CN112979530B - 核酸检测探针及其制备方法 - Google Patents

核酸检测探针及其制备方法 Download PDF

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CN112979530B
CN112979530B CN202110532356.XA CN202110532356A CN112979530B CN 112979530 B CN112979530 B CN 112979530B CN 202110532356 A CN202110532356 A CN 202110532356A CN 112979530 B CN112979530 B CN 112979530B
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孟英才
周文虎
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Shanghai Giza Gene Technology Co ltd
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Abstract

本发明公开了一种核酸检测探针及其制备方法，所述核酸检测探针由聚五羟色胺纳米粒吸附荧光标记的DNA而成，所述聚五羟色胺纳米粒由5‑羟色胺在在氢氧化钠溶液中氧化自聚而成，可吸附单链脱氧核糖核酸分子，且吸附的单链DNA分子可被互补DNA特异性解吸附；其制备方法为：向一定浓度的5‑羟色胺水溶液中加入一定量的氢氧化钠溶液，室温搅拌一定时间后，形成聚五羟色胺纳米粒；将该纳米粒吸附荧光标记的DNA可制成核酸检测探针。本发明纳米粒制备方法简单，且具有独特的DNA吸附与解吸附特性，可用于核酸分子的检测，细胞内分子成像，疾病诊断等，具有广阔的应用前景。

Description

核酸检测探针及其制备方法
技术领域
本发明涉及纳米材料和纳米生物技术领域，具体涉及一种核酸检测探针及其制备方法与应用。
背景技术
肿瘤是严重威胁人类健康的重大疾病之一，若肿瘤发展到中、晚期，会出现病灶的扩大和肿瘤转移，即使采用多种治疗方式的联合也难达到较好的疗效。如果能早期诊断出肿瘤并进行合理的治疗，即可有效控制其发展，甚至实现肿瘤的根治。因此，肿瘤的早期诊断对其有效防治非常重要。目前，临床上主要通过对体内肿瘤相关核酸或蛋白进行检测、分析以获取生命相关信息来实现肿瘤的诊断，多数时候需要快速且灵敏的对样本进行检测。此时，操作简单、成本低、灵敏快速且高特异性的生物检测器则显示出较大的临床应用价值。然而现有的生物检测器仍存在一定的局限，如较难实现快速检测，易受复杂生物环境的干扰，较难实时、灵敏的获取有关信息。
近年来，纳米材料吸附DNA在核酸检测或生物分子成像等方面展现出极大的临床转化潜力。其用于核酸检测的主要原理是，纳米材料吸附荧光标记的DNA使荧光发生淬灭，当其与互补DNA（cDNA）杂化后，发生解吸附并恢复荧光，通过检测荧光信号实现cDNA的检测。该方法具有操作简单、条件温和、成本低且特异性高等优点。实现这类应用的纳米材料需同时具有DNA吸附能力和荧光淬灭能力，且吸附的DNA可被cDNA特异性解吸附。目前，具有这些特点的典型纳米材料有碳纳米管、氧化石墨烯和一些金属氧化物纳米材料。然而，这些纳米材料因自身性质的限制，其吸附的DNA易被非特异性解吸附。如氧化石墨烯与DNA发生π-π堆叠和氢键相互作用而吸附，但其吸附的DNA易被蛋白解吸附，导致构建的检测器易受生物基质成分的干扰，产生假阳性信号。
