CN106633167A - 一种用于特异性识别细胞的印迹材料及其制备和应用 - Google Patents
一种用于特异性识别细胞的印迹材料及其制备和应用 Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
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Abstract
本发明涉及一种用于特异性识别细胞的印迹材料及其制备和应用。所述的细胞印迹材料以细胞为模板，采用细胞培养与细胞固定技术将其固定于基质材料表面，通过在基质材料表面固化预聚合体系，然后分离除去基质材料，再去除固化后聚合物表面的模板细胞制备得到。该细胞印迹材料具有高选择性和高稳定性，并被进一步应用于细胞的识别、捕获与富集中。
Description
技术领域
本发明属于功能性生物材料，及其制备技术及其在细胞识别、捕获、释放和富集中的应用，具体的说是一种新型的可以特异性识别细胞的印迹材料，及其制备和应用。
背景技术
细胞的高通量和高灵敏度检测在肿瘤诊断与治疗、环境检测和生物传感器研究中具有十分重要的作用(Aherne A.et al.,J.Am.Chem.Soc.,1996,118,8771-8772)。目前，在细胞浓度较低的环境中，细胞的检测十分困难，其主要原因是细胞含量较少，无法得到。因此，实现低细胞浓度环境中细胞检测的前提是能够将目标从环境中识别和富集出来。
细胞识别与富集细胞识别与富集技术是一种可以特异性识别和捕获目标细胞的技术。目前，识别与富集目标细胞的技术主要有两种类型，一种是基于免疫原理的技术。该技术一方面利用与目标细胞结合的抗体识别和富集细胞，另一方面利用与干扰细胞结合的抗体去除非目标细胞，达到间接识别和富集目标细胞的目的。另一种技术是基于目标细胞物理性质的技术，包括细胞的大小、密度、可变形性和带电性等。该技术利用目标细胞与干扰细胞在上述物理性质方面的差异达到识别和富集目标细胞的目的。然而，由于细胞表面抗原表达的不确定性，无法获得针对重要细胞的抗体，从而导致基于免疫原理的技术无法有效发挥其作用。另外，即使同一种细胞在不同条件下其物理性质也不尽相同，故而基于物理性质技术获得的细胞往往纯度和灵敏度较差(Catherine Alix-Panabières et al,Nat.Rev.Cancer,2014,14,623-631).因此，开发一种既能够替代抗体，又能够特异性识别和富集细胞的材料就显得十分重要。
分子印迹技术是一项以分子识别理论为基础，模拟抗体-抗原相互作用的方式，通过人工设计和合成具有特定结构和功能的分子识别材料的新技术。利用该技术制备的分子印迹材料实现对复杂环境中的目标分子的特异性识别和富集。其中，细胞印迹技术是一种以细胞为模板的新兴分子印迹技术，由其制备的细胞印迹材料作为一种天然抗体的替代品，不仅可以精确模拟天然抗体的特异性识别能力，而且具有环境耐受性更强，重复利用率更高以及适合规模化合成等优势(Schirhagl,R.et al,Anal.Chem.,2014,86,250-261)。
