CN114524622A - 一种环氧基修饰基片的制备方法、微阵列芯片及其应用 - Google Patents

一种环氧基修饰基片的制备方法、微阵列芯片及其应用 Download PDF

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CN114524622A
CN114524622A CN202210216772.3A CN202210216772A CN114524622A CN 114524622 A CN114524622 A CN 114524622A CN 202210216772 A CN202210216772 A CN 202210216772A CN 114524622 A CN114524622 A CN 114524622A
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张大霄
陈玉珠
刘亚飞
刘扬
徐红星
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Beijing Kangmin Biotechnology Co ltd
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Abstract

本发明公开了一种环氧基修饰基片的制备方法、微阵列芯片及其应用，将含有氧化硅以及环氧活性基团的溶胶‑凝胶通过浸渍提拉法涂覆在基底上，再经干燥成膜后得到。所述环氧基片制备成本低，简单稳定，适用于大规模生产的环氧微阵列生物芯片，且性能高于浸泡修饰的环氧基片，能够提供优异的检测灵敏度。

Description

一种环氧基修饰基片的制备方法、微阵列芯片及其应用
技术领域
本发明涉及微阵列芯片用基片制备技术领域，尤其涉及一种环氧基修饰基片的制备方法、微阵列芯片及其应用。
背景技术
微阵列技术作为一种多指标、高通量、低消耗的检测手段，在基因测序，蛋白组学等研究领域有着重要的应用。然而在临床领域该技术的应用则十分稀少，这主要是因为微阵列基底制备成本高以及良率问题制约了微阵列技术在临床领域的发展。所以低成本，高可靠性的微阵列基底制备方法是微阵列技术应用发展的关键。
目前，常用的微阵列基底制备手段主要包括以下两种：
1.硅烷修饰。该方法使用面最广，也是最为简单的方法。通常是利用强酸性洗液，例如食人鱼溶液或者铬酸洗液清洗玻片基底，然后将洗净的基底浸泡特定的硅烷修饰液进行硅烷修饰，从而得到表面带有功能化学基团的硅烷修饰层。这种方法虽然简单高效，但是由于玻片本身的差异，导致重现性并不好，而如果使用熔融石英或者硅片等基底，虽然能一定程度上提高重现性，但是基片材料成本也成倍提升。
2.高分子修饰。该方法通常也是需要在表面通过硅烷修饰一层功能化学基团，然后通过该化学基团去偶联高分化合物或者原位的进行高分子聚合反应。相较于直接的硅烷修饰方法，高分子能够均匀的覆盖玻片表面，提高重现性。但是高分子修饰的步骤繁琐，有的环境要求十分苛刻，例如需要无氧环境等，缺少成熟的产业化解决方案。
因此，为了改善传统操作复杂、成本高且重现性差的化学修饰方法，本发明提出通过物理方法获得环氧基修饰基片，以应用于微阵列芯片的制备，以进一步推广微阵列技术在临床检测中的应用。
发明内容
本发明的目的是为了提出一种环氧基修饰基片的制备方法、微阵列芯片及其应用，以至少解决现有技术中存在的诸多缺陷之一。
鉴于此，本发明的方案为：
一种环氧基修饰基片的制备方法，将含有氧化硅以及环氧活性基团的溶胶-凝胶通过浸渍提拉法涂覆在基底上，再经干燥成膜后得到。
