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CN109781705B - 一种高通量、超灵敏检测的点阵阵列增强芯片 - Google Patents

一种高通量、超灵敏检测的点阵阵列增强芯片 Download PDF

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CN109781705B
CN109781705B CN201910098748.2A CN201910098748A CN109781705B CN 109781705 B CN109781705 B CN 109781705B CN 201910098748 A CN201910098748 A CN 201910098748A CN 109781705 B CN109781705 B CN 109781705B
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皮付伟
张圆媛
刘玲
尹子叶
成玉梁
林琳
李文飞
孙秀兰
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Jiangnan University
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Abstract

本发明公开了一种高通量、超灵敏检测的点阵阵列增强芯片，属于食品安全检测领域。本发明通过在亲水基底上化学键合单层纳米金颗粒，将纳米金材料通过静电吸附作用自然沉降至芯片孔洞中，并形成规则排列的点阵。孔洞排布的金颗粒在颗粒表面活性剂(CTAB)的间隔下形成等离子体区域增强热点所排布的纳米金颗粒相互之间聚集形成长程效应，从而提高了芯片的检测效率和灵敏度。

Description

一种高通量、超灵敏检测的点阵阵列增强芯片
技术领域
本发明涉及一种高通量、超灵敏检测的点阵阵列增强芯片，属于食品安全检测领域。
背景技术
近年来，生物毒素、重金属等物质的污染、检测及控制问题是长期困扰我国食品安全的重要问题，也是国家层面人民健康长期关注的热点问题。传统毒素检测方法如高效液相色谱法、薄层色谱等在检测时间、检测精度、设备成本、可操作性等方面存在一定的不足，而且针对食品加工过程中生物毒素降解产物的毒力有效识别和高通量快速检测涉及较少，尤其缺乏与生物毒素消减、降解等潜在危害物毒力高通量快速检测相匹配的技术和检测器件。现有的检测技术虽有优点，但多存在检测灵敏度较低、检测限较高以及检测目标单一等问题。
表面等离子体增强是指吸附在特定纳米级粗糙界面的分析物结构信号被极大增强的一种现象，其增强机理已被证实为相邻纳米颗粒之间区域电磁场耦合形成表面电子云叠加“热点”，即“热点”效应。(“热点”：相邻的金属纳米颗粒由于局域电磁场耦合而形成的分析物信号极大增强的区域)。等离子体增强“热点”效应的发现和“热点”基底制备方法的迅速发展，使得区域等离子体技术在化学、分析、生物等研究领域已得到了广泛应用。近年来，研究表明，相对于由二聚体及三聚体纳米颗粒形成的一维(1D)“热点”，二维(2D)和三维(3D)结构的区域等离子体“热点”增强阵列因其长程效应和数量的增多而使得总体等离子体信号大大增强，并且信号增强效应更加稳定、持久。虽然三维“热点”阵列具有独立的光散射和众多空间增强局域场，但由颗粒自组装形成的区域有序二维“热点”阵列因具有偏振光效应和界面有序性而更易于实际操控和应用。
发明内容
本发明提供了一种高通量、多靶向信号增强检测阵列芯片的制备技术，通过在亲水基底上化学键合单层纳米金颗粒(金球、金棒、金十字等不同形态的金基纳米材料)，所用的纳米金材料通过静电吸附作用自然沉降至芯片孔洞中，并形成规则排列的点阵。孔洞排布的金颗粒在颗粒表面活性剂(CTAB)的间隔下形成等离子体区域增强热点所排布的纳米金颗粒相互之间聚集形成长程效应，从而提高检测效率和灵敏度。
本发明的第一个目的是提供一种高通量、超灵敏、多靶向检测的区域等离子体信号增强点阵阵列芯片的制备方法；所述方法包括如下步骤：
(1)将微球溶液分散在芯片基底上，形成单层微球点阵，自然风干；
(2)将固定有微球点阵的亲水基底蒸镀铝膜；
(3)超声清洗，除去微球颗粒，制得表面具有微纳孔洞铝膜的芯片基底；
(4)对步骤(3)处理后的芯片基底表面硅烷化处理，使处理后的界面带正电荷或负电荷；
(5)将金纳米材料滴在步骤(4)处理后的基底表面，获得点阵阵列芯片。
在本发明的一种实施方式中，所述芯片基底为亲水基底。
在本发明的一种实施方式中，所述芯片基底的材料包括但不限于玻璃片、硅片、半导体。
在本发明的一种实施方式中，所述纳米金颗粒的形态包括但不限于：金球、金棒、金十字。
在本发明的一种实施方式中，所述纳米金颗粒表面具有颗粒表面活性剂(CTAB)。
在本发明的一种实施方式中，所述步骤(1)是将微球溶液平铺在玻璃片上，自然风干，形成单层微球点阵。为保证所排布的纳米金颗粒之间具有稳定的连续信号增强的效应和进行进一步功能化，芯片基底的制备中微球排布于玻片并尽可能形成单层的步骤至为重要。
在本发明的一种实施方式中，所述步骤(2)在磁控溅射仪中蒸镀铝膜，铝膜的厚度为50nm～100nm。
在本发明的一种实施方式中，所述步骤(4)是将超声清洗后的玻璃片于0.08～0.12MPa真空干燥，再于70℃的条件下蒸镀APTES或APTMS 0.8～1.2小时。
