CN111830014B - 一种基于聚苯胺吸附双链dna的传感器检测dna的方法 - Google Patents
一种基于聚苯胺吸附双链dna的传感器检测dna的方法 Download PDFInfo
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Abstract
本发明涉及的是一种基于聚苯胺吸附双链DNA的化学发光传感器的制备方法及应用技术,属于化学发光传感领域。主要技术特征是:制备聚苯胺共价修饰的磁性氧化石墨烯、互补链DNA修饰的钴卟啉;并将其应用于化学发光传感器的制备,检测DNA的含量;以互补链DNA作为识别元件,双链DNA间的碱基互补配对作用提高了该传感器的选择性;以钴卟啉为化学发光催化剂,提高了该传感器的灵敏度;通过改变目标DNA和互补链DNA的序列,可以实现不同序列的目标DNA检测。
Description
技术领域
本发明涉及的是一种基于聚苯胺吸附双链DNA的化学发光传感器的制备方法及应用技术,属于化学发光传感领域,具体涉及聚苯胺共价修饰的磁性氧化石墨烯、互补链DNA修饰的钴卟啉的制备及在化学发光检测DNA中的应用。
背景技术
聚苯胺(PANI)是一种p型半导体,其特征在于通过含氮基团的芳香环连接而成,具有特殊的电学、光学性质,经掺杂后可具有导电性及电化学性能。经一定处理后,可制得各种具有特殊功能的设备和材料,如可作为生物或化学传感器的尿素酶传感器、电子场发射源、较传统锂电极材料在充放电过程中具有更优异的可逆性的电极材料、选择性膜材料、防静电和电磁屏蔽材料、导电纤维、防腐材料,等等。聚苯胺因其具有的原料易得、合成工艺简单、化学及环境稳定性好等特点而得到了广泛的研究和应用。据报道,在有过氧化氢和过氧化物酶的情况下,可以使得PANI轻松沉积到双链DNA上,而单链DNA则没有却不能,也就是说在一定条件下,可以利用聚苯胺对双链DNA进行选择性的吸附。
金属卟啉即卟吩及其衍生物(卟啉)与金属离子形成的配位化合物,是含四个吡咯分子的大环化合物,与生命科学关系密切,卟啉化合物广泛存在于自然界生命体中,具有特殊生理活性,对生命活动起着重要作用,如叶绿素、血红素、细胞色素P-450、维生素B12等都是生命体新陈代谢过程中至关重要的组成部分,被誉为生命色素。卟啉环的所有的原子都处在同一个平面上,具有11个双键的共轭体系,该特征使得卟啉具有特殊的化学性质,例如特 殊的紫外吸收峰以及刚性平面的分子识别能力,同时受共轭体系的影响,卟啉的化学性质比较稳定,卟啉衍生物一般具有较深的颜色。卟啉环的另一个结构特征是其空腔中心到四个氮原子的距离均为204 pm,该数值与第一过渡系的金属原子和氮原子的共价半径之和恰好匹配,故卟啉极易与过渡金属离子(M= 锰、铁、钴、镍、铜、锌等离子)形成稳定的金属配合物,即金属卟啉。卟啉化合物因其特殊的结构和生物活性被广泛应用于生物化学、分析化学、医学、合成化学、太阳能利用、特种材料、催化应用等领域。尤其金属卟啉类化合物因其高效的催化化学发光活性和独特的结构而吸引了科研工作者的兴趣。