本发明中，所述溶胶-凝胶将γ-缩水甘油醚氧丙基三甲氧基硅烷、正硅酸四乙酯置于酸性体系中水解、缩聚获得。
优选地，按体积比计，所述正硅酸四乙酯的用量范围为总反应体系的5-15％，所述γ-缩水甘油醚氧丙基三甲氧基硅烷的用量范围为总反应体系的1-10％。
优选地，所述溶胶-凝胶的制备方法为：将正硅酸四乙酯、γ-缩水甘油醚氧丙基三甲氧基硅烷溶解后，滴加盐酸和水，搅拌获得溶胶-凝胶溶液；盐酸浓度为1mol/L，按体积比计，盐酸为总反应体系的0.05-5％；水为总反应体系的2-20％。
本发明中，所述浸渍提拉法中提拉速度为200μm/s-3000μm/s。
本发明中，所述基底包括玻璃或高分子材料。
本发明还提出一种微阵列芯片，包括以上所述制备方法获得的环氧基片，及吸附在环氧基片上呈阵列分布的生物分子。
本发明中，所述环氧基片成膜表面的接触角为55°～65°。
进一步地，所述生物分子包括蛋白质、寡核苷酸；生物分子阵列点之间用硅胶围栏进行阻隔，形成独立的检测孔。
进一步地，所述生物分子通过点样吸附在环氧基片上，点样液中SDS浓度在0.1-10％，海藻糖浓度在0.5-5％，甘露醇浓度在0.2-2％。
相比现有技术，本发明的有益效果在于：
1.本发明所述环氧基片制备成本低，工艺简单，稳定可靠，适用于大规模生产的环氧微阵列生物芯片，进一步推广了微阵列技术在临床检测中的应用。
2.实验结果表面，该芯片的性能高于浸泡修饰的环氧基片，能够提供优异的检测灵敏度，细胞因子VEGF的检测限可达2pg/mL。
附图说明
图1为本发明所述环氧基修饰基片的制备流程示意图。
图2为本发明不同接触角的环氧芯片免疫结果荧光扫描图片。
图3为本发明不同的成膜方法获得环氧芯片免疫结果荧光扫描图片。
图4为本发明环氧芯片免疫VEGF浓度标准曲线。
图5为本发明不同接触角基片应用于抗体检测时的荧光扫描对比结果。
图6为本发明与商用基片应用于抗体检测时的荧光扫描对比结果。
图7为本发明与商用基片应用于抗体检测时的荧光强度对比结果。
具体实施方式
为了使本发明的目的、技术方案和有益技术效果更加清晰明白，以下结合具体实施方式，对本发明进行进一步详细说明。应当理解的是，本说明书中描述的具体实施方式仅仅是为了解释本发明，并不是为了限定本发明。
本发明为克服现有技术采用化学腐蚀以及表面改性处理交底表面的缺陷，提出一种环氧基修饰基片，将含有氧化硅以及环氧活性基团的溶胶-凝胶通过浸渍提拉法涂覆在基底上，再经干燥成膜后得到。通过在玻璃基片上沉积一层溶胶凝胶薄膜，然后在薄膜上点阵形式修饰的生物分子形成微阵列芯片。
制备上述溶胶-凝胶的方法为，将正硅酸四乙酯(TEOS)、γ-缩水甘油醚氧丙基三甲氧基硅烷(GPTS)溶解后，滴加酸性催化剂和水，在此过程中经搅拌完成TEOS和GPTS的水解及缩聚反应，获得溶胶-凝胶溶液，此过程GPTS可以与TEOS分开成两步加入，也可以同时在一步加入；按体积比计，正硅酸四乙酯的用量范围为总反应体系的5-15％，γ-缩水甘油醚氧丙基三甲氧基硅烷的用量范围为总反应体系的1-10％；酸为总反应体系的0.05-5％(1M HCl)；水为总反应体系的2-20％。
所述的，环氧基修饰基片浸渍在上述制备方法得到的溶胶-凝胶中(溶胶-凝胶经微孔滤膜过滤)，按照200μm/s-3000μm/s的速度进行提拉后静置，然后在静置状态下烘干得到。通过该方法得到的环氧基修饰基片成膜后的表面接触角为55°～65°，所述环氧基修饰基片的制备流程如图1所示。
本发明应用于微阵列芯片的基底材料可以选用玻璃，也可以选用高分子片材，如常用的PMMA、PS等。
