CN114034864A - 基于金纳米复合材料和多肽构建的核酸适配体荧光传感器对甲胎蛋白的检测方法 - Google Patents
基于金纳米复合材料和多肽构建的核酸适配体荧光传感器对甲胎蛋白的检测方法 Download PDFInfo
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Abstract
本发明属于生物化学、临床医学等领域中的甲胎蛋白检测技术领域，公开了基于金纳米复合材料和多肽构建的核酸适配体荧光传感器对甲胎蛋白的检测方法。本发明通过利用未经荧光修饰的适配体链与GDY@AuNPs@PCN共价固定，与AFP结合后引入携带荧光的核酸适配体链，形成类似于“三明治”结构的装置，同时加入多功能防污多肽降低背景信号，可实现适体与AFP的高效结合，提高了检测的灵敏性。本发明充分利用复合材料的优势与特点，实现甲胎蛋白的高效检测。
Description
技术领域
本发明属于生物化学、临床医学等领域中的甲胎蛋白检测方法，尤其是基于金纳米复合材料和多肽构建的核酸适配体荧光传感器对甲胎蛋白的检测方法。
背景技术
肝细胞癌(hepatocellular carcinoma，HCC)是全球十大最常见的癌症之一，通常也是导致肝硬化患者死亡的原因。甲胎蛋白(alpha-fetoprotein，AFP)作为肝癌最常用的蛋白质生物学标志物近年来引起了极大的关注。AFP通常由7-8月胎儿卵黄囊产生，从肝脏分泌出来的一种分子量约为70kDa的血浆蛋白，随着胎儿的出生以及生长，其含量逐渐下降。在健康人的血清中，AFP浓度低于25ng/mL以至于几乎检测不到，但据统计在将近75％的HCC患者中，AFP水平明显升高至500ng/mL。成人血液中高含量的AFP可能表明存在某些类型的癌症，特别是HCC、胃癌、胰腺癌、卵巢癌或睾丸癌，另外在霍奇金病、淋巴瘤、脑瘤和肾细胞癌中也可能发现高的AFP水平，更重要的是一些早期的癌症患者在临床上通常是无症状的，通常会导致晚期诊断和高死亡率。AFP作为生物标志物，对于检测男性、非孕妇和儿童中的一部分肿瘤具有重要的意义。开发一种用于检测AFP的快速、高灵敏度、选择性、低成本和高效益的方法对于人类疾病检测是至关重要的。
金纳米颗粒(gold nanoparticles，AuNPs)具有独特的电子和光子特性比如在520nm为中心的波长范围内具有宽吸收峰，被广泛应用于比色生物传感平台方面。AuNPs表面富含的Au能够与携带硫醇基团的核酸适配体形成强Au-S键，从而将核酸适配体固定在金纳米颗粒的表面。研究表明，在较高浓度的强电解质的作用下，AuNPs表面的电荷减少，纳米颗粒之间的距离缩短从而导致发生聚集，而NaCl是常用的聚集剂。
应用于医学和生物技术领域的肽，具有与两性离子材料结构(如含有氨基和羧基等亲水基团)相似的特征而被广泛关注。两性离子材料克服聚乙二醇的低性能和高剂量而成为重要防污材料。相较于两性离子，肽还具有容易修饰、灵活的形态以及对环境友好的性能。
发明内容
为了降低AFP的检测极限，提高检测AFP的灵敏性。本发明基于利用未经荧光修饰的适配体链与GDY@AuNPs@PCN共价固定，在加入具有防污性能的多肽消除生物污损等背景的作用下，与甲胎蛋白(AFP)结合后，引入携带荧光的核酸适配体链，形成类似于“三明治”结构的传感平台。在降低背景信号的同时能更高效的结合AFP，同时灵敏性也得到了提高。
此外本发明使用具有聚集诱导发光效应的另一种具有AIE效应的荧光团DSAI分子的核酸适配体，DSAI具有更加稳定的荧光性质，并且充分的利用了复合材料的具有更好的淬灭效果，通过两条适配体与AFP的结合形成的“三明治”结构，从而提高对AFP的检测效果。
基于金纳米复合材料和多肽构建的核酸适配体荧光传感器对甲胎蛋白的检测方法，步骤为：
(1)根据检测原理设计末端具有巯基的DNA序列、核酸适配体-1和多肽；
DNA-SH：5'-GTG ACG CTC CTAACG CTG ACT CAG GTG CAG TTC TCG ACT CGG TCTTGATGT GGG TCC TGT CCG TCC GAACCA ATC-SH-3'；