本发明旨在制备一种基于聚苯胺吸附双链DNA的化学发光传感器,并将其应用于检测DNA的含量。首先制备聚苯胺共价修饰的磁性氧化石墨烯、互补链DNA修饰的钴卟啉;当不存在待检测的目标DNA时,备聚苯胺共价修饰的磁性氧化石墨烯不会固定单链的互补链DNA,经过磁铁磁性分离后,上清液中互补链DNA修饰的钴卟啉的含量不变,化学发光反应的催化性能不发生变化;当待检测的目标DNA存在时,与互补链DNA进行碱基互补配对,形成双链DNA,形成的双链DNA-卟啉钴会被固定在聚苯胺共价修饰的磁性氧化石墨烯表面,经过磁铁磁性分离后,上清液中互补链DNA修饰的钴卟啉的含量会减少,使得化学发光反应的催化性能降低,以此实现目标DNA的高灵敏、高选择性检测,发明了一种检测DNA含量的新方法。
发明内容
本发明的目的之一是提供一种聚苯胺共价修饰的磁性氧化石墨烯、互补链DNA修饰的钴卟啉的制备方法。
本发明的目的通过以下技术方案实现:
(1)聚苯胺共价修饰的磁性氧化石墨烯的制备:向100 mL 三口烧瓶中加入30 ~50 mL 1.0 ~ 2.5 mol/L的盐酸溶液,随后加入纯化后的苯胺1.0 ~ 2.5 g,再加入0.1 ~0.25 g磁性氧化石墨烯,在冰浴环境下磁力搅拌0.5 ~ 2 h,使其充分分散;缓慢滴加10 ~30 mL 1.0 ~ 2.5 mol/L的过硫酸铵溶液,滴加速度控制在0.08 ~ 0.12 mL/min,滴完之后继续在冰浴环境中反应1 ~ 2 h;用超纯水洗涤产物3 ~ 5 次,直至滤液为无色;产物用外磁场收集;放入50 ~ 80 ºC真空干燥箱进行干燥8 ~ 12 h;干燥后,研磨;
(2)互补链DNA修饰的钴卟啉的制备:移取1.0 ~ 2.5 mL 0.1 ~ 0.5 mmol/L的钴卟啉溶液放入5 mL离心管中,加入0.1 ~ 0.5 mL 50 ~ 80 umol/L的互补链DNA,室温下振荡孵化2 ~ 5 h;在8000 ~ 10000 rad/min的转速下离心分离3 ~ 5 min,倒掉上清液,以除去未反应的互补链DNA;将产物转移到25 mL的容量瓶中定容,储存于4 ºC的条件下备用。
本发明的另一个目的是将聚苯胺共价修饰的磁性氧化石墨烯、互补链DNA修饰的钴卟啉应用于化学发光传感器的制备,检测DNA的含量;当不存在待检测的目标DNA时,聚苯胺共价修饰的磁性氧化石墨烯不会固定单链的互补链DNA,经过磁铁磁性分离后,上清液中互补链DNA修饰的钴卟啉的含量不变,化学发光反应的催化性能不发生变化;当待检测的目标DNA存在时,与互补链DNA进行碱基互补配对,形成双链DNA,形成的双链DNA-卟啉钴会被固定在聚苯胺共价修饰的磁性氧化石墨烯表面,经过磁铁磁性分离后,上清液中互补链DNA修饰的钴卟啉的含量会减少,使得化学发光反应的催化性能降低,以此实现目标DNA含量的检测。
本发明的优点及效果是:
(1)本发明制备的一种基于聚苯胺吸附双链DNA的化学发光传感器,聚苯胺共价修饰的磁性氧化石墨烯的合成条件简单、形貌可控性强;
(2)本发明制备的一种基于聚苯胺吸附双链DNA的化学发光传感器,以互补链DNA作为识别元件,双链DNA间的碱基互补配对作用提高了该传感器的选择性;
(3)本发明制备的一种基于聚苯胺吸附双链DNA的化学发光传感器,以钴卟啉为化学发光催化剂,提高了该传感器的灵敏度;
(4)本发明制备的一种基于聚苯胺吸附双链DNA的化学发光传感器,通过改变目标DNA和互补链DNA的序列,可以实现不同序列的目标DNA检测。
附图说明
图1是聚苯胺共价修饰的磁性氧化石墨烯的扫描电镜表征图。
具体实施方式
实施例1