在本发明的一种实施方式中，所述微球为SiO2微球。
在本发明的一种实施方式中，所述芯片基底经过清洗，所述清洗具体是：
(1)分别用无水乙醇和超纯水超声清洗20～30分钟；
(2)将步骤(1)清洗后的芯片基底烘干后，在等离子体清洗机中进行清洗10～15分钟；
在本发明的一种实施方式中，所述微球清洗步骤：用移液枪吸取150μl的SiO2微球，采用降速梯度离心法用超纯水清洗5次，将清洗后的微球稀释至浓度为0.5～1.0mol/L。
本发明的第二个目的是提供应用所述方法制备的芯片。
本发明还要求保护所述芯片在化学、分析、生物领域检测方面的应用。
有益效果：本发明提供了一种高通量、超灵敏、多靶向检测的点阵阵列增强芯片，该芯片能够将检测信号的灵敏度提高102倍，并将检测限降低至aM级。
附图说明
图1(a)为基于微球刻蚀点阵技术的“热点”增强阵列芯片基底制备流程图；(b)为除去微球后芯片孔洞扫描电镜(SEM)图；
图2左图为玻璃基质等离子体“热点”增强阵列芯片示意图；右图为纳米颗粒区域等离子体增强模拟图；
图3FITC-BSA标记的芯片孔洞倒置荧光显微镜图(10x100)。
图4纳米金“热点”点阵阵列扫描电镜图。
图5金棒和金球“热点”点阵阵列芯片拉曼信号扫描图。
图6用本芯片来检测添加各种浓度的miRNA-21的荧光反应，图中由下至上的曲线分别代表添加的miRNA-21浓度0,0.00001,0.00008,0.0002,0.0003,0.0006,0.0008,0.0013,0.001,0.0016，0.005,0.1,1,10,50,100,150,200,400fM。
图7荧光强度对miRNA-21浓度的对数的依赖性，线性范围为0.0006至0.0016和0.1至100fM。每个数据是三次重复的平均值(N＝3)，误差条表示测量的标准偏差。
图8金棒和金球溶液的拉曼信号扫描图。
具体实施方式
实施例1芯片的制备
如图1和图2所示，本发明的芯片制备流程如下：
(1)玻片和微球的清洗：分别用无水乙醇和超纯水超声清洗玻片，清洗时间为30分钟；将清洗后的玻片烘干后在等离子体清洗机中进行清洗，时间为15分钟；将清洗后的玻片保存于超纯水中。
(2)微球在玻璃界面上定点排布,即用移液枪吸取50微升浓度为1mol/L微球溶液滴在玻片上，当微球溶液扩散到玻片底部时，将玻片底部旋转180°至顶部，使部分微球溶液回流，以形成面积更大的单层微球点阵；
(3)将玻片在磁控溅射仪中镀铝以固定微球的位置,通过调节蒸镀时间和蒸镀速率调控铝膜的厚度为50nm；
(4)将蒸镀铝膜的玻片在超纯水中进行超声清洗，除去界面上的微球得到芯片孔洞，将此玻片拍摄扫面电镜(SEM)。
实施例2芯片上金棒(AuNR)和金球(AuNP)的拉曼信号检测
将合成的金棒(AuNR)和金球(AuNP)沉降至纳米孔洞中，金棒即在孔洞中自聚形成信号增强“热点”点阵阵列，再加入200微升拉曼探针分子血红蛋白超声摇匀并检测拉曼信号，计算点阵增强效果。检测结果如图5所示，结果显示，相比于纯的血红蛋白分子，AuNR和AuNP点阵在波数600cm-1和1121cm-1均有增强的信号，且AuNP在波数1600cm-1处也有增强的信号。经过计算，本芯片的增强效果可达102倍。由此可见，本芯片具有良好的信号增强性能。
实施例3病原微生物microRNA检测
金棒或金十字偶联DNA步骤：
1.在PCR管中加入等体积数的100uM ssDNA和1mM TCEP，反应条件：静置避光3h
2.吸取0.1M的金纳米棒或金十字1mL；
3.吸取与步骤1体积和等体积数的SDS-HCL(pH＝3)加入步骤2中；
4.室温震荡12h,转速为1000rpm；
5.1000rpm离心10min，吸取上清液，向沉淀中加入0.005M CTAB备用。
将连接DNA2的金纳米十字沉降至孔洞中，此DNA链末端修饰有cy5荧光染料。另有金棒上连接DNA1，此DNA链与DNA2部分碱基互补配对，此时cy5荧光染料不发光，处于猝灭状态；当加入病原微生物miRNA-21时，由于此RNA与DNA1部分碱基互补配对，此时金棒上偶联的DNA1与RNA杂交，释放出金十字上偶联的DNA2的cy5荧光，根据cy5的荧光强度可对病原微生物的miRNA-21进行检测。检测结果如图6，图7所示，由荧光强度对miRNA-21浓度的对数的曲线给出了由等式表示的两个线性响应(F＝1830.32log C+6349.27，R2＝0.9901；以及F＝244.41log C+1916.10，R2＝0.9984)；检测限可低至0.5aM和0.03fM，而其他荧光方法的检测限为nM，表明该芯片具有高通量、超灵敏特性。
所用的DNA1、DNA2和microRNA序列均购自上海生物工程有限公司。
DNA1:5’-HS-C6-AAAAAATCAACATCAGTCTGATAAGCTA-3’
DNA2:5’-HS-C6-AAAAAAAAAAAAAAAATAGCTTATCAGACT-cy5-3’
miRNA-21:5’-UAGCUUAUCAGACUGAUGUUGA-3’
对比例1：
具体实施方式同实施例2，区别在于，纳米金材料没有沉降至纳米孔洞中，直接将200微升拉曼探针分子血红蛋白加入等量金棒(AuNR)和金球(AuNP)溶液中，超声摇匀并检测拉曼信号，结果如图8所示，与点阵芯片(图5)相比，溶液中的拉曼信号减弱了数10倍。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (10)