因此，选择合适的纳米材料能稳定吸附DNA，同时可抵抗生物基质成分的非特异性解吸附，在临床上具有重要的应用价值。CN202010675017.2《一种酸性环境中可降解的表面涂层及其制备方法与应用 》提供了一种在氨水环境中制备聚五羟色胺涂层的方法，因为5-羟色胺在聚合过程中会产生质子，该方法通过氨水缓冲质子并提供碱性环境促进其氧化聚合，此外，氨也能以掺杂的形式修饰在聚五羟色胺结构中。该方法得到的聚五羟色胺其结构中的氨基以非质子化的形式存在且可包裹于各种材料的表面，该聚五羟色胺以涂层的形式包裹于纳米材料，其表面带负电，并与DNA存在静电排斥，不能高效吸附DNA，不适用于核酸检测等应用，且该涂层在酸性环境中可发生降解，可作为酸敏材料进行应用。
发明内容
为了解决现有技术中存在的问题，本发明提供一种可吸附DNA的纳米粒、核酸检测探针及其制备方法与应用。所述纳米粒可吸附各种碱基类型的单链DNA，并具有超强的荧光淬灭能力；该纳米粒吸附的单链DNA可抵抗高至5 mM的磷酸盐或5%的胎牛血清（FBS）所引起的解吸附；该纳米粒吸附的单链DNA可被cDNA解吸附；吸附荧光标记的探针DNA可实现核酸检测或细胞内基因成像。所述纳米粒制备方法简单，且具有独特的DNA吸附与解吸附特性，应用前景广泛。
为了实现上述目的，本发明首先提供一种可吸附DNA的纳米粒，所述纳米粒为聚五羟色胺纳米粒（PHT NPs），所述聚五羟色胺纳米粒由5-羟色胺（5-HT）在在氢氧化钠溶液中氧化自聚而成。
作为优选，反应液中所述5-羟色胺（5-HT）的质量浓度为0.5 ~ 5 mg/mL，所述氢氧化钠的摩尔浓度为1 mM~5 mM。
作为优选，所述氧化自聚反应时间为1~5天。
基于一个总的技术构思，本发明还提供了一种所述纳米粒的制备方法，包括以下步骤：
S1、分别配置5-羟色胺盐酸盐溶液、氢氧化钠溶液；
S2、将氢氧化钠溶液加入5-羟色胺盐酸盐溶液中，室温避光搅拌一定时间，离心，洗涤，即得聚五羟色胺纳米粒;所述5-羟色胺盐酸盐溶液的质量浓度为0.5 ~ 5 mg/mL，所述氢氧化钠的摩尔浓度为1 mM~5 mM。
作为优选，所述反应时间为1~5天。
基于一个总的技术构思，本发明还提供了一种核酸检测探针，所述核酸检测探针由上述纳米粒吸附荧光标记的DNA而成。
基于一个总的技术构思，本发明还提供了一种所述核酸检测探针的制备方法，包括以下步骤：
S1、将荧光标记的DNA分散于缓冲液中得到分散液；
S2、将所述分散液与所述纳米粒混合后进行吸附，室温孵育过夜、离心，收集沉淀即得。
作为优选，所述缓冲液为HEPES缓冲液，所述纳米粒质量浓度为200 μg/mL。
基于一个总的技术构思，本发明还提供了上述纳米粒在核酸检测中的应用。所述纳米粒吸附荧光标记的探针DNA，然后与cDNA孵育30 min，体系的荧光恢复值与cDNA浓度呈线性关系，可用于cDNA的定量检测。
基于一个总的技术构思，本发明还提供了上述纳米粒在细胞内分子成像中的应用。所述纳米粒吸附荧光标记的探针DNA后，进入细胞内，被互补核酸解吸附，发生荧光点亮，可实现胞内基因成像。
本发明发现5-羟色胺在低浓度的氢氧化钠溶液中能发生氧化聚合并形成纳米粒，而不能形成涂层包裹于基底材料表面。该反应中，5-羟色胺聚合产生的质子不能完全被氢氧化钠中和，多余的会被自身结构中的氨基中和掉，且反应中没有其他物质掺杂聚五羟色胺，可得到一种带正电的纳米粒（pH 5-8环境中均带正电），与氨水中制备的聚五羟色胺涂层具有完全不同的性质。该纳米粒能够高效吸附DNA，并抵抗生物干扰成分（磷酸盐和血清）的解吸附，且能特异性被互补DNA解吸附，实现核酸的特异性检测，具有重要的应用价值。
本发明上述方案有如下有益效果：
1、本发明提供了一种可吸附DNA的纳米粒，本发明创新地发现了5-羟色胺（5-HT）在氢氧化钠溶液中可以氧化自聚形成聚五羟色胺纳米粒（PHT NPs），该纳米粒可吸附各种碱基类型的单链DNA，并具有超强的荧光淬灭能力，并且该纳米粒吸附的单链DNA可抵抗高至5 mM的磷酸盐或5%的FBS所引起的解吸附。
2、本发明提供了一种可吸附DNA的纳米粒，该纳米粒吸附的单链DNA可被cDNA解吸附，且cDNA浓度越高，引起单链DNA解吸附的量越多。
