CN104931684A - 一种纳米荧光传感器及其制备方法和应用 - Google Patents
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Abstract
本发明提供了一种可用于生物医学及生命科学领域的纳米荧光传感器及其制备方法和应用。本发明将金纳米笼与核酸适体及表面正电荷修饰技术结合在一起,提出一种全新的纳米荧光传感器。该传感器的特点是:以金纳米笼作为纳米容器,在其表面修饰了一层聚阳离子,将荧光染料填充到金纳米笼中,利用静电作用将核酸适体组装到金纳米笼表面,形成具有生物响应作用的分子门。一旦加入待检测的靶分子如ATP,则发生靶分子及其适体的特异性识别反应,导致适体链从金纳米笼表面脱离,使封堵笼孔的分子门被打开,孔内的荧光分子被释放出来。因此,利用该荧光传感器,能够实现对靶分子的高灵敏、高选择性检测,为癌症等重大疾病的早期诊断、治疗等提供新方法。
Description
技术领域
本发明属于生物分析及生命科学领域,具体涉及一种检测ATP的纳米荧光传感器及其制备方法和应用。
背景技术
三磷酸腺苷(ATP)是广泛存在于生物细胞内的一种辅酶,由腺苷和三个磷酸基组成。作为细胞内能量传递的“分子通货”,ATP储存和传递化学能,是体内组织细胞所需能量的主要来源,参与蛋白质、脂肪、糖和核苷酸的合成,对细胞许多代谢过程均有重要的调节作用,ATP的含量变化可以反映细胞的变异和损伤。因此,ATP作为重大疾病的标志物之一得到了广泛的研究和关注。通过检测恶性肿瘤ATP浓度变化,可以了解其细胞代谢情况,为恶性肿瘤药敏的异质性和个体化疗提供可行性治疗方案。因此,建立快速、灵敏、准确的ATP分析方法不仅有助于癌症等重大疾病的早期诊断及治疗,同时对于临床医学、生命科学、食品卫生、环境监测、医药及化妆品等诸多领域也有着重要的实际意义。
目前,国内外ATP的检测方法除了作为主流技术的高效液相色谱法以外,其它还有电泳法、分光光度法和生物发光法等。这些方法在仪器设备、时间、灵敏度、准确性等方面都或多或少地存在不足,尤其是灵敏度和选择性尚待提高。为此,迫切需要开发高灵敏、高选择性的检测技术用于ATP的检测。
近几年来,金纳米笼作为一种新兴的纳米材料得到了广泛的关注。该纳米材料是一种中空多孔的结构,即笼状颗粒的内部为空心结构,光滑的笼壁表面上分布着小孔。与传统的球型金纳米颗粒相比,金纳米笼的局域表面等离子共振峰位于近红外区700~900nm,该波段对于生物医学意义重大,尤其是对于活体研究。将金纳米笼与核酸适体和金纳米笼表面正电荷修饰技术相结合的技术还未见文献报道。
发明内容
为了克服现有技术存在的不足,同时,也为了更加有效地利用具有特殊结构的纳米材料的显著优点,针对检测ATP的纳米荧光传感器少有报道,因此,本发明的第一目的:构建并制备一种新型的可用于检测ATP的纳米荧光传感器,即将金纳米笼与核酸适体和表面正电荷修饰技术结合在一起,使该纳米荧光传感器具有可生物响应的分子门,一旦加入需要检测的靶分子如ATP,则发生靶分子及其适体的特异性识别反应,导致适体链从金纳米笼表面脱离,使封堵笼孔的分子门被打开,并使孔内被封堵的荧光分子被释放出来,因此,利用该荧光传感器,能够实现对靶分子的高灵敏、高选择性检测,为癌症等重大疾病的早期诊断、治疗、理论研究等提供新方法;本发明的第二目的:提供一种该纳米荧光传感器的制备方法;本发明的第三目的:提供一种应用该纳米荧光传感器检测ATP的方法。将本发明提出的纳米荧光传感器用于ATP的荧光检测,能够显著地提高ATP检测的灵敏度和准确度。
本发明是通过以下技术方案实现发明目的的。本发明的纳米荧光传感器是以金纳米笼作为载体,利用其空心多孔的结构特性,在其内部装载荧光分子,为了防止荧光分子的外泄,通过在其表面组装核酸适体分子门,将金纳米笼表面的孔封堵;其中,所述的核酸适体可以通过静电作用被组装到金纳米笼表面,具体可以通过在金纳米笼表面修饰正电荷的方法而将其组装到金纳米笼表面,优选地采用聚二烯丙基二甲基氯化铵即PDDA作为正电荷修饰剂在金纳米笼表面修饰一层聚阳离子。