1.一种高通量、超灵敏、多靶向检测的区域等离子体信号增强点阵阵列芯片的制备方法，其特征在于包括如下步骤：
(1)将微球溶液分散在芯片基底玻片上，当微球溶液扩散到玻片底部时，将玻片底部旋转180°至顶部，使微球溶液回流，进行微球二次界面重排，自然风干，形成单层微球点阵；
(2)将排布有微球点阵的亲水基底蒸镀铝膜，印记微球点阵排布形貌；
(3)超声清洗，除去微球颗粒，制得表面具有微-纳孔洞铝膜覆盖的芯片基底；
(4)对步骤(3)处理后的芯片基底表面硅烷化处理，使处理后的微-纳孔洞内的基底界面带正电荷或负电荷；
(5)将纳米金材料滴在步骤(4)处理后的基底表面，使纳米金材料沉降自聚于微-纳孔洞内的基底上，除去铝膜，获得具有微米级等离子体增强效应的金纳米颗粒集聚的点阵阵列芯片。
2.根据权利要求1所述的方法，其特征在于，所述步骤(1)是将微球溶液平铺在玻璃片上，自然干燥，形成单层微球点阵。
3.根据权利要求1所述的方法，其特征在于，所述步骤(2)在磁控溅射仪中蒸镀铝膜，铝膜的厚度为50nm～100nm。
4.根据权利要求1所述的方法，其特征在于，所述芯片基底为亲水基底。
5.根据权利要求1或4所述的方法，其特征在于，所述芯片基底的材料包括但不限于玻璃片、硅片、半导体。
6.根据权利要求1所述的方法，其特征在于，所述纳米金材料的形态包括：金球、金棒或金十字。
7.根据权利要求1或6所述的方法，其特征在于，所述纳米金材料表面具有颗粒表面活性剂。
8.根据权利要求1或2所述的方法，其特征在于，所述微球的浓度为0.5～1.0mol/L。
9.应用权利要求1～8任一所述方法制备获得的高通量、超灵敏、多靶向检测的区域等离子体信号增强点阵阵列芯片。
10.权利要求9所述的高通量、超灵敏、多靶向检测的区域等离子体信号增强点阵阵列芯片在化学、分析、生物领域检测方面的应用。
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