(1)聚苯胺共价修饰的磁性氧化石墨烯的制备:向100 mL 三口烧瓶中加入30mL1.0 mol/L的盐酸溶液,随后加入纯化后的苯胺1.0 g,再加入0.1 g磁性氧化石墨烯,在冰浴环境下磁力搅拌1 h,使其充分分散;缓慢滴加10 mL 1.0 mol/L的过硫酸铵溶液,滴加速度控制在0.08 mL/min,滴完之后继续在冰浴环境中反应1 h;用超纯水洗涤产物3次,滤液变为无色;产物用外磁场收集;放入50 ºC真空干燥箱进行干燥10 h;干燥后,研磨;
(2)互补链DNA修饰的钴卟啉的制备:移取1.0 mL 0.2 mmol/L的钴卟啉溶液放入5mL离心管中,加入0.1 mL 80 umol/L的互补链DNA,室温下振荡孵化2 h;在8000 rad/min的转速下离心分离5 min,倒掉上清液,以除去未反应的互补链DNA;将产物转移到25 mL的容量瓶中定容,储存于4 ºC的条件下备用。
实施例2
(1)聚苯胺共价修饰的磁性氧化石墨烯的制备:向100 mL 三口烧瓶中加入40mL1.5 mol/L的盐酸溶液,随后加入纯化后的苯胺1.5 g,再加入0.15 g磁性氧化石墨烯,在冰浴环境下磁力搅拌2 h,使其充分分散;缓慢滴加10 mL 2.5 mol/L的过硫酸铵溶液,滴加速度控制在0.1 mL/min,滴完之后继续在冰浴环境中反应1.5 h;用超纯水洗涤产物4 次,滤液为无色;产物用外磁场收集;放入60 ºC真空干燥箱进行干燥10 h;干燥后,研磨;
(2)互补链DNA修饰的钴卟啉的制备:移取2.5 mL 0.25 mmol/L的钴卟啉溶液放入5 mL离心管中,加入0.25 mL50 umol/L的互补链DNA,室温下振荡孵化4 h;在8000 rad/min的转速下离心分离5 min,倒掉上清液,以除去未反应的互补链DNA;将产物转移到25 mL的容量瓶中定容,储存于4 ºC的条件下备用。
实施例3
(1)聚苯胺共价修饰的磁性氧化石墨烯的制备:向100 mL 三口烧瓶中加入50mL2.5 mol/L的盐酸溶液,随后加入纯化后的苯胺2.5 g,再加入0.25 g磁性氧化石墨烯,在冰浴环境下磁力搅拌2 h,使其充分分散;缓慢滴加30 mL 2.5 mol/L的过硫酸铵溶液,滴加速度控制在0.12 mL/min,滴完之后继续在冰浴环境中反应2 h;用超纯水洗涤产物5 次,滤液为无色;产物用外磁场收集;放入80 ºC真空干燥箱进行干燥8 h;干燥后,研磨;
(2)互补链DNA修饰的钴卟啉的制备:移取2.5 mL 0.1 mmol/L的钴卟啉溶液放入5mL离心管中,加入0.2 mL80 umol/L的互补链DNA,室温下振荡孵化5 h;在10000 rad/min的转速下离心分离3 min,倒掉上清液,以除去未反应的互补链DNA;将产物转移到25 mL的容量瓶中定容,储存于4 ºC的条件下备用。
实施例4
将聚苯胺共价修饰的磁性氧化石墨烯、互补链DNA修饰的钴卟啉应用于化学发光传感器的制备,检测DNA的含量;当不存在待检测的目标DNA时,聚苯胺共价修饰的磁性氧化石墨烯不会固定单链的互补链DNA,经过磁铁磁性分离后,上清液中互补链DNA修饰的钴卟啉的含量不变,化学发光反应的催化性能不发生变化;当待检测的目标DNA存在时,与互补链DNA进行碱基互补配对,形成双链DNA,形成的双链DNA-卟啉钴会被固定在聚苯胺共价修饰的磁性氧化石墨烯表面,经过磁铁磁性分离后,上清液中互补链DNA修饰的钴卟啉的含量会减少,使得化学发光反应的催化性能降低,以此实现目标DNA含量的检测。