本发明应用于微阵列芯片采用环氧基修饰基片并点阵有生物分子，且分子阵列点之间用硅胶围栏进行阻隔，形成独立的检测孔，所述生物分子包括寡核酸、抗体、蛋白等等；所述微阵列芯片适用于大规模生产，能够提高优异的检测灵敏度。
以下是本发明作为优选的实验实施例，用于说明并验证本发明上述方案及其技术效果，所选案例仅为较佳的实验实施例，并非对本发明的限定。特别地，本发明制备及实验实施例采用的原料均为市售。
以下是溶胶-凝胶溶液的制备例：
制备例1
量取102mL异丙醇(IPA)于250mL锥形瓶中，室温下，电磁搅拌，加入5％即6mL正硅酸四乙酯(TEOS)，室温搅拌10分钟后，然后注射泵1mL/min加入盐酸(HCl)和水的混合溶液，其具体成分为0.05％即0.075mL 1M的盐酸(HCl)水溶液和2％即2.4mL的水，室温搅拌12h。随后加入1％即1.2mLγ-缩水甘油醚氧丙基三甲氧基硅烷(GPTS)，然后注射泵1mL/min的速度加入盐酸和水的混合物，其具体成分为0.15mL 1M的盐酸(HCl)水溶液和5.1mL的水，室温搅拌12h，即得到溶胶-凝胶溶液1。
制备例2
量取102mL异丙醇(IPA)于250mL锥形瓶中，室温下，电磁搅拌，加入10％即12mL正硅酸四乙酯(TEOS)，室温搅拌10分钟后，然后注射泵1mL/min加入盐酸(HCl)和水的混合溶液，其具体成分为1.25％即1.5mL 1M的盐酸(HCl)水溶液和12.75％即15.3mL的水，室温搅拌12h。随后加入1％即1.2mLγ-缩水甘油醚氧丙基三甲氧基硅烷(GPTS)，然后注射泵1mL/min的速度加入盐酸和水的混合物，其具体成分为0.15mL 1M的盐酸(HCl)水溶液和5.1mL的水，室温搅拌12h，即得到溶胶-凝胶溶液2。
制备例3
量取102mL异丙醇(IPA)于250mL锥形瓶中，室温下，电磁搅拌，加入15％即18mL正硅酸四乙酯(TEOS)，室温搅拌10分钟后，然后注射泵1mL/min加入盐酸(HCl)和水的混合溶液，其具体成分为5％即6mL 1M的盐酸(HCl)水溶液和20％即24mL的水，室温搅拌12h。随后加入10％即12mLγ-缩水甘油醚氧丙基三甲氧基硅烷(GPTS)，然后注射泵1mL/min的速度加入盐酸和水的混合物，其具体成分为0.15mL 1M的盐酸(HCl)水溶液和5.1mL的水，室温搅拌12h，即得到溶胶-凝胶溶液3。
制备例4
量取82.5mL异丙醇(IPA)于250mL锥形瓶中，室温下，电磁搅拌，加入5％即5mL正硅酸四乙酯(TEOS)，加入5％即5mLγ-缩水甘油醚氧丙基三甲氧基硅烷(GPTS)，室温搅拌10分钟后，然后注射泵1mL/min加入盐酸(HCl)和水的混合溶液，其具体成分为2.5％即2.5mL 1M的盐酸(HCl)水溶液和4.25％即4.25mL的水，室温搅拌12h，即得到溶胶-凝胶溶液4。
将上述制备例1-4所得溶胶凝胶溶液对基底进行浸渍提拉获得环氧基化玻片，如实施例1-4。
实施例1
取50mL制备例1得到的溶胶凝胶溶液1，使用0.22μm微孔滤膜过滤加入大小为7cm×5cm×2cm的石英方缸，利用浸渍提拉仪在200μm/s的速度对玻片进行浸渍提拉。待过程完毕后静置1min，取下后常温下静置4min，在100摄氏度烘箱中烘烤5min，冷却至室温后即得到环氧基修饰玻片1。
实施例2