因此，在细胞印迹技术的基础上，我们以细胞为模板，首先采用细胞培养与细胞固定技术将模板细胞固定于基质材料上，其次引发预聚合体系在基质材料表面的固化作用得到印迹层，然后分离基质材料与印迹层，最后去除印迹层表面的模板细胞。当模板细胞除去后，材料表面就会形成与模板细胞发生多重作用的印迹位点。从而获得了具有特异性识别和富集目标细胞能力的印迹材料，并实现了细胞的识别、捕获、释放和富集。
发明内容
以待特异性识别的细胞为模板，采用细胞培养与细胞固定技术将其固定于基质材料上，通过在基质材料表面固化聚合预聚合体系形成印迹层，分离印迹层与基质材料，然后去除印迹层表面的模板细胞，得到一种能够特异性识别模板细胞的印迹材料，并将该材料应用于细胞的识别、捕获、释放和富集过程中。为实现上述目的，本发明采用的技术方案为：
(1)采用物理处理或化学修饰方法改造基质材料表面，使其利于细胞在其表面固定。其中，基质材料包括细胞培养瓶、细胞培养板、细胞培养皿、载玻片、盖玻片中的一种或二种以上，基质材料的基质为硅氧烷类基质、聚苯乙烯类基质和金属基质。基质材料表面的改性方法包括物理涂层、表面热处理、机械打磨和等离子体表面处理的一种或二种以上；以及将能与模板细胞发生相互作用的抗体、核酸适配体、多肽和蛋白质中的一种或二种以上固载在基质材料表面。
(2)在不需处理或已处理的基质材料表面采用生长贴附、物理吸附、免疫作用等方式固定模板细胞。模板细胞是指动物细胞、植物细胞、真菌、细菌、和孢子。
(3)随后将预聚合体系加入到固定有细胞的基质材料表面，以光引发、热引发或自由基引发等方式引发预聚合体系固化。其中，预聚合体系中的单体是丙烯酰胺、N,N-二甲基丙烯酰胺、N,N-亚甲基双丙烯酰胺、N-异丙基丙烯酰胺、N-羟甲基丙烯酰胺、N-叔丁基丙烯酰胺、石墨烯掺杂丙烯酰胺、石墨烯掺杂N,N-二甲基丙烯酰胺、石墨烯掺杂N,N-亚甲基双丙烯酰胺、石墨烯掺杂N-异丙基丙烯酰胺、石墨烯掺杂N-羟甲基丙烯酰胺、石墨烯掺杂N-叔丁基丙烯酰胺、甲基氯硅烷、苯基氯硅烷、甲基乙烯基氯硅烷、乙基三氯硅烷、丙基三氯硅烷、乙烯基三氯硅烷、γ-氯丙基三氯硅烷和氟硅单体、聚二甲基硅氧烷，多巴胺、烯丙基胺、乙烯、丙烯、丁二烯、苯乙烯、4-乙烯基吡啶、衣康酸、丙烯酸、丙烯酸钠、甲基丙烯酸、甲基丙烯酸甲酯、对苯乙烯、乙二醇二甲基丙烯酸甲酯、氯苯乙烯、2-(三氟甲基)丙烯酸、丙烯腈、乙烯醇、乙酸乙烯、丙烯醛、聚氨基甲酸酯和2-羟基丙酸中的一种或二种以上；能通过自组装方式形成高分子材料的聚合物，包括聚砜、聚醚砜和聚芳砜中的一种或二种以上；用于溶解或分散单体或聚合物的溶剂包括水、磷酸盐缓冲液、氯化钠溶液、葡萄糖溶液、甲醇、乙醇、甲苯、乙醚、石油醚、氯仿、二氯甲烷、乙腈、乙酸乙酯、丙酮、正己烷、环己烷、苯、乙酸、苯甲醚、二甲基亚砜、溴苯、二氯化碳、四氯化碳、N,N-二甲基甲酰胺和三氟乙酸中的一种或二种以上；由功能单体或聚合物溶解或分散于溶剂中形成的溶液的质量浓度范围为5％-90％。
(4)预聚合体系固化后，将基质材料与印迹层分离。接着用胰酶消化法或溶剂萃取法等将印迹层表面的模板细胞彻底去除，直至检测不到模板细胞的存在，进而得到识别和富集细胞的印迹材料(MIP)。
(5)在基质材料上不固载模板细胞，按照上述从(1)到(4)的步骤制备得到非印迹材料(NIP)。
(6)将该细胞印迹材料用于生物分析、生物化工、生物医学和生物技术领域内细胞的选择性识别、捕获、释放和富集。
