CN109239040A - 一种基于适体链-黑磷纳米片荧光能量共振转移的砷离子检测方法 - Google Patents

一种基于适体链-黑磷纳米片荧光能量共振转移的砷离子检测方法 Download PDF

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CN109239040A
CN109239040A CN201811171454.XA CN201811171454A CN109239040A CN 109239040 A CN109239040 A CN 109239040A CN 201811171454 A CN201811171454 A CN 201811171454A CN 109239040 A CN109239040 A CN 109239040A
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李晓春
李海琴
张校亮
于化忠
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Abstract

一种基于适体链‑黑磷纳米片荧光能量共振转移的砷离子检测方法，属于砷离子检测技术领域，可解决现有砷离子的检测步骤较复杂，费时费力，费用高的问题，该检测方法利用黑磷纳米片荧光淬灭的特性，结合黑磷纳米片与标记有荧光染料ROX的As3+适体链组合成的As3+探针，根据反应体系中荧光染料ROX的荧光强度恢复程度的变化对待测溶液中的As3+进行定量检测。该检测方法具有检测速度快、检测步骤简便、检测灵敏度高、选择性好等优点。

Description

一种基于适体链-黑磷纳米片荧光能量共振转移的砷离子检 测方法
技术领域
本发明属于砷离子检测技术领域，具体涉及一种基于适体链-黑磷纳米片荧光能量共振转移的砷离子检测方法。
背景技术
近年来随着工业化的发展，重金属污染越来越严重，尤其是砷的污染引起了人们的高度关注。砷是一种高度有毒的致癌物质，其中三价砷化合物比其他砷化合物毒性更强，三价砷的毒性大约是五价砷的60倍。砷污染分布广泛，据报道有二十多个国家遭受砷污染，由此引发了许多急性和慢性健康问题，如皮肤损伤，循环系统疾病等高风险的疾病。更严重的是，砷不能从体内除去，并积聚在人体组织中。因此，对环境中As3+的检测尤其重要。
已经有多种方法可以检测不同环境样品如空气，水，土壤和沉积物中的As3+。传统的检测As3+的方法有高效液相色谱法（HPLC）、原子荧光光谱法（AFS）、电感耦合等离子体原子发射光谱法（ICP-AES）、电感耦合等离子体质谱法（ICP-MS）等。虽然这些检测方法的检测精度较高，然而这些都是高度复杂的分析技术，需要在仪器测量之前进行长时间的样品制备过程，检测步骤较复杂，需要检测人员具备一定的专业知识，价格昂贵，费时费力。所以急需开发一些比较快速且简便的检测砷离子的方法。
2009年，Mina Kim等人（Arsenic Removal from VietnameseGroundwater UsingtheArsenic-Binding DNA Aptamer, Environ. Sci. Technol., 2009, 43, 9335–9340.）第一个利用SELEX的方法筛选出了As3+的含有100个碱基的适体链（5’-GGTAATACGACTCACTATAGGGAGATACCAGCTTATTCAATTTTACAGAACAACCAACGTCGCTCCGGGTACTTCTTCATCGAGATAGTAAGTGCAATCT-3’），并且该适体链还被用于去除越南地下水中的砷。随后，国内外许多课题组便开展了利用这个含有100个碱基的砷离子适体链实现了As3+的定量检测。Wu等人（Wu Y, LiuL, Zhan S, et al. Ultrasensitive aptamer biosensor for arsenic(III) detectionin aqueous solution based on surfactant-induced aggregation of goldnanoparticles[J]. Analyst, 2012, 137(18):4171-4178.）报道了一种基于比色和共振散射（RS）的生物传感器，通过砷离子结合适体，靶和阳离子表面活性剂之间的特殊相互作用，通过金纳米粒子（AuNPs）的聚集实现对水溶液中的As3+超灵敏检测。Song等人（Song L,Mao K, Zhou X, et al. A novel biosensor based on Au@Ag core-shellnanoparticles for SERS detection of arsenic (III)[J]. Talanta, 2016, 146:285-290.）通过合成Au @ Ag壳核纳米粒子，提出了一种基于SERS和As3+适体（100个碱基）的简单而新颖的方法，用于定量检测砷离子。