3、本发明提供了一种可吸附DNA的纳米粒的制备方法，只需将氢氧化钠溶液加入5-羟色胺盐酸盐溶液中，室温避光搅拌一定时间，离心，洗涤，即得聚五羟色胺纳米粒，该纳米粒制备简便，能稳定吸附单链DNA，并能被cDNA特异性解吸附。
4、本发明提供了一种核酸检测探针，该核酸检测探针用上述纳米粒吸附荧光标记的DNA而成，可构建荧光传感的核酸检测器，用于对目标cDNA的高敏检测。
5、本发明提供了上述纳米粒在核酸检测中的应用，所述纳米粒吸附荧光标记的探针DNA，然后与cDNA孵育30 min，体系的荧光恢复值与cDNA浓度呈线性关系，可用于cDNA的定量检测，用于疾病的诊断。
6、本发明提供了上述纳米粒在细胞内分子成像中的应用，该纳米粒吸附荧光标记的探针DNA，可被细胞内的目标分子解吸附，导致荧光点亮，可实现胞内分子成像。
7、本发明提供了上述纳米粒在核酸药物递送中的应用，只需将单链DNA与该纳米粒在溶液中混合即可发生吸附，可用于核酸药物的递送。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为本发明实施例1中聚五羟色胺纳米粒（PHT NPs）的合成示意图和表征。其中，（A）纳米粒的合成示意图；（B）TEM表征；（C）紫外吸收光谱，图中插图为纳米粒实物图；（D）红外光谱表征；（E）不同pH环境中的zeta电位。
图2为本发明实施例2中聚五羟色胺纳米粒（PHT NPs）吸附荧光标记DNA的示意图、荧光淬灭能力和吸附动力学曲线。其中，（A）纳米粒吸附荧光素（FAM）标记DNA的示意图；（B）PHT NPs淬灭荧光的能力；（C）纳米粒吸附FAM标记A15 DNA的动力学曲线；（D）纳米粒吸附FAM标记DNA的动力学曲线。
图3为本发明实施例3中聚五羟色胺纳米粒（PHT NPs）吸附DNA的稳定性。其中，（A）磷酸盐或血清对DNA的解吸附示意图；（B）cDNA对DNA的解吸附示意图；（C）磷酸盐解吸附动力学曲线；（D）血清解吸附动力学曲线；（E）cDNA对DNA的解吸附动力学曲线。
图4为本发明实施例4中DNA/PHT NPs探针用于cDNA的检测。
图5为本发明实施例5中DNA/PHT NPs探针用于细胞内核酸分子的成像。其中（A）DAPI染A549细胞核成像；（B）DNA/PHT NPs探针用于A549细胞内miR-21成像；(C) DAPI染HEK293细胞核成像；(D) DNA/PHT NPs探针用于HEK293细胞内miR-21成像。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的修改或替换，均属于本发明的范围。
若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段；若未特别指明，实施例中所用试剂均为市售。
本发明涉及到的百分号“%”，若未特别说明，是指质量百分比；但溶液的百分比，除另有规定外，是指100ml溶液中含有溶质的克数。
本发明所述重量份可以是μg、mg、g、kg等本领域公知的重量单位，也可以是其倍数，如1/10、1/100、10倍、100倍等。
以下实施例中，所采用的仪器及生产厂家的详细信息参见表1：
表1 主要仪器名称及生产厂家
Figure 200772DEST_PATH_IMAGE001
以下实施例中，所采用的主要试剂名称及生产厂家参见表2：
表2 主要试剂名称及生产厂家
Figure 218406DEST_PATH_IMAGE002
实施例1
聚五羟色胺纳米粒（PHT NPs）的制备与表征：
本实施例的PHT NPs采用以下方法制备得到：
S1、配置氢氧化钠溶液：将80 mg氢氧化钠溶解于100 mL超纯水中，即得20 mM的氢氧化钠溶液，备用。