取50mL制备例2得到的溶胶凝胶溶液2，使用0.22μm微孔滤膜过滤加入大小为7cm×5cm×2cm的石英方缸，利用浸渍提拉仪在2000μm/s的速度对透明亚克力板进行浸渍提拉。待过程完毕后静置1min，取下后常温下静置4min，在100摄氏度烘箱中烘烤5min，冷却至室温后即得到环氧基修饰亚克力板2。
实施例3
取50mL制备例3得到的溶胶凝胶溶液3，使用0.22μm微孔滤膜过滤加入大小为7cm×5cm×2cm的石英方缸，利用浸渍提拉仪在3000μm/s的速度对透明聚苯乙烯板进行浸渍提拉。待过程完毕后静置1min，取下后常温下静置4min，在100摄氏度烘箱中烘烤5min，冷却至室温后即得到环氧基修饰聚苯乙烯板3。
实施例4
取50mL制备例4得到的溶胶凝胶溶液，使用0.22μm微孔滤膜过滤加入大小为7cm×5cm×2cm的石英方缸，利用浸渍提拉仪在2500μm/s的速度对玻片进行浸渍提拉。待过程完毕后静置1min，取下后常温下静置4min，在100摄氏度烘箱中烘烤5min，冷却至室温后即得到环氧基修饰玻片4。
实验例1环氧基修饰玻片的检测
使用东莞市晟鼎精密仪器有限公司静态水接触角测量仪，在已制成的基片上滴15μL纯水，使用配套软件进行拍照，测出对应接触角，检测结果如表1所示。
表1：
实施例 实施例1 实施例2 实施例3 实施例4
接触角 63°-65° 58°-60° 55°-57° 58°-62°
实验例2微阵列芯片的制备及抗体检测
1)取10μL 2mg/mL IgG抗体小鼠于200μL离心管内，加入5μL 0.5M(NH4)2SO4，加入5μL甘油，加入10μL PBS，充分混匀后即得点样液。
2)使用北京博奥晶典生物技术有限公司生产的点样仪点样于实施例1-4中制备所得的环氧基修饰，用注射泵20mL/min加入PBS+1％BSA进行清洗，吹干后，贴上自制硅胶围栏，即得环氧基片。
3)使用40μL 20ng/mL山羊抗小鼠IgG偶联CY3孵育2h，纯水清洗后，使用北京博奥晶典生物技术有限公司生产的扫描仪，扫描得荧光扫描图片用
Figure BDA0003535210320000071
Figure BDA0003535210320000072
LuxScanTM10K系列微阵列软件得到强度值如图2所示，不难发现上述实施例1-4在应用于抗体检测时强度值较高且比较接近。
实验例3微阵列芯片的制备及细胞因子VEGF的检测
1)取10μL 2mg/mL VEGF抗体于200μL离心管内，加入5μL 0.5M(NH4)2SO4，加入5μL甘油，加入10μL PBS+0.1％SDS，充分混匀后即得点样液。
2)使用北京博奥晶典生物技术有限公司生产的点样仪点样于实施例1中制备所得的环氧基修饰的玻片表面，用注射泵20mL/min加入PBS+1％BSA进行清洗，吹干后，贴上自制硅胶围栏，即得环氧基片。
3)向载玻片里独立的检测孔分别加入40μL 2ng/mL、500pg/mL、125pg/mL、31.25pg/mL、7.8125pg/mL VEGF抗原，孵育2h，后清洗，加入40μL VEGF检测抗体孵育2h，纯水清洗后，使用北京博奥晶典生物技术有限公司生产的扫描仪进行扫描，即得荧光扫描图片如图3所示，图3中a-e对应样本浓度由高到底的5个扫描结果，采用
Figure BDA0003535210320000081
LuxScanTM10K系列微阵列软件得到强度值，并拟合获取标准曲线如图4所示，从图中读取最小检测极限为2pg/mL。
对比例1
调整制备例1的配比和实施例1的提拉速度取不同接触角的环氧基修饰玻片，分别获得接触角75°-80°、50°-52°两个对比例，采用实验例2相同的手段，进行点样制备环氧基片、然后使用山羊抗小鼠IgG偶联CY3孵育并扫描仪进行扫描。获得荧光扫描图片如图5所示，图5a、5b分别代表接触角较大，以及较小的两个对比例，很明显看出，两者扫描清晰度整体均受较大影响，不是理想的扫描结果。
