CN105352919B - 双色荧光含金碳点的制备及该碳点在可视化检测的应用 - Google Patents
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Abstract
本发明公开了一种双色荧光含金碳点的制备及该碳点在可视化检测的应用。该含金碳点的制备主要是：将氯金酸溶液和谷胱甘肽溶液混合，并用纯净水稀释；将混合溶液在80～100℃油浴条件下边搅拌边加热，加热时间1.5～2h，趁热将其转移到盛有葡萄糖的锥形瓶中，家用微波炉高火档加热20～25min；将生成的黄褐色产物用纯净水溶解、离心，将上清液透析24h，即得产品。基于含金碳点的过氧化物模拟酶性质，通过DNA适配体和前列腺癌抗原的特异性作用对双色荧光含金碳点催化表面位点的覆盖来检测前列腺癌抗原患者血清中前列腺癌抗原的含量，实现对前列腺癌抗原的快速可视化检测。该方法快速简便、易于操作、灵敏度高，在临床医学具有重要意义。
Description
技术领域
本发明属于应用化学技术领域，具体涉及一种双色荧光含金碳点的制备，基于这种含金碳点具有过氧化物酶的催化性能，实现对前列腺癌抗原的可视化检测。
背景技术
随着越来越多纳米材料制备方法的出现，含金碳点成为了越来越重要的一类材料。由于相比于单一组分纳米材料表现出的独特性质，含金碳点越来越受到人们的重视，且使得他们应用广泛，如催化、传感、电子应用、医疗诊断等等。到目前为止，有很多关于含金碳点的制备方法被研究出来，但是大多数这些制备方法都是相当复杂繁琐的，且他们通常都涉及到半导体材料和新型金属。所以能开发出一种简单的含金碳点制备方法受到人们的广泛期待。
碳点(Carbon Dots，CDs)作为一种近些年来发现的新型碳纳米材料，受到了人们极大的关注。碳点具有荧光性质，其粒径通常小于10nm，于2004年Xu等在使用凝胶电泳制备分离纳米管时偶然发现，并于2006年由Sun制备出。与其它量子点相比，碳点除具有优良的荧光性能外，还具有低分子量、粒径小、生物相容性好、无毒、对环境友好、良好的催化性等优点，且制备碳点的原料富足、价格低廉。
贵金属纳米团簇的催化性能是一个很有前途的应用。例如，金最初被认为是具有催化惰性的，但纳米级别的金目前已经被证明在一个广泛的化学反应中具有良好的催化活性。金纳米簇的另一个重要的应用就是作为过氧化物人工模拟酶，由于天然酶存在着稳定性差和催化活性的灵敏度容易受环境影响等缺陷，人们慢慢致力于人工酶模拟的研究，金属纳米簇拥有与天然酶相似的内在酶活性，因其超微小的体积、低毒等优点在分子成像、生物传感、医药以及催化领域有着潜在的应用价值。
目前，前列腺癌已超过皮肤癌成为男性最常见的癌症，在癌致死死亡因素中排名第二。前列腺癌抗原在大多数有临床意义的前列腺癌病人血清中都会升高，也是其最重要的早期检测指标。因此，快速而准确地检测前列腺癌抗原对临床医学具有非同寻常的意义。
发明内容
本发明的目的之一在于提供一种新型的双色荧光含金碳点的制备，该双色荧光含金碳点的制备，包括如下步骤：
(1)将氯金酸溶液和谷胱甘肽溶液混合，并用纯净水将混合溶液稀释；
(2)将上一步所得混合溶液在80～100℃油浴条件下边搅拌边加热，加热时间1.5～2h，趁热将其转移到盛有葡萄糖的锥形瓶中，家用微波炉高火档加热20～25min，生成黄褐色产物；
(3)将生成的黄褐色产物用纯净水分散、离心，将上清液透析24h，即得纯净的含金碳点分散液。
具体的，步骤(1)所述氯金酸溶液和谷胱甘肽溶液分别是5mL、20mM的氯金酸溶液和1.5mL、100mM的谷胱甘肽溶液，所述用纯净水将混合溶液稀释是稀释到50mL；步骤(2)所述葡萄糖为2g。
本发明的双色荧光含金碳点的制备方法操作简单，现象明显，材料易得。通过这种双色荧光可进行生物细胞的双色标记；基于这种含金碳点具有过氧化物酶的催化性能，可实现对前列腺癌抗原(PSA)的可视化检测。