近年来，二维（2D）纳米材料黑磷（BP）因其特有的物理化学性质，以及在光学、能量转换、生物医学诊断等方面表现出的优异性能，得到了广大科研工作者的广泛研究。研究发现，黑磷具有良好的淬灭荧光的性能，且黑磷可以很好的与生物分子或待检无机离子的核酸适配体通过范德华力结合在一起，因此可以将黑磷与核酸适配体的结合物看作待测物的有效探针，实现待测物的定量检测。如闫武娟等人（闫武娟, 王秀翃, 姚倩,等. 基于黑磷荧光淬灭特性的肿瘤细胞探测[J]. 中国激光, 2018, 45(2).）利用黑磷纳米片可淬灭荧光染料荧光的这一特性，合成了可特异性识别人乳腺癌细胞MCF7的标记有荧光染料FAM的DNA适体，实现了人乳腺癌细胞的定量检测。Zhou等人（Zhou J, Li Z, Ying M, et al.Black phosphorus nanosheets for rapid microRNA detection[J]. Nanoscale, 2018,10, 5060-5064.）利用黑磷纳米片作为荧光淬灭材料，开发了一种快速检测microRNA的灵敏传感平台，生物传感器显示出对microRNA的良好线性响应，线性范围从10nM~1000nM。此外，该生物传感器还可以区分三核苷酸多态性。
发明内容
本发明针对现有砷离子的检测步骤较复杂，费时费力，费用高的问题，提供一种基于适体链-黑磷纳米片荧光能量共振转移的砷离子的检测方法。将待测溶液加入到含有ROX荧光染料标记的As3+适体链和黑磷纳米片分散液的反应体系中进行反应，在没有加入As3+时，标有ROX荧光的As3+适体链与黑磷纳米片通过范德华力结合在一起，ROX与黑磷纳米片发生能量共振转移使得ROX的荧光淬灭；加入As3+后，根据反应体系中荧光染料ROX的荧光强度恢复程度的变化建立荧光染料ROX的荧光强度的变化与As3+浓度的线性关系，实现了As3+的快速灵敏、选择性好的定量检测。
本发明采用如下技术方案：
一种基于适体链-黑磷纳米片荧光能量共振转移的砷离子的检测方法，包括如下步骤：
第一步，反应体系的制备，将黑磷纳米片分散液与ROX荧光染料标记的As3+适体链溶液在避光条件下进行混合，使用快速混匀器充分混匀，混匀后在避光条件下静置大约8~10min，使得ROX荧光染料的荧光淬灭，形成反应体系；
第二步，砷离子的检测，将不同浓度的砷离子溶液，加入到反应体系中反应后，通过荧光光谱仪检测608nm处的峰值变化，建立As3+浓度与608nm处荧光强度的线性关系，来对As3+进行定量检测。
第一步中，砷离子适体链的DNA序列为ATG CAA ACC CTT AAG AAA GTG GTC GTCCAA AAA ACC ATTG。
第一步中，黑磷纳米片分散液的浓度为100µg/mL，ROX荧光染料标记的As3+适体链的浓度为100nM。
第一步中，黑磷纳米片分散液与ROX荧光染料标记的As3+适体链溶液的体积比为5:1。
第二步中，砷离子溶液，加入反应体系中，反应温度为25~30℃，反应时间为30min。
本发明的有益效果如下：
本发明开发了一种基于适体链-黑磷纳米片荧光能量共振转移的砷离子的检测方法，利用黑磷纳米片的荧光淬灭性质，合成了可特异性识别As3+的核酸适配体，并在适体链上标记ROX荧光染料，建立ROX荧光强度与As3+浓度之间的线性关系，实现了As3+的定量检测。本检测方法简单快捷，可以实现“一步式”检测，检测结果只需观察ROX染料的发射峰信号的恢复值即可。
本发明提供了一种重金属As3+的定量检测方法，本发明的反应体系由100 µg/mL的黑磷纳米片分散液与100nM的标记有ROX荧光染料的As3+适体链溶液构成，二者的体积比为5:1。在没有加入As3+时，标有ROX荧光的As3+适体链与黑磷纳米片通过范德华力结合在一起，ROX与黑磷纳米片发生能量共振转移使得ROX的荧光淬灭；加入As3+后，根据反应体系中ROX荧光强度的变化建立ROX荧光强度的变化与As3+浓度的线性关系，实现了As3+的快速灵敏、选择性好的定量检测。在本发明中，（1）检测过程操作简单，可以实现一步式的反应，检测时间短，且成本较低；（2）黑磷纳米片对As3+适体链的吸附力强，可以有效的吸附更多的适体链，使得ROX的荧光强度有效的淬灭，提高检测灵敏度及检测范围。
附图说明
图1为本发明实施例中黑磷纳米片分散液浓度优化的光谱图及分散点图，其中，A为光谱图，B为分散点图；
图2为本发明实施例中加入不同浓度砷离子溶液后，ROX荧光染料608nm处发射光谱图和分散点图，其中，A为发射光谱图，B为分散点图。
具体实施方式
实施例1
一种基于适体链-黑磷纳米片荧光能量共振转移的砷离子的检测方法，该检测方法包括下步骤：
步骤一、As3+适体链溶液的配制：取原始浓度为100µM的标记有ROX染料的适体链溶液4µL，加入一定体积的超纯水配制成100nM的适体链溶液备用。
步骤二、黑磷纳米片分散液浓度的优化：取1mL原始浓度为200µg/mL的黑磷纳米片分散液四等份，分别加入不同体积的超纯水，配制成浓度分别为200µg/mL、100µg/mL、50µg/mL、25µg/mL和0µg/mL的黑磷纳米片分散液；取40µL不同浓度的黑磷纳米片分散液分别加入到200µL 100nM的As3+适体链溶液中，使用快速混匀器混匀，混匀后在避光条件下静置大约8~10 min，使得ROX荧光染料的荧光淬灭，如图所示，当黑磷纳米片分散液的浓度为100µg/mL时，ROX荧光染料的荧光淬灭率最高，所以优化后的黑磷纳米片分散液的浓度为100µg/mL。
步骤三、不同浓度As3+溶液的配制：称取1.7344 mg AsNaO2，加入20mL的超纯水，使其充分溶解配制成浓度为50 ppm的As3+溶液。将50ppm的As3+溶液加入不同体积的超纯水进行稀释，配制成浓度为200ppb、100ppb、50ppb、20ppb、10ppb、5ppb、2ppb、1ppb、0ppb的As3+溶液。
步骤四、As3+的定量检测：将上述已知浓度的As3+溶液加入到所述反应体系中，通过荧光光谱仪检测608nm处的峰值变化，建立As3+浓度与608nm处荧光强度的线性关系，来对As3+进行定量检测。