本发明是一种基于模板细胞与印迹位点之间的多重作用的细胞印迹材料，该材料能对目标细胞进行特异性识别、捕获、释放和富集。
当模板细胞与预聚合体系接触时会形成多重作用位置，通过在预聚合体系的固化过程中记忆二者之间的多重作用，当模板细胞除去后，材料表面就会形成与模板细胞发生多重作用的印迹位点。
模板细胞与印迹位点之间在空间结构、大小、形状和官能团上，通过空间构型和氢键作用、静电作用以及范德华作用表现出的互补亲和能力。本发明具有如下优点：
(1)本发明利用细胞印迹技术制备得到了能用于特异性识别细胞的印迹材料。利用印迹材料表面形成的与细胞发生多重作用的印迹位点，提高了细胞识别和富集的纯度与灵敏度，降低了富集细胞时非特异性作用的干扰。
(2)本发明中的印迹材料对目标细胞的识别是多重作用，包括空间构型和氢键作用、静电作用以及范德华作用，对目标细胞表现更强出的互补亲和能力。
(3)本发明中所用的基质材料和预聚合体系具有良好的生物兼容性和稳定性，利于保持细胞的活性与结构以及重复使用，进而有利于再识别目标细胞。
(4)本发明中细胞印迹材料的制备流程简单，易于操作。相较于其他细胞识别与富集方法，采用该材料能简化识别过程，提高识别效率，应用范围更广。
附图说明
图1：SMMC-7721细胞在细胞培养板表面的贴附效果图；
图2：印迹层表面的SMMC-7721细胞；
图3：以SMMC-7721细胞为模板制备得到的细胞印迹材料(MIP1)；
图4：MIP1和NIP识别与捕获SMMC-7721细胞的效果图；
图5：以MCF-7细胞为模板制备得到的细胞印迹材料(MIP2)。
具体实施方式
实施例1
基于SMMC-7721细胞模板的细胞印迹材料(MIP1)的制备
将SMMC-7721细胞培养于十二孔板基质材料表面中，当约80％的培养板底部被铺满时(图1)，用PH＝7.2的磷酸盐缓冲液(PBS)溶液清洗细胞，然后加入质量浓度为4％的多聚甲醛溶液固定细胞，5分钟后，用PH＝7的PBS溶液清洗去除多聚甲醛溶液。然后向十二孔板中的每孔加入1毫升由质量浓度为30％丙烯酰胺和质量浓度为1％的亚甲基双丙烯酰胺组成的预聚合水溶液，然后再加入1微升四甲基乙二胺和10微升质量浓度为10％的过硫酸铵水溶液引发预聚合溶液的固化反应。20分钟后，将固化形成的印迹层与基质材料分离并将印迹层翻转，在印迹层表面有SMMC-7721细胞存在(图2)。将印迹层反复浸泡于质量浓度0.25％的胰酶溶液中，直至采用DAPI染色法在印迹层表面检测不到细胞存在，用PBS溶液将胰酶溶液清洗后即得到用于特异性识别SMMC-7721细胞的细胞印迹材料(图3)。如图3所示，在印迹材料表面有大量印迹位点存在，这些印迹位点通过利用与目标细胞在空间结构、大小、形状和官能团上表现出的互补亲和作用，达到快速识别和捕获目标细胞的目的。
采用相同的方法，在不存在SMMC-7721细胞的十二孔板中，制备得到非印迹材料(NIP)。
实施例2
细胞印迹材料(MIP1)用于识别与捕获SMMC-7721细胞
将两份细胞浓度为1×106cells/ml的SMMC-7721细胞分别接种至MIP1印迹面和NIP表面，2小时后，细胞贴附于MIP1表面上的印迹位点处，而非印迹位点处和NIP表面无SMMC-7721细胞的贴附(图4)，说明MIP1的印迹位点通过与SMMC-7721细胞发生多重作用，能够快速、高校和高灵敏的识别与捕获目标细胞。进一步将SMMC-7721细胞加入到人体血液样本中，按照上述方法，MIP1也实现了从血液中特异性的识别和富集SMMC-7721细胞。