S2、配置5-HT溶液：将10 mg 5-HT溶解于4.5 mL超纯水中，备用。
S3、PHT NPs的制备：将0.5 mL氢氧化钠溶液加入到4.5 mL 5-HT溶液中，在室温环境下搅拌反应2天，然后离心（20000 rpm，10 min），收集沉淀。将沉淀分散于水中，即得PHTNPs分散液，备用。
通过场发射透射电子显微镜表征纳米粒形貌；测定PHT NPs在350 nm~900 nm之间的紫外吸收光谱；将PHT NPs冷冻干燥后，进行红外光谱表征；测定PHT NPs在不同pH环境中的zeta电位，使用的缓冲液浓度为10 mM，分别为：pH 5的醋酸钠缓冲液、pH 6的MES缓冲液、pH 7的HEPES缓冲液、pH 8的Tris缓冲液。
由图1可知：本发明制备的PHT NPs形貌呈球形，粒径在100 nm左右；紫外光谱显示，PHT NPs在350 nm到900 nm之间具有很强的紫外吸收，而5-HT原料在这个波长之间却没有明显的紫外吸收；红外光谱显示，与5-HT对应的特征峰在PHT NPs的红外光谱中均合并成宽大的吸收峰；接下来，用马尔文粒径/电位测定仪测纳米粒的zeta电位，结果显示PHT NPs在pH 5~8的范围内带正电，且pH越低的环境下，纳米粒的正电性越强。
以上表明本发明提供的方法可成功制备正电性的PHT NPs，在纳米材料领域具有一定的应用前景。
实施例2
考察实施例1中PHT NPs对荧光的淬灭能力和对DNA的吸附。
（1） 荧光淬灭能力：将10 μL浓度为2 mg/mL的PHT NPs加入含有20 nM FAM标记A15 DNA的HEPES缓冲溶液（10 mM，pH 7.6）中，混匀后室温孵育2 h，然后用酶标仪扫描激发波长470 nm，发射波长510 nm-600 nm处的荧光光谱，并计算荧光淬灭效率。
（2） DNA吸附动力学：将10 μL各种浓度的PHT NPs加入含有20 nM FAM标记A15DNA的HEPES缓冲溶液（10 mM，pH 7.6）中，混匀后立即用酶标仪测定体系的动态荧光值，测定时间为20 min，完毕后绘制荧光随时间变化的曲线。用终浓度为200 μg/mL的PHT NPs吸附FAM标记的A15、T15、C15和G15，比较这四种DNA的吸附情况。
由图2可知：本发明制备的PHT NPs吸附FAM标记的A15 DNA后，可高效淬灭FAM的荧光，且淬灭效率（QE）达到97.23%。此外，随着PHT NPs浓度的增加，体系荧光淬灭比例越大，表明更多的DNA发生吸附。接下来，用PHT NPs吸附四种不同碱基组成的DNA，结果四种DNA都能快速与纳米粒发生吸附，导致体系荧光快速被淬灭。
以上表明本发明提供的PHT NPs可快速吸附DNA，且不同碱基类型的DNA都能发生吸附。
实施例3
考察实施例1、2中PHT NPs对DNA吸附的稳定性。
将20 nM FAM标记的随机DNA在HEPES缓冲液（10 mM，pH 7.6）中吸附于200 μg/mLPHT NPs，然后加入10 μL各种浓度的磷酸盐、血清蛋白（FBS）或cDNA，接下来用酶标仪测定体系的动态荧光值，测定时间为40 min，绘制荧光-时间变化曲线。
由图3可知：本发明制备的PHT NPs吸附FAM-DNA后，向体系中加入各种浓度的磷酸盐，体系的荧光没有恢复，说明DNA没有发生解吸附。此外，加入各种浓度的FBS后，整体DNA的释放量低于10%，表明DNA能够稳定吸附于PHT NPs表面，抵抗血清蛋白引起的解吸附。当向体系中加入各种浓度的cDNA时，体系荧光很快发生了恢复，且cDNA浓度越高，荧光恢复值越大。
以上表明本发明提供的PHT NPs吸附的DNA可抵抗磷酸盐或血清的解吸附，因此该纳米粒吸附DNA具有较高的稳定性；相反，cDNA可使吸附的DNA发生解吸附，可用于cDNA的特异性检测。
实施例4
DNA/PHT NPs探针的制备和该探针用于DNA的检测。
