CN106834440A - 一种利用肽核酸和纳米金免标记比色检测原癌基因c‑mycmRNA的方法 - Google Patents
一种利用肽核酸和纳米金免标记比色检测原癌基因c‑mycmRNA的方法 Download PDFInfo
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Abstract
本发明涉及一种利用肽核酸和纳米金免标记比色检测原癌基因c‑myc mRNA的方法，其特征在于包括下述步骤：首先在合成的酒红色纳米金溶液中加入PNA，此时溶液的颜色为蓝色，同时观察紫外吸收光谱，当加入原癌基因c‑myc mRNA的有效序列与PNA杂交后，溶液的颜色又变回酒红色，同时紫外吸收光谱也恢复，由此，低达50nM靶RNA肉眼即能够检测出。本法简单、快速、成本低、纳摩尔级的样品无需仪器就可以肉眼观察到颜色的变化，而且该法可以区分与靶标单个碱基错配的情况。
Description
技术领域
本发明属原癌基因c-myc mRNA分析检测领域，具体涉及一种利用肽核酸和纳米金免标记比色检测原癌基因c-myc mRNA的方法。
背景技术
mRNA(messenger RNA)与人类许多基因疾病密切相关，在癌症的早期阶段中往往能观测到mRNA的异常表达。因此，对特定mRNA的灵敏检测对于实现遗传性疾病(肿瘤或癌症)的早期临床诊断具有重要意义。目前mRNA的检测主要包括基于生物传感原理的荧光分析、电化学、电化学发光技术及聚合酶链式反应扩增技术等。尽管这些检测技术灵敏度较高、实用性较强，但繁琐的实验过程和昂贵的仪器装置阻碍了其在疾病诊断、环境及食品安全监测等分析检测中的应用。因此，成本低廉、操作简单、能实现现场快速测定的方法逐渐受到人们的密切关注。特别是纳米技术与DNA检测技术相结合的比色基因检测方法得到了迅猛发展，尤其是纳米金(AuNPs)凭借制备简单、粒径可控、生物兼容性好等优点在生物检测中得到广泛应用。近年来，许多工作者利用功能化AuNPs实现了对核酸，小分子和细胞的灵敏检测。这些检测基于“交联聚集”方法：利用功能化AuNPs表面的修饰配体(DNA)与目标分析物间的相互作用来克服粒子间的斥力发生聚集，由于AuNPs颜色对聚集比较敏感，通过观察颜色变化实现对目标分析物的检测。由于RNA极易被核酸酶降解，若构建基于DNA为捕获探针的“交联聚集”比色法，不仅操作步骤繁琐，而且在形成的DNA-RNA在检测过程中容易被酶分解掉。
肽核酸(PNA)是一类新的以肽键连接的寡核苷酸模拟物，N-(2-氨基乙基)甘氨酸替代糖-磷酸酯的骨架在结构上很好地模拟了DNA，空间大小与天然核酸相近，加上结构方面的其他特性，PNA既保持了对核酸的特异识别能力，又比寡核苷酸具有更多的优点，主要表现如下三个方面：(1)由于PNA呈电中性，与RNA的杂交不存在静电斥力，因而PNA与RNA之间亲和性较DNA更强，结合的稳定性和特异性都大大提高；(2)PNA识别单碱基的能力强于RNA和DNA；(3)PNA抗酶解能力增强。PNA的酰胺键不同于氨基酸的肽键，其生物学性质极为稳定，而且与RNA形成的复合物不是RNA酶H的底物，不易被蛋白酶或核酸酶降解。用PNA替代DNA分子对目标RNA进行检测，具有无法比拟的优势，因此，成为本领域一直渴望解决的技术难题。
发明内容
本发明针对上述现有技术中存在的问题，提供一种利用肽核酸和纳米金免标记比色检测原癌基因c-myc mRNA的方法，解决了现有技术中操作步骤繁琐、检测过程中容易被酶分解掉的问题。
本发明的技术方案包括下述步骤：
首先在合成的酒红色纳米金溶液中加入PNA，此时溶液的颜色为蓝色，同时观察紫外吸收光谱，当加入原癌基因c-myc mRNA的有效序列与PNA杂交后，溶液的颜色又变回酒红色，同时紫外吸收光谱也恢复，由此，低达50nM靶RNA肉眼即能够检测出。
所述的纳米金(AuNPs)的合成过程为：
步骤1、取HAuCl4·3H2O配制50mL浓度为0.01％HAuCl4水溶液；
步骤2、取柠檬酸钠配制1mL浓度为1％柠檬酸钠水溶液；
步骤3、将50mL 0.01％HAuCl4水溶液加热到沸腾，剧烈搅拌下加入1.0mL1％柠檬酸钠水溶液，恒温加热搅拌15min，停止加热继续搅拌至室温，即得酒红色AuNPs溶液。
所述的AuNPs溶液的浓度为0.45nM；AuNPs粒径为13.2±0.5nm。
所述的PNA为在C端和N端未修饰的12个碱基的肽核酸序列：