对比例2
1)取10μL 2mg/mL IgG抗体小鼠于200μL离心管内，加入5μL 0.5M(NH4)2SO4，加入5μL甘油，加入10μL PBS，充分混匀后即得点样液。
2)使用北京博奥晶典生物技术有限公司生产的点样仪点样于商业化环氧修饰玻片(使用CN1588006A文献中采用化学浸泡法制备获得)，用注射泵20mL/min加入PBS+1％BSA进行清洗，吹干后，贴上自制硅胶围栏，即得环氧基片。
3)使用40μL 20ng/mL山羊抗小鼠IgG偶联CY3孵育2h，纯水清洗后，使用北京博奥晶典生物技术有限公司生产的扫描仪，扫描，即得荧光扫描图片，如图6所示，采用
Figure BDA0003535210320000091
LuxScan TM10K系列微阵列软件得到强度值结果如图7所示。在其他条件相同的情况下，图6a代表本发明实验例2应用于抗体检测扫描结果(同图2)，6b代表商用基片应用于抗体检测时的扫描结果，图6a清晰度明显更好；对应的，图7显示在其他条件相同的情况下，本发明提供的基片在应用于检测时强度信号明显大于商用，由此可判断本发明提供的环氧基修饰基片相比市售产品在应用于检测时在信号强度的优越性和灵敏度，因而可获取更低的检测极限值。
本发明并不仅仅限于说明书和实施方式中所描述，因此对于熟悉领域的人员而言可容易地实现另外的优点和修改，故在不背离权利要求及等同范围所限定的一般概念的精神和范围的情况下，本发明并不限于特定的细节、代表性的方案和这里描述的示例。

Claims (10)

1.一种环氧基修饰基片的制备方法，其特征在于，将含有氧化硅以及环氧活性基团的溶胶-凝胶通过浸渍提拉法涂覆在基底上，再经干燥成膜后得到。
2.根据权利要求1所述的制备方法，其特征在于，所述溶胶-凝胶将γ-缩水甘油醚氧丙基三甲氧基硅烷、正硅酸四乙酯置于酸性体系中水解、缩聚获得。
3.根据权利要求2所述的制备方法，其特征在于，按体积比计，所述正硅酸四乙酯的用量范围为总反应体系的5-15％，所述γ-缩水甘油醚氧丙基三甲氧基硅烷的用量范围为总反应体系的1-10％。
4.根据权利要求2所述的制备方法，其特征在于，所述溶胶-凝胶的制备方法为：将正硅酸四乙酯、γ-缩水甘油醚氧丙基三甲氧基硅烷溶解后，滴加盐酸和水，搅拌获得溶胶-凝胶溶液；盐酸浓度为1mol/L，按体积比计，盐酸为总反应体系的0.05-5％；水为总反应体系的2-20％。
5.根据权利要求1所述的制备方法，其特征在于，所述浸渍提拉法中提拉速度为200μm/s-3000μm/s。
6.根据权利要求1所述的制备方法，其特征在于，所述基底包括玻璃或高分子材料。
7.一种微阵列芯片，包括权利要求1-6任意一项所述制备方法获得的环氧基片，及吸附在环氧基片上呈阵列分布的生物分子。
8.根据权利要求7所述的微阵列芯片，其特征在于，所述环氧基片成膜表面的接触角为55°～65°。
9.根据权利要求7所述的微阵列芯片，其特征在于，所述生物分子包括蛋白质、寡核苷酸；生物分子阵列点之间用硅胶围栏进行阻隔，形成独立的检测孔。
10.根据权利要求7所述的微阵列芯片，其特征在于，所述生物分子通过点样吸附在环氧基片上，点样液中SDS浓度在0.1-10％，海藻糖浓度在0.5-5％，甘露醇浓度在0.2-2％。
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