Claims (6)

1.一种基于适体链-黑磷纳米片荧光能量共振转移的砷离子的检测方法，其特征在于：将待测溶液加入到含有ROX荧光染料标记的As3+适体链和黑磷纳米片分散液的反应体系中进行反应，在没有加入As3+时，标有ROX荧光的As3+适体链与黑磷纳米片通过范德华力结合在一起，ROX与黑磷纳米片发生能量共振转移使得ROX的荧光淬灭；加入As3+后，根据反应体系中荧光染料ROX的荧光强度恢复程度的变化建立荧光染料ROX的荧光强度的变化与As3+浓度的线性关系，实现了As3+的快速灵敏、选择性好的定量检测。
2.一种如权利要求1所述的基于适体链-黑磷纳米片荧光能量共振转移的砷离子的检测方法，其特征在于：包括如下步骤：
第一步，反应体系的制备，将黑磷纳米片分散液与ROX荧光染料标记的As3+适体链溶液在避光条件下进行混合，使用快速混匀器充分混匀，混匀后在避光条件下静置大约8~10min，使得ROX荧光染料的荧光淬灭，形成反应体系；
第二步，砷离子的检测，将不同浓度的砷离子溶液，加入到反应体系中反应后，通过荧光光谱仪检测608nm处的峰值变化，建立As3+浓度与608nm处荧光强度的线性关系，来对As3+进行定量检测。
3.根据权利要求2所述的一种基于适体链-黑磷纳米片荧光能量共振转移的砷离子的检测方法，其特征在于：第一步中，砷离子适体链的DNA序列为ATG CAA ACC CTT AAG AAAGTG GTC GTC CAA AAA ACC ATTG。
4.根据权利要求2所述的一种基于适体链-黑磷纳米片荧光能量共振转移的砷离子的检测方法，其特征在于：第一步中，黑磷纳米片分散液的浓度为100µg/mL，ROX荧光染料标记的As3+适体链的浓度为100nM。
5.根据权利要求2所述的一种基于适体链-黑磷纳米片荧光能量共振转移的砷离子的检测方法，其特征在于：第一步中，黑磷纳米片分散液与ROX荧光染料标记的As3+适体链溶液的体积比为5:1。
6.根据权利要求2所述的一种基于适体链-黑磷纳米片荧光能量共振转移的砷离子的检测方法，其特征在于：第二步中，砷离子溶液，加入反应体系中，反应温度为25~30℃，反应时间为30min。
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