12-mer PNA(N'-GCATCG TCG CGG-C')。
所述的原癌基因c-myc mRNA的有效序列为：
5’-CCG CGA CGA UGC-3’(RNAcom)。
所述的单个碱基错配的RNA序列：
5’-CCG CGA GGA UGC-3’(RNAmis)。
本发明的优点效果如下：
与荷负电的DNA相比，电中性的PNA具有优越的杂交特性，用PNA替代DNA与核酸靶标所形成的双链具有高度稳定性和不易被核酸酶降解的特性。PNA具有较强的金纳米聚集能力，而当加入核酸靶标RNA时，PNA与核酸靶标RNA形成双链，又能使团聚的金纳米再次分散的独特光学特性。基于此，利用裸AuNPs和PNA构建“非交联聚集”且“防核酸酶降解”的免标记比色检测原癌基因c-mycmRNA的方法，操作简单、快速、成本低、灵敏度极高，纳摩尔级的样品无需仪器肉眼即可观察到颜色的变化，而且该法能够区分与靶基因单碱基错配的情况。
附图说明
图1a为AuNPs溶液颜色随PNA浓度变化的光学照片；
图1b为AuNPs溶液颜色随PNA浓度变化的紫外吸收光谱；
图1c为AuNPs A600/A520值随PNA浓度变化。
图2a为PNA/AuNPs免标记比色检测原癌基因c-myc mRNA及识别单个碱基错配RNAmis的光学照片；
图2b为PNA/AuNPs免标记比色检测识别原癌基因c-myc mRNA及单个碱基错配RNAmis的AuNPs溶液的紫外吸收光谱；
图2c为高浓度的NaCl存在下，PNA/AuNPs免标记比色检测原癌基因c-mycmRNA及识别单个碱基错配RNAmis的紫外吸收光谱。
具体实施方式
以下结合附图和具体实施方式对本发明做进一步的阐述。
实施例
首先在合成的酒红色纳米金溶液中加入PNA，此时溶液的颜色为蓝色，同时观察紫外吸收光谱，当加入原癌基因c-myc mRNA的有效序列与PNA杂交后，溶液的颜色又变回酒红色，同时紫外吸收光谱也恢复，由此，低达50nM靶RNA肉眼即能够检测出。
所述的纳米金(AuNPs)的合成过程为：
步骤1、取HAuCl4·3H2O配制50mL浓度为0.01％HAuCl4水溶液；
步骤2、取柠檬酸钠配制1mL浓度为1％柠檬酸钠水溶液；
步骤3、将50mL 0.01％HAuCl4水溶液加热到沸腾，剧烈搅拌下加入1.0mL1％柠檬酸钠水溶液，恒温加热搅拌15min，停止加热继续搅拌至室温，即得酒红色AuNPs溶液。
将所制备AuNPs溶液用紫外-可见分光光度计测定，根据Beer-Lambert定律可知AuNPs溶液的浓度为0.45nM；根据TEM表征，AuNPs粒径为13.2±0.5nm。
PNA浓度对AuNPs聚集程度的影响
在不存在NaCl的情况下，将不同浓度PNA加入到200μL的AuNPs溶液中(0.45nM)，在室温下孵育10min后，记录AuNPs溶液的颜色(图1a)及相应的紫外吸收光谱变化(图1b)。如图1a所示，随着PNA浓度逐渐变大，AuNPs溶液颜色由酒红色向蓝紫色转变，这说明PNA能够有效诱导AuNPs聚合，这一点在相应的紫外吸收光谱(图1b)中也得到体现。加入PNA后的AuNPs溶液，其在520nm的特征吸收峰强度逐渐下降，而在600nm处吸光度逐渐增加。用AuNPs在600nm的吸光度与在520nm处吸光度的比值A600/A520可定量表征AuNPs聚集程度。如图1c所示，A600/A520值越大表明AuNPs聚集程度越高，A600/A520值越小表明聚集程度愈小，即分散性越好。AuNPs的聚集程度与PNA浓度密切相关。图1表明：在PNA浓度较低时(≤0.1μM)，AuNPs没有明显的聚集，颜色变化不明显也不容易被肉眼观察到。与此相反，PNA在高浓度(≥0.2μM)时，可以直接观测到AuNPs颜色由酒红色逐渐转变为蓝紫色；相应的在520nm的特征吸收峰强度逐渐下降，而在600nm处吸光度逐渐增加；相应的A600/A520值增大，AuNPs聚集程度增大。
PNA/AuNPs免标记比色检测原癌基因c-myc mRNA的平台的构建及单个碱基错配的识别：
(1)所采用的PNA为在C端和N端未修饰的12个碱基的肽核酸序列：12-mer PNA(N'-GCA TCG TCG CGG-C')；所采用的原癌基因c-myc mRNA的有效序列为5’-CCG CGA CGA UGC-3’(RNAcom)；单个碱基错配的RNA序列：5’-CCG CGA GGA UGC-3’(RNAmis)均购于康卫生物技术有限公司。NaCl为分析纯，不用纯化直接使用。