优选地,上述纳米荧光传感器,所述的荧光分子是罗丹明B。
一种制备上述纳米荧光传感器的制备方法,包括如下步骤:
(1)在磁珠-金纳米笼的复合物中加入PDDA溶液,室温振荡过夜;
(2)磁分离上述溶液后加入罗丹明B溶液,室温振荡过夜;
(3)向上述溶液中加入核酸适体溶液,室温振荡过夜,磁分离,用PBS缓冲溶液清洗后即制得荧光传感器;
其中,所述的磁珠-金纳米笼的复合物按如下方法制备而成:将磁珠与金纳米笼的混合溶液置于室温振荡反应10h,磁分离,用PBS缓冲溶液清洗,移除上清夜,即得。
一种利用本发明的纳米荧光传感器用于ATP的检测,方法如下:
(1)将ATP样品溶液加入到本发明的纳米荧光传感器的PBS悬浮液中,37℃恒温振荡反应1h,发生靶分子ATP与其适体的特异性结合,使ATP适体从金纳米笼表面脱离,分子门被打开;
(2)磁分离上述溶液,收集上清液,检测其荧光信号。
本发明的有益效果:本发明提出的纳米荧光传感器将纳米技术与适体分子生物门相结合,通过分子识别及特异性反应,使适体分子门被打开,释放出荧光分子,实现了荧光信号的检测,该方法使ATP的检测灵敏度得到显著提高,可实现对ATP高灵敏、高选择性的检测。本发明的纳米荧光传感器具有结构简单,稳定性好,可控性强,荧光信号灵敏等优点,同时,不受其它常见干扰物质影响,具有高的选择性,可应用于生物体系中ATP的荧光检测。实验结果表明,采用本发明提出的纳米荧光传感器可在1.0×10-10~8.0×10-9M范围内实现对ATP的高灵敏、高选择性检测。该传感器及其检测技术在生物医学、生命科学等领域具有较大的应用潜力和广阔的应用前景,可用于ATP的特异性检测,为癌症等重大疾病的早期诊断及治疗提供新的途径和方法。
附图说明
图1不同ATP浓度的荧光信号强度。
图2ATP浓度与荧光信号强度的线性关系。
具体实施方式
以下是本发明涉及的具体实施例,对本发明的技术方案做进一步描述,但是本发明的保护范围并不限于这些实施例。凡是不背离本发明构思的改变或等同替代均包括在本发明的保护范围之内。
下面通过实施例具体地说明本发明,但本发明不受下述实施例的限定。
实验仪器:磁性分离架(天津倍思乐色谱技术开发中心);F-4600荧光分光光度计(日立,日本);THZ-82A气浴恒温振荡器(金坛市医疗器械厂)。
实验试剂:3-4μm巯基修饰磁珠(天津市倍思乐色谱技术开发中心);罗丹明B(阿拉丁);三磷酸腺苷(ATP);ATP适体链:ACC TGG GGG AGT ATT GCG GAG GAA GGT,PBS溶液为0.01M(pH 7.4,Na2HPO4-NaH2PO4)。
实施例1:
一种制备本发明纳米荧光传感器的制备方法,包括如下步骤:
(1)在磁珠-金纳米笼的复合物中加入200μL浓度为5.832mg/mL的Pdda溶液,室温振荡过夜;
(2)磁分离上述溶液,用PBS缓冲溶液清洗后加入100μL1.0×10-5mol/L罗丹明B的PBS溶液,室温振荡过夜;
(3)向上述溶液加入10μL浓度为1.0×10-5M的ATP适体溶液,室温振荡过夜,磁分离,移除上清液,纳米荧光传感器制备完成;
其中,所述的磁珠-金纳米笼的复合物按如下方法制备而成:将10μL巯基磁珠与200μL金纳米笼均匀混合,室温振荡反应10h,磁分离,用PBS溶液清洗,移除上清液,即得;所述的巯基磁珠是购买的商品(天津市倍思乐色谱技术开发中心);所述的金纳米笼按文献方法获得(G.D.Moon,S.W.Choi,X.Cai,W.Y.Li,E.C.Cho,U.Jeong,L.V.Wang and Y.N.Xia.J.Am.Chem.Soc.2011,133,4762–4765)。
