实施例二 结合图4，柔性阵列微芯片电极的开发
将聚二甲基硅氧烷（PDMS）与适量的固化剂按质量比9：1比例混合，固化后形成透明的电极基底材料。制作镂空有机玻璃板（镂空部分为电极形状），将其紧密覆盖于PDMS薄片之上。将含有1% HAuCl4, 200 g L-1 KHCO3及2% 葡萄糖（体积比为2：1：1）的化学镀金液注入上述镂空部分，排空气泡后于避光条件下静置3 h，然后去除镀金液，在PDMS表面可形成一层质地均匀的纳米金薄膜。纳米金层具有优良的导电性，同时借助基底PDMS材料的柔韧性，得到的柔性微芯片电极在制备、储存、携带及使用等各方面都具有独特优势。同时设计灵活的阵列电极形貌，制作满足实际检测需要的多种阵列分布的微芯片金膜电极。发展高通量、多通道的分析新方法，满足同一微芯片上多种肿瘤标志物或多种样品的同时检测，为癌症等重大疾病的快速分析和诊断提供检测平台。
实施例三 结合图5，基于新型信号探针的高灵敏、高通量电化学发光肿瘤标志物检测
在设计的柔性微芯片表面，利用界面修饰技术构建仿生传感界面。将10 μL 的一抗(100 μg L-1, 0.01 M pH 7.4 PBS) 滴到电极表面4 ℃孵育12 h，没有结合的抗体用缓冲液清洗去除。然后将所制得的电极浸于1 %（w/w）BSA 溶液中，室温条件下浸泡1 h，封闭电极表面可能存在的活性位点和非特异性吸附，最后用缓冲溶液多次清洗电极。修饰好的电极置于4 ℃冰箱中保存待用。将制备好的免疫电极浸泡在100 μL 不同浓度的肿瘤标志物溶液中，37 ℃下孵育40 min，取出清洗数次，除去非特异性吸附的抗原。然后滴加10 μL二抗修饰的信号探针室温下孵育40 min，然后PBS洗涤。然后通过探针上携带的大量铱配合物发光材料，实现肿瘤标志物高灵敏、高通量的电化学发光检测。如图4所示，制备的64位点高通量微芯片电极可对64个血液样本进行分析，促进了电化学发光技术在临床肿瘤疾病筛查、诊断中应用和发展。
本实验是在MPI-E型电化学发光分析系统上进行的。三电极系统：以制备的64位点高通量微芯片电极为工作电极，以Ag/AgCl（饱和KCl）)为参比电极，以铂丝电极为对电极。缓冲溶液为含有5 mM TPA的0.1 mol/L PBS缓冲溶液(pH7.4, 0.1 mol/L NaCl)，采用循环伏安扫描(0.2-1.25 V)，扫速：100 mV s−1，光电倍増管高压为800 V。
Claims (3)
1.一种肿瘤标志物电化学发光检测方法，其特征在于：大量的具有高的电化学发光效率的铱配合物分子被装入介孔硅作为电化学发光信号探针，而介孔硅表面的金纳米颗粒在这里被用来作为肿瘤标志物抗体的载体并且改善介质的电子传递从而提高电化学发光信号；将制备的导电性能极佳且表面易于修饰的金膜微芯片电极设计成灵活的阵列电极形貌，在其表面修饰不同的肿瘤标志物第一抗体，然后与待测样本以及电化学发光信号探针反应，利用抗原与抗体的特异性结合，形成三明治的构型，通过探针上携带的大量铱配合物发光材料，实现肿瘤标志物的检测；金膜微芯片电极的研制：以聚二甲基硅氧烷(PDMS)为基底材料，采用电沉积或者化学沉积技术在基底材料上修饰上纳米金导电材料，制备导电性能极佳且表面易于修饰的金膜微芯片电极，同时设计灵活的阵列电极形貌。
2.根据权利要求1所述的一种肿瘤标志物电化学发光检测方法，其特征在于检测方法为电致化学发光信号检测，电化学发光物质为铱的配合物，电化学发光强度是传统的钌配合物的24倍。
3.根据权利要求1所述的一种肿瘤标志物电化学发光检测方法，其特征在于所用电极为柔性阵列微芯片电极。
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