(2)在不加入NaCl的情况下，PNA能够引起AuNPs聚集而变色。进而向该体系中加入50nM与PNA完全互补的原癌基因c-myc mRNA(RNAcom)，如图2a所示，PNA与RNA杂交生成双链PNA-RNAcom，此时AuNPs溶液由蓝紫色转变酒红色。若向该体系中加入单个碱基错配RNAmis，溶液的颜色也发生了相应的转变，但效果不如全匹配的RNA明显。从紫外吸收光谱图2b可知：此时含有PNA-RNAcom和PNA-RNAmis的AuNPs溶液在600nm的紫外吸收峰已经消失，只有520nm的特征吸收峰。这说明PNA-RNA复合物的生成使得团聚的金纳米粒子又再次分散。有趣的是，在无NaCl存在下，PNA-RNAcom和PNA-RNAmis的AuNPs溶液颜色差别不大，但随着NaCl浓度的不断变化，二者的差异逐渐变大，即使在高浓度的NaCl影响下，PNA-RNAcom仍旧能有效的稳定纳米金，而单个碱基错配的PNA-RNAmis却不能抵抗高盐离子对其的干扰(图2a)。由图2c也可以看出，随着NaCl浓度增加到0.15mol/L，PNA-RNAcom仍旧能有效的稳定AuNPs，溶液在600nm的紫外吸收峰仍能保持稳定；而PNA-RNAmis已不能稳定AuNPs，此时溶液在600nm的紫外吸收峰亦明显降低。因此，在NaCl存在的情况下，可利用PNA引起金纳米聚集变色，而PNA-RNA的复合物使团聚的金纳米粒子再次分散的独特光学特性，构建一种新型的原癌基因c-mycmRNA的免标记比色检测方法，这种简单的体系具有极高的灵敏度，肉眼能够检测出50nM靶RNA，而且该法能够区分与靶基因单碱基错配的情况。
发明名称: 一种利用肽核酸和纳米金免标记比色检测原癌基因c-myc mRNA的方法
申请人名称: 辽宁石油化工大学
序列名称: 原癌基因c-myc mRNA
原癌基因c-myc mRNA有效序列:
5’-CCG CGA CGA UGC-3’（RNAcom）
单个碱基错配的RNA序列：
5’-CCG CGA GGA UGC-3’（RNAmis）
肽核酸序列：
12-mer PNA (N'-GCA TCG TCG CGG-C')
Claims (6)
1.一种利用肽核酸和纳米金免标记比色检测原癌基因c-myc mRNA的方法，其特征在于包括下述步骤：首先在合成的酒红色纳米金溶液中加入PNA，此时溶液的颜色为蓝色，同时观察紫外吸收光谱，当加入原癌基因c-myc mRNA的有效序列与PNA杂交后，溶液的颜色又变回酒红色，同时紫外吸收光谱也恢复，由此，低达50nM靶RNA肉眼即能够检测出。
2.根据权利要求1所述的一种利用肽核酸和纳米金免标记比色检测原癌基因c-mycmRNA的方法，其特征在于所述的纳米金(AuNPs)的合成过程为：
步骤1、取HAuCl4·3H2O配制50mL浓度为0.01％HAuCl4水溶液；
步骤2、取柠檬酸钠配制1mL浓度为1％柠檬酸钠水溶液；
步骤3、将50mL 0.01％HAuCl4水溶液加热到沸腾，剧烈搅拌下加入1.0mL1％柠檬酸钠水溶液，恒温加热搅拌15min，停止加热继续搅拌至室温，即得酒红色AuNPs溶液。
3.根据权利要求2所述的一种利用肽核酸和纳米金免标记比色检测原癌基因c-mycmRNA的方法，其特征在于所述的AuNPs溶液的浓度为0.45nM；AuNPs粒径为13.2±0.5nm。
4.根据权利要求1所述的一种利用肽核酸和纳米金免标记比色检测原癌基因c-mycmRNA的方法，其特征在于所述的PNA为在C端和N端未修饰的12个碱基的肽核酸序列：
12-mer PNA(N'-GCA TCG TCG CGG-C')。
5.根据权利要求1所述的一种利用肽核酸和纳米金免标记比色检测原癌基因c-mycmRNA的方法，其特征在于所述的原癌基因c-myc mRNA的有效序列为：
5’-CCG CGA CGA UGC-3’(RNAcom)。
6.根据权利要求1所述的一种利用肽核酸和纳米金免标记比色检测原癌基因c-mycmRNA的方法，其特征在于所述的单个碱基错配的RNA序列：
5’-CCG CGA GGA UGC-3’(RNAmis)。
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