基于Pd/COF-LZU1非标记型C-反应蛋白免疫传感器的研制

doi:10.7503/cjcu20150344

摘要/Abstract

摘要：

采用溶剂热法, 通过有机单体合成了一种亚胺键连接的共价有机框架材料(COF-LZU1); 在常温常压条件下, 通过后合成的方法将贵金属钯(Ⅱ)引入到COF材料中, 合成了复合材料Pd/COF-LZU1, 该材料具有优良的催化性能. 利用Pd/COF-LZU1多孔复合材料将C-反应蛋白(CRP)抗体(anti-CRP)固定在玻碳电极表面, 构建了一种非标记型CRP免疫传感器. 当抗体与抗原发生免疫反应时, 形成的免疫复合物会阻碍电化学探针[Fe(CN)₆]^4-/3-的电子传递, 降低其响应电流, 从而实现CRP的快速检测. 采用交流阻抗和差示脉冲伏安法(DPV)考察了免疫传感器的电化学特性, 同时考察了测试底液的pH值、抗原培育时间和抗体固定浓度等实验条件对传感器性能的影响. 在最优的实验条件下, 采用DPV法对CRP进行检测的线性范围为5~180 ng/mL, 检出限为1.66 ng/mL, 线性相关系数为0.992.

关键词: 钯负载共价有机框架材料, C-反应蛋白, 免疫传感器, 非标记型

Abstract:

A new imine-linked covalent organic framework(COF) material was synthesized using organic monomer via solvothermal methods. Pd(Ⅱ)-coordinated COF, Pd/COF-LZU1, was accordingly synthesized through simple post-treatment at ambient conditions, which showed excellent catalytic activity. Then, a label-free C-reactive protein(CRP) immunosensor was constructed using the Pd/COF-LZUI porous composite material to immobilize anti-CRP on the surface of GCE. When anti-CRP binds with CRP antigen, the formed immuno complex obstruct the electric signal of Fe(CN)₆^4-/3-, which can be used to realize label-free detection of CRP. Electrochemical impedence spectroscopy(EIS) and differential pulse voltammetry(DPV) were employed to characterize the immunosensor. The experimental conditions such as scanning rate, pH value of testing solution, the concentration of anti-CRP and incubation time were also optimized. Under the optimal experimental conditions, the linear range was 5—180 ng/mL, the detection limit was 1.66 ng/mL, with the linear correlation coefficient of 0.992. This immunosensor provides a novel and simple way to detect CRP, and develops the application of the COFs in the electrochemical immunosensor.

Key words: Pd/covalent organic frameworks(COFs), C-Reactive protein(CRP), Immunosensor, Label-free

中图分类号:

O657.1

TrendMD:

刘婷知, 夏介仁, 李瑶, 陈文凯, 张帅, 刘仪, 郑丽, 杨云慧. 基于Pd/COF-LZU1非标记型C-反应蛋白免疫传感器的研制. 高等学校化学学报, 2015, 36(10): 1880.

LIU Tingzhi, XIA Jieren, LI Yao, CHEN Wenkai, ZHANG Shuai, LIU Yi, ZHENG Li, YANG Yunhui. Preparation of Label-free C-Reactive Protein Immunosensor Based on the Palladium-coordinated Covalent Organic Frameworks(Pd/COF-LZU1) Material^†. Chem. J. Chinese Universities, 2015, 36(10): 1880.

图/表 14

Flow chart of the synthesis of COF-LZU1 and Pd/COF-LZU1

Scheme 2 Stepwise procedure of immunosensor

Fig.1 13C CP/MAS NMR spectrum of COF-LZU1

Fig.2 IR spectrum of COF-LZU1

Fig.3 PXRD patterns of COF-LZU1(a) and Pd/COF-LZU1(b)

Fig.4 TEM images of COF-LZU1(A, B) and Pd/COF-LZU1(C, D)

Fig.5 Differential pulse voltammetries(A) and electrochemical impedance spectroscopies(B) of different electrodesa. Pd/COF-LZU1/CHIT/GCE; b. Pd/CHIT/GCE; c. COF-LZU1/CHIT/GCE; d. bare GCE.

Fig.6 Electrochemical impedance spectroscopies(EIS) using different modified electrodes in 5 mmol/L [Fe(CN)6]4-/3- solutiona. Bare GCE; b. Pd/COF-LZU1/CHIT/GCE; c. anti-CRP/Pd/COF-LZU1/CHIT/GCE; d. CRP/anti-CRP/Pd/COF-LZU1/CHIT/GCE.

Fig.7 Electrochemical impedance spectroscopies of the immunosensor after incubated 40 μg/mL anti-CRP containing different concentrations of CRPConcentration of CRP/(ng·mL-1), a—i: 0, 5, 10, 20, 30, 60, 80, 100, 150.

Fig.8 CV curves of the immunosensor at various scan rates after incubated 40 ng/mL CRPScan rate/(mV·s-1), a—e: 20, 40, 60, 80, 100. Inset is plots of peak current vs. v1/2.

Fig.9 Influences of pH(A), concentration of anti-CRP(B)and CRP antigen incubation time(C) on the current responds of the immunosensor after incubated 40 ng/mL CRP

Fig.10 Calibration curve of immunosensor(A) and DPV response of different concentrations of CRP(B) Base solution: PBS(0.01 mol/L, pH=7.4) containing 0.5 mmol/L [Fe(CN)6]3-/4-.Concentration of CRP/(ng·mL-1): a—h. 0; 5; 30; 60; 90; 120; 150; 180.

Table 1 Selectivity of the immunosensor

Interferent	I_{interferent+CRP}/μA	I_CRP /μA	I_{interferent+CRP}/I_CRP
BSA	-3.748	-3.618	1.035
HCG	-3.821	-3.618	1.056
Gly	-3.501	-3.618	0.967
Glu	-3.815	-3.618	1.054

Table 2 Recovery of the immunosensor

Sample	Added/(ng·mL^-1)	Found/(ng·mL^-1)	Recovery(%)	Average recovery(%)
1	5.0	4.5	90.0
2	40.0	37.6	94.0	94.47
3	50.0	49.7	99.4

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