DNA-1：5'-GTG ACG CTC CTA ACG CTG ACT CAG GTG CAG TTC TCG ACT CGG TCTTGA TGT GGG TCC TGT CCG TCC GAA CCA ATC-3'；
多肽：EKEKEKEPPPC；
(2)石墨二炔(GDY)二维材料溶液的配制：
将石墨二炔固体粉末称取10mg溶于20mL超纯水中,超声处理2-3h，得到0.5mg/mL的GDY溶液。
(3)GDY@AuNPs复合物的制备：
将步骤(2)超声均匀的GDY溶液添加到HAuCl4溶液中连续搅拌的条件下孵育30min，以促进进金(III)与石墨二炔表面的π-π堆积作用，并混合物加热至80℃；将柠檬酸钠添加至体系中混合均匀并在80℃继续搅拌4h；然后将所得复合物离心去除过量的HAuCl4等离子，并在冷冻干燥机中干燥得到最终的GDY@AuNPs复合物，溶于超纯水中，并超声使其分散均匀，备用。
步骤(3)中，GDY溶液、HAuCl4溶液和柠檬酸钠溶液的体积比为10:50:1；其中，GDY溶液的浓度为0.5mg/mL，HAuCl4溶液的浓度为1mM；柠檬酸钠溶液的浓度为300mM；离心的速度为6000rpm，离心时间为2h；
(4)GDY@AuNPs@PCN复合物的制备：
将卟啉基金属有机骨架材料中多孔配位网络结构PCN溶液缓慢滴加到步骤(3)超声均匀的GDY@AuNPs复合物溶液中；
步骤(4)中，PCN溶液和GDY@AuNPs复合物溶液的体积比为400μL：1mL；其中，PCN溶液的浓度为0.1mg/mL，GDY@AuNPs复合物溶液的浓度为2mg/mL。
(5)淬灭：
将末端修饰巯基的核酸适配体DNA-SH与GDY@AuNPs@PCN复合物在PBS缓冲液中孵育12h，经13000rpm,30min离心洗脱后，测其荧光强度F0。
步骤(5)中，孵育总体系中末端修饰巯基的核酸适配体DNA-SH的浓度范围为5-40nM,GDY@AuNPs@PCN的浓度为5-25μg/mL；所述PBS缓冲液的浓度为10mM，pH＝7.4。
(6)检测：
将多肽和一定浓度梯度的AFP加入到步骤(5)所得的总体系中，在室温下反应30min，再加入经DSAI修饰的DNA-1，得到反应液，继续反应30min，经过离心后，测其荧光强度F。
步骤(6)中，所述反应液中，多肽的浓度为50～300ng/mL，经DSAI修饰的DNA-1的终浓度为10nM；所述AFP的浓度为0.01～8ng/mL。
与步骤(5)淬灭后的荧光数值F0进行比较，按照F/F0-1公式查看荧光恢复率，加入的AFP浓度与荧光回复率呈一定的正比关系。
AFP的浓度为0.01～8ng/mL内，F/F0-1与AFP浓度呈现明显的线性关系，基于3S/N，计算其检测极限值为1.528ng/L。
本发明具有以下优点：
(1)本发明中所用到的金纳米易于获得，原理简单、操作方便，性质稳定。
(2)本发明利用金纳米颗粒表面富含的Au与核酸适配体在一定盐浓度的诱导下发生“Au-S”共价结合，解决了因物理吸附可能出现的假阳性信号。
(3)本发明利用复合纳米材料检测AFP，这比单一的纳米材料检测的灵敏性要高，这对以后优化AFP检测提供了一定的基础，也为AFP检测开辟了新的研究道路。
(4)本发明为了使传感器具有较高的信噪比，消除生物污损等背景。生物污损主要通过非特异性相互作用进行对传感器造成干扰，因此，在体系中加入具有防污性能的多肽，用来抵抗AFP在该传感界面上的非特异性吸附。
(5)本发明通过两条适体与AFP的结合形成的“三明治”结构，从而更高效的结合AFP，提高AFP检测的灵敏性。
附图说明：
图1为基于GDY@AuNPs@PCN复合物和防污多肽形成的三明治结构示意图；
图2为DSAI动力学图。
图3为GDY@AuNPs@PCN复合物的浓度选择图。
图4为核酸适配体的浓度选择图。
图5为防污多肽的浓度选择图。
图6为核酸适配体中加入不同浓度AFP后荧光强度(A)和荧光恢复率(F/F0-1)的变化(B)。
图7为核酸适配体溶液中加入不同种类蛋白后荧光强度的变化图，其中每种蛋白的终浓度为0.5ng/mL。
具体实施方式：
以下结合实施例对本发明做进一步说明，实施例是用于说明本发明，而不是用于限制本发明的范围。