本发明的目的之二在于提供上述双色荧光含金碳点在可视化检测的应用，它包括如下步骤：
(1)基于巯基和金之间的键合作用，以链端修饰巯基的单链DNA适配体对含金碳点分散液进行修饰；
(2)基于适配体和前列腺癌抗原特异性作用对催化位点的掩盖，以3,3',5,5'-四甲基联苯胺即TMB和H2O2为显色底物，在TMB和H2O2量一定时，加入和前列腺癌患者血清作用后的修饰了单链DNA适配体的含金碳点分散液，适配体和前列腺癌抗原的特异性作用会掩盖含金碳点表面的部分催化位点，掩盖的量和前列腺癌抗原的量有关，检测蓝色氧化态TMB的生成含量，以此来检测前列腺癌抗原的含量。
具体的，步骤(1)所述以链端修饰巯基的单链DNA适配体对含金碳点分散液进行修饰的过程是：将30μL、0.1μM的链端修饰巯基的单链DNA适配体加入到3mL含金碳点分散液中，反应24h后，再进行透析以除去未反应的DNA适配体。
具体的，步骤(2)所述和前列腺癌患者血清作用后的修饰了单链DNA适配体的含金碳点分散液的过程是：将前列腺癌患者血清稀释105～106倍加入到1～10mg/L的修饰了DNA适配体的含金碳点分散液中充分反应30min。
本发明的双色荧光含金碳点在可视化检测的应用方法，基于含金碳点的过氧化物模拟酶性质，通过DNA适配体和前列腺癌抗原的特异性作用对双色荧光含金碳点催化表面位点的覆盖来检测前列腺癌抗原患者血清中前列腺癌抗原的含量，实现对前列腺癌抗原的快速可视化检测。该方法快速简便、易于操作、灵敏度高，在临床医学具有重要意义。
附图说明
图1是本发明实施例1中含金碳点的紫外-可见吸收光谱图。
图2是本发明实施例1中含金碳点的荧光光谱图，其中，曲线a为荧光激发光谱图，曲线b为荧光发射光谱图。
图3是本发明实施例1中含金碳点的TEM图。
图4是本发明实施例1中含金碳点的能谱分析图。
图5、图6、图7、图8是本发明实施例2中含金碳点乳腺癌细胞中激光共聚焦荧光显微镜双色成像图；其中，图5为含金碳点在细胞中绿色荧光成像，图6为含金碳点在细胞中的红色荧光成像，图7为细胞的明场成像，图8为图5、图6、图7合并后图。
图9、图10是本发明实施3中含金碳点与适配体结合前、后的zeta点位图。
图11是本发明实施3中适配体和适配体-含金碳点的圆二色谱图；图中，曲线1是适配体的圆二色谱图，曲线2是适配体-含金碳点的圆二色谱图。
图12、图13分别是本发明实施3中，不同浓度的PSA加入到TMB、H2O2和修饰DNA适配体的含金碳点中表现出的颜色渐变图和相应的可见吸收光谱图。
图14、图15、图16是本发明实施4中，改变含金碳点的量达到调控不同浓度前列腺癌抗原的可视化检测图。
具体实施方式
下面结合附图和实施例对本发明作进一步详细的描述。以下实施例旨在说明本发明而不是对本发明的进一步限定。
实施例1：
含金碳点的制备及成分分析。将5mL、20mM新配置的氯金酸溶液和1.5mL、100mM的谷胱甘肽溶液混合，用超纯水将其稀释到50mL。溶液在80℃油浴条件下边搅拌边加热，加热时间2h，趁热将其转移到盛有2g葡萄糖的锥形瓶中，家用微波炉高火档加热20min。将生成的黄褐色产物用50mL超纯水再次分散、离心(16,000转/分钟，30min)，将上清液进行透析(截留分子量为8000～14000)24h，即可得到纯净的含金碳点分散液。用紫外-可见分光光度计和荧光分光光度计测得含金碳点的紫外-可见光谱图和荧光光谱图，如图1、图2所示；并对其进行TEM表征，如图3所示，可知含金碳点的大小在2nm左右，又对含金碳点颗粒进行了元素分析，结果如图4所示。从图中可知：这种含金碳点由碳和金元素组成(谱图中的铜来自实验中铜网中的铜)。
实施例2：
含金碳点在细胞中双色成像。取含金碳点分散于水中，配制成5mg/mL的分散液，然后取1mL含金碳点分散液和1mL乳腺癌的磷酸缓冲溶液，再加9mL细胞培养液，于37℃培养4小时，再用激光共聚焦荧光显微镜成像，选用405nm激发波长，得到细胞的双色成像，如图5、图6、图7、图8所示。
