基于硫代黄素T诱导G-四联体构成的生物传感器检测铅离子

doi:10.7503/cjcu20190689

摘要/Abstract

摘要：

硫代黄素T(ThT)荧光分子在自由状态下荧光强度很弱, 通过在Tris-HCl缓冲液中加入Pb²⁺的适配体即富含G的DNA序列, 可与ThT荧光分子形成G-四联体结构, 使荧光信号迅速增强; 向溶液中加入Pb²⁺, Pb²⁺与其适配体有很好的结合特异性, 可生成更牢固的G-四联体结构, 使ThT分子被释放出来, 导致溶液的荧光强度降低, 基于此可检测溶液中的Pb²⁺离子. 实验中优化了缓冲溶液组成、 ThT荧光分子浓度、 Pb²⁺适配体浓度及反应时间等条件. 结果表明, 在10 mmol/L Tris-HCl(pH=8.3, 含2 mmol/L MgCl₂)缓冲溶液中, ThT荧光分子和Pb²⁺适配体的浓度分别为10 μmol/L和200 nmol/L, 反应10 min时, 随着溶液中Pb ²⁺浓度的增加, 荧光强度减弱. Pb²⁺浓度在20~1000 nmol/L范围内时, 荧光强度与Pb²⁺的浓度呈现良好的线性关系(R²=0.9941), 检出限为1 nmol/L. 实际水样测试结果表明, 该方法的回收率在98.8%~101.3%之间. 该传感器灵敏、快速、无需化学修饰荧光分子且成本低.

关键词: 硫代黄素T, G-四联体, 铅离子

Abstract:

The simple and rapid Pb²⁺ detection method is constructed by using a fluorescent aptasensor that is based on G-quadruplex formation in Tris-HCl buffer solution. In the absence of Pb²⁺, the Thioflavin T (ThT) dye binds to the G-rich Pb²⁺ aptamer and forms a Pb²⁺ aptamer/ThT G-quadruplex complex, which results in high fluorescence intensity. Upon addition of Pb²⁺, the Pb²⁺ aptamer/ThT complex will be replaced by the formation of a Pb²⁺ aptamer/Pb²⁺ complex and the fluorescence intensity of the solution decreases dramatically.The experimental conditions such as the buffer solution, the concentration of ThT fluorescent molecules, the concentration of Pb²⁺ aptamer, and the reaction time were optimized. When the concentrations of ThT fluorescent molecules and Pb²⁺ aptamers were 10 μmol/L and 200 nmol/L, respectively, the fluorescence intensity decreased with the increase of Pb ²⁺ concentration in 10 mmol/L Tris-HCl(pH=8.3, containing 2 mmol/L MgCl₂) buffer solution. When the concentration of Pb²⁺ was in the range of 20—1000 nmol/L, there was a good linear relationship between the fluorescence intensity and the concentration of Pb²⁺(R²=0.9941), and this fluorescence aptasensor was highly sensitive and rapid, with a detection limit of 1 nmol/L and a total reaction time of only 10 min. Furthermore, this method is cost-effective and simple, removing the requirement for labeling the detection reagents with a fluorophore-quencher pair.

Key words: Thioflavin T, G-quadruplex, Lead ion

中图分类号:

O655

TrendMD:

夏姣云,徐彤,卿静,熊艳,刘俊杰,龚福春. 基于硫代黄素T诱导G-四联体构成的生物传感器检测铅离子. 高等学校化学学报, 2020, 41(5): 1004.

XIA Jiaoyun,XU Tong,QING Jing,XIONG Yan,LIU Junjie,GONG Fuchun. Highly Sensitive Sensor for Lead Ion Based on Thioflavin T-induced G-quadruplex Formation ^†. Chem. J. Chinese Universities, 2020, 41(5): 1004.

图/表 8

Scheme 1 Scheme of the fluorescence Pb2+ aptasensor based on G-quadruplex formation

Fig.1 Fluorescence emission spectra of different samples in 10 mmol/L Tris-HCl buffer(pH=8.3) containing 2 mmol/L MgCl2 a. Only 10 μmol/L ThT; b. 10 μmol/L ThT+1 μmol/L Pb2+; c. 10 μmol/L ThT+100 nmol/L Pb2+aptamer; d. 10 μmol/L ThT+100 nmol/L Pb2+ aptamer+1 μmol/L Pb2+; e. buffer.

Fig.2 Optimization of experimental conditions (A) Effect of the concentration of Pb2+ aptamer(10 mmol/L Tris-HCl, pH=8.3, 2 mmol/L MgCl2, 10 μmol/L ThT+1 μmol/L Pb2+); (B) effect of the concentration of ThT(10 mmol/L Tris-HCl, pH=8.3, 2 mmol/L MgCl2, 200 nmol/L Pb2+ aptamer); (C) effect of incubation time on fluorescence reduction(10 mmol/L Tris-HCl, pH=8.3, 2 mmol/L MgCl2, 200 nmol/L Pb2+ aptamer+10 μmol/L ThT+1 μmol/L Pb2+).

Fig.3 Fluorescence emission spectra of ThT in the presence of increasing amounts of Pb2+ c(ThT): 10 μmol/L; c(Pb2+ aptamer): 200 nmol/L.

Fig.4 Fluorescence spectra of the proposed platform at 500 nm vs. Pb2+ concentration Inset: dependence of fluorescence intensity on the Pb2+ concentration.

Fig.5 Selectivity of the proposed method for Pb2+ detection Without(A) and with(B) masking reagent SCN- for Hg2+. The concentrations of the Pb2+ analogues were 1 μmol/L in the presence of 200 nmol/L Pb2+ aptamer and 10 μmol/L ThT, while the concentration of Pb2+ was 1 μmol/L. The blank was the sample without Pb2+ and only contains 200 nmol/L ATP aptamer and 10 μmol/L ThT.

Table 1 Comparison of analytical methods for the detection of Pb2+

Method	LOD/(nmol·L^-1)	Time/min	Ref.
Gold Nano Alloy	96	20	[15]
Phage-AuNPs	45	70	[16]
DNA aptamers	60.7	60	[17]
Gold nanoparticles	13	20	[18]
DNA aptamers	3.0	22	[19]
G-quadruplex	1.0	10	This work

Table 2 Determination of Pb2+ in real samples

Sample	Added/(nmol·L^-1)	Found/(nmol·L^-1)	Recovery(%)	Standard deviation (%, n=3)
Tap water 1	50.0	49.4	98.8	1.1
Tap water 2	100.0	99.4	99.4	1.6
Tap water 3	200.0	202.5	101.3	3.2

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