实施例3
基于MCF-7细胞模板的细胞印迹材料(MIP2)的制备
同实施例1，以MCF-7细胞为模板制备得到了可以特异性识别和捕获MCF-7细胞的印迹材料(MIP2)，如图5所示。
实施例4
基于光引发石墨烯掺杂预聚合体系的大肠杆菌细胞印迹材料(MIP3)的制备
将大肠杆菌培养在培养皿表面，当80％的培养皿底部被大肠杆菌铺满后，加入掺杂有石墨烯的丙烯酰胺和亚甲基双丙烯酰胺组成的预聚合水溶液，利用紫外光引发预聚合体系的固化反应，用含乙酸和十二烷基硫酸钠(SDS)的水溶液充分冲洗印迹层以去除表面的大肠杆菌，制备得到的印迹材料(MIP3)不仅可以特异性识别和捕获大肠杆菌，还可以在近红外光的照射下杀死大肠杆菌，起到灭菌的效果。
实施例5
基于氨酯单体的孢子细胞印迹材料(MIP4)的制备
将苏云金芽胞杆菌的孢子细胞固定于聚二甲基硅氧烷(PDMS)基质材料表面，同时将混有4,4’-亚甲基双(异氰酸苯酯)、双酚A和间苯三酚的四氢呋喃溶液在65℃温度下搅拌溶液至凝胶点，然后将上述固定有孢子的基质材料轻压于凝胶溶液表面，待其固化后，去除基质材料和模板孢子，制备得到对苏云金芽胞杆菌的孢子具有特异性识别和捕获能力的印迹材料(MIP4)。
实施例6
基于聚醚砜自组装的酵母细胞印迹材料(MIP5)的制备
将酿酒酵母细胞固定在载玻片上，加入浓度为15％的聚醚砜的二甲基亚砜溶液，然后迅速加入去离子水使聚醚砜自组装成印迹层，然后用SDS溶液冲洗去除酿酒酵母细胞得到细胞印迹材料(MIP5)。
实施例7
基于适配体修饰的有机硅基质材料的MCF-7细胞印迹材料(MIP6)的制备
将特异性识别MCF-7细胞的适配体固定在PDMS基质材料表面，然后在其表面加入MCF-7细胞的PBS溶液使细胞与适配体充分作用后，去除未被特异性结合的细胞。然后按照实施例1的方法制备能够特异性识别MCF-7细胞的印迹材料(MIP6)。
实施例8
基于等离子处理的金属基质材料的SMMC-7721细胞印迹材料(MIP7)的制备
将钛合金基质材料表面做等离子体处理，然后将SMMC-7721细胞固定于处理后的基质材料，按照实施例1的方法，制备能够特异性识别MCF-7细胞的印迹材料(MIP7)。
Claims (10)
1.一种用于特异性识别细胞的印迹材料，其特征在于：
其可按如下过程制备获得，以待特异性识别的细胞为模板细胞，将模板细胞附着或固定于基质材料表面，再将其置于预聚合体系中，通过在基质材料表面固化预聚合体系，然后分离除去基质材料，再去除固化后聚合物表面的模板细胞，得到可用于模板细胞特异性识别的印迹材料。
2.如权利要求1所述的模板细胞，其特征在于：
模板细胞为生物学中构成生物体的基本单位，包括动物细胞、植物细胞、真菌、细菌和孢子中的一种或二种以上。
3.如权利要求1所述的预聚合体系，其特征在于：
预聚合体系由能引发聚合或自组装形成高分子材料的单体或聚合物组成的溶液。
4.如权利要求3所述的由能引发聚合或自组装形成高分子材料的单体或聚合物组成的溶液，其特征在于：