（1） DNA检测探针的制备：将1 μM FAM荧光标记的DNA分散于HEPES缓冲液（10 mM，pH 7.6）中得到分散液；将分散液与200 μg/mL PHT NPs混合，室温孵育过夜，离心（20000rpm，10 min），去除上清液，洗涤，沉淀分散到HEPES缓冲液中，得到DNA/PHT NPs探针，备用。
（2） DNA的检测：将90 μL的DNA/PHT NPs探针与10 μL各种浓度的cDNA孵育30min，接下来用酶标仪测定体系的荧光值，绘制荧光-cDNA浓度变化曲线。
以上表明本发明提供的PHT NPs吸附FAM-DNA构建的检测器可实现靶DNA的检测，用于疾病的诊断。
实施例5
考察实施例4中DNA/PHT NPs探针用于细胞内分子的成像。
（1） 细胞成像探针的制备：将100 nM的FAM-miR21 DNA与100 μg/mL的PHT NPs在HEPES缓冲液（10 mM，pH 7.6）中孵育24 h，然后离心（20000 rpm，10 min），去除上清液，洗涤，沉淀分散于RPMI-1640培养基中，即得DNA/PHT NPs探针，备用。
（2） 细胞培养：人非小细胞肺癌细胞（A549细胞）和人胚胎肾细胞（HEK293细胞）购自于中南大学湘雅医学实验中心。细胞用含有10%（v/v）FBS和1%双抗（penicillin-streptomycin）的RPMI-1640培养基培养于37 ℃，5% CO2的培养箱中。
（3） 取对数生长的A549细胞，消化，接种到24孔板，每孔5×104个细胞。将细胞放入培养箱中培养过夜，细胞完全贴壁生长，去掉培养基，用PBS洗涤，然后加入含有DNA/PHTNPs探针（100 μg/mL）的细胞培养基，孵育8 h后，去掉孵育过的培养基，用PBS轻轻洗涤三次，DAPI（1 μg/mL）染细胞核，然后在荧光显微镜下观察并拍照。
miR21在多种肿瘤细胞中高表达，而在正常细胞中低表达，因此可作为一种肿瘤标记物用于肿瘤的诊断与治疗。由图5细胞内miR-21成像结果可知，DNA/PHT NPs与A549细胞孵育8 h后出现很强的绿色荧光，而与正常组织细胞（HEK293细胞）孵育后，荧光保持在背景水平。说明探针上吸附的DNA与A549细胞中高表达的miR-21发生了互补配对反应，并解吸附荧光恢复，而正常细胞中miR-21低表达，探针上的DNA没有解吸附，进而实现细胞内miR-21的成像。
以上所述是本发明的优选实施方案，应当指出，对于本领域的普通技术人员来说，在不脱离本发明所述原理的前提下，还可做出若干改进和润饰，这些改进和润饰也在本发明的保护范围内。

Claims (4)

1.一种核酸检测探针，其特征在于，所述核酸检测探针由聚五羟色胺纳米粒吸附荧光标记的DNA而成，所述聚五羟色胺纳米粒由5-羟色胺在在氢氧化钠溶液中氧化自聚而成，所述聚五羟色胺纳米粒形貌呈球形，粒径在100 nm左右；所述聚五羟色胺纳米粒具体制备方法为：
S1、分别配置5-羟色胺盐酸盐溶液、氢氧化钠溶液；
S2、将氢氧化钠溶液加入5-羟色胺盐酸盐溶液中，室温避光搅拌一定时间，离心，洗涤，即得聚五羟色胺纳米粒；所述5-羟色胺盐酸盐溶液的质量浓度为0.5 ~ 5 mg/mL，所述氢氧化钠的摩尔浓度为1 mM~5 mM。
2.根据权利要求1所述的核酸检测探针，其特征在于，所述氧化自聚反应在室温避光搅拌条件下进行，所述氧化自聚反应时间为1~5天。
3.一种权利要求1所述的核酸检测探针的制备方法，其特征在于，包括如下步骤：
S1、将荧光标记的DNA分散于缓冲液中得分散液；
S2、将所述分散液与所述纳米粒混合后进行吸附，室温孵育过夜、离心，收集沉淀即得。
4.根据权利要求3所述的核酸检测探针的制备方法，其特征在于，所述缓冲液为HEPES缓冲液。
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