实施例3：
PSA的可视化检验：将30μL、0.1μM的链端修饰巯基的单链DNA适配体加入到3mL实施例1制备的含金碳点分散液中，反应24h后，透析(截留分子量8000～14000)，以除去未反应的DNA适配体。由于DNA带负电荷，含金碳点的zeta电位明显降低，如图9、图10所示；图11所示的圆二色光谱的变化进一步说明了DNA成功连接到含金碳点表面。取10μL不同浓度的PSA(1，5，10，50，100，500，1000ng/mL)加入到90μL连接DNA适配体的含金碳点分散液中，37℃反应2h，再加入100μL、5mM的TMB和100μL、400μM的H2O2反应15min，出现随浓度变化的颜色梯度变化，如图12所示；并检测其紫外-可见吸收光谱，如图13所示。
实施例4：
改变体系中含金碳点的量来调控不同浓度PSA的可视化检测。将已知前列腺癌抗原浓度的前列腺癌患者血清稀释，使得前列腺癌抗原浓度分别为49和50ng/mL、19和20ng/mL、9和10ng/mL。将10μL含49和50ng/mL的PSA血清加入到90μL、0.138mg/mL的含金碳点分散液中，同样，将含19和20ng/mL、9和10ng/mL的PSA血清分别加入到浓度为0.0579mg/mL和0.0264mg/mL的含金碳点分散液中，37℃反应2h后再加入到100μL、5mM的TMB和100μL、0.1M的H2O2混合液中反应15min，出现不同颜色，如图14、图15、图16所示。
Claims (5)
1.一种双色荧光含金碳点的制备，其特征在于包括如下步骤：
(1)将氯金酸溶液和谷胱甘肽溶液混合，并用纯净水将混合溶液稀释；
(2)将上一步所得混合溶液在80～100℃油浴条件下边搅拌边加热，加热时间1.5～2h，趁热将其转移到盛有葡萄糖的锥形瓶中，家用微波炉高火档加热20～25min，生成黄褐色产物；
(3)将生成的黄褐色产物用纯净水分散、离心，将上清液透析24h，即得纯净的含金碳点分散液。
2.根据权利要求1所述双色荧光含金碳点的制备，其特征在于：步骤(1)所述氯金酸溶液和谷胱甘肽溶液分别是5mL、20mM的氯金酸溶液和1.5mL、100mM的谷胱甘肽溶液，所述用纯净水将混合溶液稀释是稀释到50mL；步骤(2)所述葡萄糖为2g。
3.一种如权利要求1所述双色荧光含金碳点在可视化检测的应用，其特征在于包括如下步骤：
(1)基于巯基和金之间的键合作用，以链端修饰巯基的单链DNA适配体对含金碳点分散液进行修饰；
(2)基于适配体和前列腺癌抗原特异性作用对催化位点的掩盖，以3,3',5,5'-四甲基联苯胺即TMB和H2O2为显色底物，在TMB和H2O2量一定时，加入和前列腺癌患者血清作用后的修饰了单链DNA适配体的含金碳点分散液，适配体和前列腺癌抗原的特异性作用会掩盖含金碳点表面的部分催化位点，掩盖的量和前列腺癌抗原的量有关，检测蓝色氧化态TMB的生成含量，以此来检测前列腺癌抗原的含量。
4.根据权利要求3所述双色荧光含金碳点在可视化检测的应用，其特征在于：步骤(1)所述以链端修饰巯基的单链DNA适配体对含金碳点分散液进行修饰的过程是：将30μL、0.1μM的链端修饰巯基的单链DNA适配体加入到3mL含金碳点分散液中，反应24h后，再进行透析以除去未反应的DNA适配体。
5.根据权利要求3所述双色荧光含金碳点在可视化检测的应用，其特征在于：步骤(2)所述和前列腺癌患者血清作用后的修饰了单链DNA适配体的含金碳点分散液的过程是：将前列腺癌患者血清稀释105～106倍加入到1～10mg/L的修饰了DNA适配体的含金碳点分散液中充分反应30min。
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