能发生聚合反应形成高分子材料的单体，包括丙烯酰胺、N,N-二甲基丙烯酰胺、N,N-亚甲基双丙烯酰胺、N-异丙基丙烯酰胺、N-羟甲基丙烯酰胺、N-叔丁基丙烯酰胺、石墨烯掺杂丙烯酰胺、石墨烯掺杂N,N-二甲基丙烯酰胺、石墨烯掺杂N,N-亚甲基双丙烯酰胺、石墨烯掺杂N-异丙基丙烯酰胺、石墨烯掺杂N-羟甲基丙烯酰胺、石墨烯掺杂N-叔丁基丙烯酰胺、甲基氯硅烷、苯基氯硅烷、甲基乙烯基氯硅烷、乙基三氯硅烷、丙基三氯硅烷、乙烯基三氯硅烷、γ-氯丙基三氯硅烷和氟硅单体、聚二甲基硅氧烷，多巴胺、烯丙基胺、乙烯、丙烯、丁二烯、苯乙烯、4-乙烯基吡啶、衣康酸、丙烯酸、丙烯酸钠、甲基丙烯酸、甲基丙烯酸甲酯、对苯乙烯、乙二醇二甲基丙烯酸甲酯、氯苯乙烯、2-(三氟甲基)丙烯酸、丙烯腈、乙烯醇、乙酸乙烯、丙烯醛、聚氨基甲酸酯和2-羟基丙酸中的一种或二种以上；能通过自组装方式形成高分子材料的聚合物，包括聚砜、聚醚砜和聚芳砜中的一种或二种以上；用于溶解或分散单体或聚合物的溶剂包括水、磷酸盐缓冲液、氯化钠溶液、葡萄糖溶液、甲醇、乙醇、甲苯、乙醚、石油醚、氯仿、二氯甲烷、乙腈、乙酸乙酯、丙酮、正己烷、环己烷、苯、乙酸、苯甲醚、二甲基亚砜、溴苯、二氯化碳、四氯化碳、N,N-二甲基甲酰胺和三氟乙酸中的一种或二种以上；由功能单体或聚合物溶解或分散于溶剂中形成的溶液的质量浓度范围为5％-90％。
5.如权利要求1所述的固化预聚合体系的方法，其特征在于：能引发预聚合体系固化的方法，包括光引发、热引发、电场处理、自由基引发和溶剂交换中的一种或二种以上；将由能聚合的单体或能自组装的聚合物组成的预聚合体系加入到固定有模板细胞的基质材料表面，引发聚合。
6.如权利要求1所述的去除模板细胞的方法，其特征在于：将固化后的高分子材料表面的模板细胞去除的方法，包括胰酶消化法、紫外或红外光杀灭去除法和溶剂萃取法中的一种或二种以上。
7.如权利要求1所述的基质材料，其特征在于：
细胞在其表面正常贴附的无机或有机高分子材料，包括细胞培养瓶、细胞培养板、细胞培养皿、载玻片、盖玻片中的一种或二种以上；
在未处理或已处理的基质材料表面，细胞通过生长贴附、物理吸附、亲疏水作用、静电作用、氢键作用、免疫作用或化学处理方式固定中的一种或二种以上。
8.如权利要求7所述的无机或有机高分子材料，其特征在于：无机或有机高分子材料的材质为硅氧烷类基质、聚苯乙烯类基质和金属基质中的一种或二种以上。
9.如权利要求1或7所述的基质材料，其特征在于：基质材料应用前能进行表面改性处理，通过物理处理或化学修饰方法改造基质材料表面，改造方式为下述中的一种或二种以上，包括物理涂层、表面热处理、机械打磨和等离子体表面处理、以及将能与模板细胞发生相互作用的抗体、核酸适配体、多肽和蛋白质中的一种或二种以上固载在基质材料表面。
10.一种权利要求1-9任一所述特异性识别细胞的印迹材料用于细胞的选择性识别、捕获、释放和富集中的一种或二种以上。
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| CN111909407A (zh) * | 2020-07-31 | 2020-11-10 | 华中科技大学 | 一种选择性分离耐药杆菌的印迹薄膜材料及其制备与应用 |
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