MRAB OXA-23基因检测电化学DNA传感器的构建及初步评价

doi:10.3969/j.issn.1673-8640.2019.04.013

摘要/Abstract

摘要：

目的构建用于检测多重耐药鲍曼不动杆菌（MRAB）OXA-23突变基因的新型电化学DNA传感器。方法采用多分枝长针海胆形铂纳米树枝（Pt-LSSUs@PAA）修饰电化学DNA传感器界面,以六氨合钌（RuHex）为信号指示剂,通过差分脉冲伏安法检测传感器杂交前后与DNA骨架中带负电的磷酸基团结合的RuHex产生的电化学信号差异。用构建完成的电化学DNA传感器定量检测MRAB OXA-23突变基因。结果构建的电化学DNA传感器具有良好的稳定性和重现性,Pt-LSSUs@PAA的修饰能显著增大电极的有效面积,加快电极表面的电子传导速率,达到信号放大的目的。构建完成的电化学DNA传感器测定OXA-23浓度的线性范围为10 fmol/L ~10 nmol/L,检测限为3.3 fmol/L（信噪比为3）,且具有良好的特异性。结论构建的电化学DNA传感器可灵敏、特异地定量检测MRAB OXA-23基因。

关键词: OXA-23基因, 多重耐药鲍曼不动杆菌, 多分枝长针海胆形铂纳米树枝, DNA传感器

Abstract:

Objective To establish a new electrochemical DNA sensor for determining multidrug-resistant Acinetobacter baumannii（MRAB） OXA-23 gene. Methods Polyallylamine-functionalized Pt nanostructures with long-spined sea-urchin-like morphology（Pt-LSSUs@PAA） was used to modify electrochemical DNA sensor interface. RuHex was used as a signal indicator. Using the established electrochemical DNA sensor,the differential pulse voltammetry was used to determine the presence of negatively charged phosphorus-bound RuHex electrochemical signal differences generated by OXA-23 mutant gene to be determined quantitatively. Results The sensor had good stability and reproducibility. The modification of Pt-LSSUs@PAA can increase the effective area of electrode,speed up the electron conduction rate on the surface of electrode and achieve signal amplification. The linear range of OXA-23 was 10 fmol/L-10 nmol/L. The determination limit was 3.3 fmol/L（signal-to-noise ratio was 3）,and the sensor had good specificity. Conclusions The established sensor can achieve sensitive and specific quantitative determination of OXA-23 mutant gene.

Key words: OXA-23 gene, Multidrug-resistant Acinetobacter baumannii, Polyallylamine-functionalized Pt nanostructures with long-spined sea-urchin-like morphology, DNA sensor

中图分类号:

R446.1

郭宏波, 戴飘飘, 黄华亮, 郭彦伟. MRAB OXA-23基因检测电化学DNA传感器的构建及初步评价[J]. 检验医学, 2019, 34(4): 346-350.

GUO Hongbo, DAI Piaopiao, HUANG Hualiang, GUO Yanwei. Establishment on electrochemical DNA sensor for MRAB OXA-23 gene determination[J]. Laboratory Medicine, 2019, 34(4): 346-350.

图/表 7

图1 电化学DNA传感器构建示意图

图2 DNA传感器的检测原理示意图注：RuHex结合在双链DNA上,结合的钌离子为三价（Ru3+）,在-0.2 V电压下Ru3+转变为Ru2+,进而被Fe3+氧化为Ru3+,形成电流循环

图3 Pt-LSSUs@PAA的表征图谱注：（a）场发射扫描电子显微镜图;（b）透射电子显微镜图;（c）能量衍射光电子光谱元素组成图;（d）氮元素图;（e）铂元素图

图4 Pt-LSSUs@PAA 电化学表征注：（a）Pt-LSSUs@PAA在0.5 mol/L H2SO4溶液中的循环伏安图;铂纳米粒子;Pt-LSSUs@PAA;（b）5 mmol/L铁氰化钾溶液中裸玻碳电极、电化学DNA传感器、与目标DNA结合的电化学DNA传感器的循环伏安图;裸玻碳电极;电化学DNA传感器;与目标DNA结合的电化学DNA传感器;（c）电极构建过程电化学阻抗光谱图;Pt-LSSUs@PAA修饰电极;Pt-LSSUs@PAA修饰电极+DNA探针;电化学DNA传感器;与目标DNA结合的电化学DNA传感器

图5 实验条件的优化注：（a）Pt-LSSUs@PAA浓度;（b）探针的固定时间;（c）目标核酸与探针结合时间

图6 电化学DNA传感器检测OXA-23基因的性能评价注：（a）不同浓度OXA-23标准品的差分脉冲伏安法表征曲线;按箭头方向,OXA-23标准品浓度从下到上依次为10 fmol/L、100 fmol/L、1 pmol/L、10 pmol/L、100 pmol/L、1 nmol/L、10 nmol/L;（b）不同浓度OXA-23标准品的线性关系图;（c）DNA传感器的稳定性

表1 MRAB OXA-23基因实际样本检测

样本编号	OXA-23基因		回收率（%）	RSD（%）
样本编号	加入浓度（pmol/L）	回收浓度（pmol/L）	回收率（%）	RSD（%）
1	0.01	0.010 8	108.00	3.63
2	1.00	0.997 4	99.74	4.29
3	10.00	10.113 0	101.13	2.97

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