(1)合成特异的DNA序列如下：
DNA-SH：5'-GTG ACG CTC CTA ACG CTG ACT CAG GTG CAG TTC TCG ACT CGG TCTTGA TGT GGG TCC TGT CCG TCC GAA CCA ATC-SH-3'；
DNA-1：5'-GTG ACG CTC CTA ACG CTG ACT CAG GTG CAG TTC TCG ACT CGG TCTTGA TGT GGG TCC TGT CCG TCC GAA CCA ATC-3'；
多肽：EKEKEKEPPPC；
(2)石墨二炔(GDY)二维材料溶液的配制：将得到的石墨二炔固体粉末称取10mg溶于20mL超纯水中,超声处理2-3h，得到0.5mg/mL的GDY溶液
(3)GDY@AuNPs复合物的制备：
将超声均匀的GDY溶液添加HAuCl4溶液中连续搅拌的条件下孵育30min，以促进进金(III)与石墨二炔表面的π-π堆积作用，并混合物加热至80℃。将柠檬酸钠添加至体系中混合均匀并在80℃继续搅拌4h。
GDY溶液、HAuCl4溶液和柠檬酸钠溶液的体积比为10:50:1；其中，GDY溶液的浓度为0.5mg/mL，HAuCl4溶液的浓度为1mM；柠檬酸钠溶液的浓度为300Mm。然后将所得复合物离心去除过量的HAuCl4等离子，并在冷冻干燥机中干燥得到最终的GDY@AuNPs复合物，溶于超纯水中，并超声使其分散均匀，备用。
(4)GDY@AuNPs@PCN复合物的制备：
将PCN溶液缓慢滴加到步骤(3)超声均匀的GDY@AuNPs复合物溶液中。
其中，PCN溶液和GDY@AuNPs复合物溶液的体积比为400μL：1mL；PCN溶液的浓度为0.1mg/mL，GDY@AuNPs复合物溶液的浓度为2mg/mL。(5)向20nM DNA-SH体系中加入梯度浓度的金纳米复合材料GDY@AuNPs@PCN(5-25μg/mL)，选出最佳淬灭率的GDY@AuNPs@PCN浓度，复合物浓度为15μg/mL时荧光变化率达到最大值；当浓度继续增大时，荧光变化率反而逐渐降低。因此，选择的最佳浓度为15μg/mL。
(6)将不同浓度的DNA-SH(5-40nM)加入到包含15μg/mL的GDY@AuNPs@PCN的体系当中，再加入等浓度的AFP，结果显示加入20nM DNA的溶液引起的荧光增长率最高。
(7)不同浓度的多肽(50-500ng/mL)加入到包含15μg/mL的GDY@AuNPs@PCN和20nMDNA-SH的体系中，当多肽浓度浓度为300ng/mL时，该反应体系最终的效果最好。
(8)将不同浓度的AFP(-0.01-8ng/mL)加入到含有纳米材料GDY@AuNPs@PCN(15μg/mL)、DNA-SH(20nM)、携带DSAI的核酸适配体-1(10nM)以及多肽(300ng/mL)的体系中，吸取200μL测其荧光光谱并记录，查看荧光的恢复情况。
(9)检测了几种与AFP类似的物质(BSA、CEA、HSA、IgG和Thrombin)在同等条件下与待测目标AFP作比较，从而分析该传感器的选择性。在(8)的相同条件下，评估该传感器的灵敏性。
图1基于GDY@AuNPs@PCN复合物和防污多肽形成的三明治结构示意图。
由图2DSAI的动力学图，可以看出，一定浓度的DSAI分子在所测量时间范围内的荧光强度没有明显的变化，表明该荧光分子在PBS缓冲体系中能够保持良好的稳定性。
由图3GDY@AuNPs@PCN复合材料最佳浓度选择图可以看出，F/F0-1随着GDY@AuNPs@PCN复合物的浓度的增加逐渐增大，该复合物浓度为15μg/mL时荧光变化率达到最大值；当浓度继续增大时，荧光变化率反而逐渐降低。故GDY@AuNPs@PCN最佳浓度为15μg/mL。
由图4核酸适配体浓度的选择图可以看出，不同浓度核酸适配体溶液在反应环境中的荧光回复强度变化，最佳浓度为20nM。
由图5防污多肽浓度的选择图可以看出，当多肽浓度浓度为300ng/mL时，荧光的变化率最好。
由图6核酸适配体中加入不同浓度AFP后荧光强度和荧光恢复率(F/F0-1)的变化可以看出，A图中，随着AFP的浓度增大，荧光值在不断增大；从B图中可以看到AFP的浓度在0.01-0.08ng/mL范围内，F/F0-1与AFP的浓度呈现明显的线性关系。