实施例2:
一种利用本发明的纳米荧光传感器用于ATP的检测,方法如下:
(1)将ATP样品溶液加入到本发明的纳米荧光传感器中,用PBS缓冲溶液(PH=7.4)稀释至200μL,37℃恒温振荡反应1h,发生靶分子ATP与其适体的特异性结合,使ATP适体从金纳米笼表面脱离,分子门被打开;
(2)磁分离上述溶液,收集上清液,检测其荧光信号,荧光检测条件:激发波长和发射波长分别为530、573nm。
图1为不同ATP浓度对应的荧光信号强度,ATP的浓度分别为(0,1.0×10-10,5.0×10-10,8.0×10-10,1.0×10-9,2.0×10-9,5.0×10-9,6.0×10-9,8.0×10-9,1.0×10-8,2.0×10-8,5.0×10-8mol/L);图2为ATP浓度与荧光信号强度的线性关系。结果表明,ATP浓度在1.0×10-10~8.0×10-9mol/L时,荧光信号强度与ATP的浓度呈现良好的线性关系,其线性方程为:FL=159.44257+20.13502×CATP(10-9mol/L),线性相关系数为0.9935。
本发明将纳米技术与分子生物技术相结合,通过分子识别及特异性反应,可实现对ATP高灵敏、高选择性的检测。本发明将金纳米笼与核酸适体和表面正电荷修饰技术结合在一起,使该纳米荧光传感器具有可生物响应的分子门,一旦加入需要检测的靶分子如ATP,则发生靶分子及其适体的特异性识别反应,导致适体链从金纳米笼表面脱离,使封堵笼孔的分子门被打开,并使孔内被封堵的荧光分子被释放出来,因此,利用该荧光传感器,能够实现对靶分子的高灵敏、高选择性检测。
本发明的纳米荧光传感器结构简单,稳定性好,可控性强,荧光信号灵敏,同时,不受其它常见干扰物质影响,具有高的选择性。该纳米荧光传感器及其检测技术在生物医学、生命科学等领域具有较大的应用潜力和广阔的应用前景,可用于ATP的特异性检测,为癌症等重大疾病的早期诊断及治疗提供新的途径和方法。
Claims (7)
1.一种纳米荧光传感器,其特征在于:以金纳米笼作为载体,其内部载有荧光分子,其表面被组装上核酸适体,可将金纳米笼孔封堵,防止荧光分子外泄。
2.一种如权利要求1所述的纳米荧光传感器,其特征在于:所述的核酸适体可以通过静电作用被组装到金纳米笼表面。
3.一种如权利要求1-2中任一项所述的纳米荧光传感器,其特征在于:所述的核酸适体可以通过在金纳米笼表面修饰正电荷而将其组装到金纳米笼表面。
4.一种如权利要求1-3中任一项所述的纳米荧光传感器,其特征在于:所述的正电荷可通过在金纳米笼表面修饰一层聚阳离子PDDA,即聚二烯丙基二甲基氯化铵而产生。
5.一种如权利要求1-4中任一项所述的纳米荧光传感器,其特征在于:所述的荧光分子是罗丹明B。
6.一种如权利要求1-5中任一项所述的纳米荧光传感器的制备方法,其特征在于步骤如下:
(1)在磁珠-金纳米笼的复合物中加入PDDA溶液,室温振荡过夜;
(2)磁分离上述溶液后加入罗丹明B溶液,室温振荡过夜;
(3)向上述溶液中加入核酸适体溶液,室温振荡过夜,磁分离,用PBS缓冲溶液清洗后即制得纳米荧光传感器;
其中,所述的磁珠-金纳米笼的复合物按如下方法制备而成:将磁珠与金纳米笼的混合溶液置于室温振荡反应10h,磁分离,用PBS缓冲溶液清洗,移除上清夜,即得。
7.一种如权利要求1-5中任一项所述的纳米荧光传感器的应用,其特征在于用于ATP的检测,方法如下:
(1)将ATP样品溶液加入到如权利要求1-5中任一项所述的纳米荧光传感器的PBS悬浮液中,37℃恒温振荡反应1h,发生ATP与其适体的特异性结合,使ATP适体从金纳米笼表面脱离,分子门被打开;
(2)磁分离上述溶液,收集上清液,检测其荧光信号。
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