Claims (6)
1.一种基于聚苯胺吸附双链DNA的化学发光传感器检测DNA的方法,其特征在于,该方法具有以下检测原理:
当不存在待检测的目标DNA时,聚苯胺共价修饰的磁性氧化石墨烯不会固定单链的互补链DNA,经过磁铁磁性分离后,上清液中互补链DNA修饰的钴卟啉的含量不变,化学发光反应的催化性能不发生变化;当待检测的目标DNA存在时,与互补链DNA进行碱基互补配对,形成双链DNA,形成的双链DNA-卟啉钴会被固定在聚苯胺共价修饰的磁性氧化石墨烯表面,经过磁铁磁性分离后,上清液中互补链DNA修饰的钴卟啉的含量会减少,使得化学发光反应的催化性能降低,以此实现目标DNA含量的检测;
其中所述的聚苯胺共价修饰的磁性氧化石墨烯和互补链DNA修饰的钴卟啉的制备具有以下工艺步骤:
(1)聚苯胺共价修饰的磁性氧化石墨烯的制备:向100mL三口烧瓶中加入30~50mL 1.0~2.5mol/L的盐酸溶液,随后加入纯化后的苯胺1.0~2.5g,再加入0.1~0.25g磁性氧化石墨烯,在冰浴环境下磁力搅拌0.5~2h,使其充分分散;缓慢滴加10~30mL 1.0~2.5mol/L的过硫酸铵溶液,滴加速度控制在0.08~0.12mL/min,滴完之后继续在冰浴环境中反应1~2h;用超纯水洗涤产物3~5次,直至滤液为无色;产物用外磁场收集;放入50~80℃真空干燥箱进行干燥8~12h;干燥后,研磨;
(2)互补链DNA修饰的钴卟啉的制备:移取1.0~2.5mL 0.1~0.5mmol/L的钴卟啉溶液放入5mL离心管中,加入0.1~0.5mL 50~80umol/L的互补链DNA,室温下振荡孵化2~5h;在8000~10000rad/min的转速下离心分离3~5min,倒掉上清液,以除去未反应的互补链DNA;将产物转移到25mL的容量瓶中定容,储存于4℃的条件下备用。
2.根据权利要求1所述的一种基于聚苯胺吸附双链DNA的化学发光传感器检测DNA的方法,其特征是:以互补链DNA作为识别元件,双链DNA间的碱基互补配对作用提高了该传感器的选择性。
3.根据权利要求1所述的一种基于聚苯胺吸附双链DNA的化学发光传感器检测DNA的方法,其特征是:以钴卟啉为化学发光催化剂,提高了该传感器的灵敏度。
4.根据权利要求1所述的一种基于聚苯胺吸附双链DNA的化学发光传感器检测DNA的方法,其特征是:通过改变目标DNA和互补链DNA的序列,可以实现不同序列的目标DNA检测。
5.根据权利要求1所述的一种基于聚苯胺吸附双链DNA的化学发光传感器检测DNA的方法,其特征是:聚苯胺共价修饰的磁性氧化石墨烯的工艺步骤(1)中所述的纯化后的苯胺由质量分数75%~80%的苯胺经二次减压蒸馏提纯获得。
6.根据权利要求1所述的一种基于聚苯胺吸附双链DNA的化学发光传感器检测DNA的方法,其特征是:聚苯胺共价修饰的磁性氧化石墨烯的工艺步骤(1)中所述的聚苯胺共价修饰的磁性氧化石墨烯的合成条件简单、形貌可控性强,粒径范围为80nm~100nm。
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