图7为核酸适配体溶液中加入不同种类蛋白后荧光强度的变化，其中每种蛋白的终浓度为0.5ng/mL，由图可以看出核酸适配体溶液对AFP优异的选择性。
Claims (9)
1.基于金纳米复合材料和多肽构建的核酸适配体荧光传感器对甲胎蛋白的检测方法，其特征在于，包括如下步骤：
(1)根据检测原理设计末端具有巯基的DNA序列、核酸适配体-1和多肽；
DNA-SH：5'-GTG ACG CTC CTA ACG CTG ACT CAG GTG CAG TTC TCG ACT CGG TCT TGATGT GGG TCC TGT CCG TCC GAA CCA ATC-SH-3'；
DNA-1：5'-GTG ACG CTC CTA ACG CTG ACT CAG GTG CAG TTC TCG ACT CGG TCT TGATGT GGG TCC TGT CCG TCC GAA CCA ATC-3'；
多肽：EKEKEKEPPPC；
(2)配制石墨二炔GDY二维材料溶液，备用；
(3)GDY@AuNPs复合物的制备：
将步骤(2)超声均匀的GDY二维材料溶液添加到HAuCl4溶液中连续搅拌的条件下孵育一定时间，混合物加热至一定温度；将柠檬酸钠添加至体系中混合均匀并在该温度下继续搅拌一定时间；然后将所得复合物离心去除过量的HAuCl4等离子，并在冷冻干燥机中干燥得到最终的GDY@AuNPs复合物，溶于超纯水中，并超声使其分散均匀，备用；
(4)GDY@AuNPs@PCN复合物的制备：
将卟啉基金属有机骨架材料中多孔配位网络结构PCN溶液缓慢滴加到步骤(3)超声均匀的GDY@AuNPs复合物溶液中；
(5)将末端修饰巯基的核酸适配体DNA-SH与GDY@AuNPs@PCN复合物在PBS缓冲液中孵育一定时间，经离心洗脱后，测其荧光强度F0；
(6)将多肽和一定浓度梯度的AFP加入到步骤(5)所得的总体系中，在室温下反应一定时间，再加入经DSAI修饰的DNA-1，得到反应液，继续反应一定时间，经过离心后，测其荧光强度F；与步骤(5)淬灭后的荧光数值F0进行比较，按照F/F0-1公式查看荧光恢复率。
2.如权利要求1所述的检测方法，其特征在于，步骤(2)中，石墨二炔GDY二维材料溶液的配制步骤为：将石墨二炔固体粉末称取10mg溶于20mL超纯水中，超声处理2-3h，得到0.5mg/mL的GDY溶液。
3.如权利要求1所述的检测方法，其特征在于，步骤(3)中，GDY溶液、HAuCl4溶液和柠檬酸钠溶液的体积比为10:50:1；其中，GDY溶液的浓度为0.5mg/mL，HAuCl4溶液的浓度为1mM；柠檬酸钠溶液的浓度为300mM；离心的速度为6000rpm，离心时间为2h。
4.如权利要求1所述的检测方法，其特征在于，步骤(3)中，孵育时间为30min；混合物加热至80℃，搅拌时间为4h。
5.如权利要求1所述的检测方法，其特征在于，步骤(4)中，PCN溶液和GDY@AuNPs复合物溶液的体积比为400μL：1mL；其中，PCN溶液的浓度为0.1mg/mL，GDY@AuNPs复合物溶液的浓度为2mg/mL。
6.如权利要求1所述的检测方法，其特征在于，步骤(5)中，孵育总体系中末端修饰巯基的核酸适配体DNA-SH的浓度范围为5-40nM,GDY@AuNPs@PCN的浓度为5-25μg/mL；所述PBS缓冲液的浓度为10mM，pH＝7.4。
7.如权利要求1所述的检测方法，其特征在于，步骤(5)中，孵育时间为12h；离心洗脱的转速为13000rpm,时间为30min。
8.如权利要求1所述的检测方法，其特征在于，步骤(6)中，所述反应液中，多肽的浓度为50～300ng/mL，经DSAI修饰的DNA-1的终浓度为10nM；所述AFP的浓度为0.01～8ng/mL。
9.如权利要求1所述的检测方法，其特征在于，步骤(6)中，室温下反应的时间为30min，再加入经DSAI修饰的DNA-1，得到反应液，继续反应的时间为30min。
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