基于纳米金复合探针-蛋白芯片检测心肌损伤标志物

doi:10.7503/cjcu20150133

高等学校化学学报 ›› 2015, Vol. 36 ›› Issue (9): 1687.doi: 10.7503/cjcu20150133

基于纳米金复合探针-蛋白芯片检测心肌损伤标志物

黄丽君^1,², 张丽华¹(), 毛红菊²(), 王萍², 蒋友旭¹, 李盼盼¹, 金庆辉², 赵建龙²

1. 郑州大学第二附属医院, 郑州 450014
2. 中国科学院上海微系统与信息技术研究所, 传感技术联合国家重点实验室, 上海 200050

收稿日期:2015-02-06 出版日期:2015-09-10 发布日期:2015-07-17
作者简介:联系人简介: 张丽华, 女, 教授, 主要从事冠心病及相关防治方面的研究. E-mail:zlhxp@126.com; 毛红菊, 女, 博士, 研究员, 主要从事生物传感器及纳米技术在重大疾病诊断治疗方面的研究. E-mail:hjmao@mail.sim.ac.cn
基金资助:
国家“九七三”计划项目(批准号: 2012CB933303)和上海市科学技术委员会生物医药项目(批准号: 12441902600)资助

Detection of Myocardial Injury Markers Based on Double Gold Nanoparticles Probes and Protein Chip^†

HUANG Lijun^1,², ZHANG Lihua^1,^*(), MAO Hongju^2,^*(), WANG Ping², JIANG Youxu¹, LI Panpan¹, JIN Qinghui², ZHAO Jianlong²

1. The Second Affiliated Hospital of Zhengzhou University, Zhengzhou 450014, China
2. Shanghai Institute of Microsystem and Information Technology, State Key Laboratories of Transducer Technology,Chinese Academy of Science, Shanghai 200050, China

Received:2015-02-06 Online:2015-09-10 Published:2015-07-17
Contact: ZHANG Lihua,MAO Hongju E-mail:zlhxp@126.com;hjmao@mail.sim.ac.cn

摘要/Abstract

摘要：

运用纳米金复合探针结合蛋白芯片, 建立了一种检测心肌损伤标志物的新方法. 构建了2种纳米金探针: 标记有检测抗体和DNA探针1的检测探针和标记有DNA探针2(与DNA探针1的碱基互补配对)的信号探针. 当目的抗原存在时, 检测探针经检测抗体和抗原, 与芯片上捕获抗体结合固定在芯片上, 信号探针通过碱基互补配对与检测探针结合使信号放大, 最后利用纳米金成核原理染色, 通过显微镜观察结果并定量分析. 该体系在40 min内可检测多种标志物, 其中肌钙蛋白Ⅰ(cTnⅠ)的检出限为10 pg/mL, 与临床电化学发光法(ECLIA)灵敏度相当; 肌红蛋白(MYO)与新型脂肪酸结合蛋白(HFABP)的检出限分别为640和10 pg/mL, 与ECLIA及酶联免疫吸附法(ELISA)相比, 灵敏度显著提高.

关键词: 纳米金探针, 蛋白芯片, 杂交, 心肌损伤标志物

Abstract:

A novel method based on gold nanoparticles(AuNPs) probes and protein chip was developed to detect myocardial injury markers. Two AuNPs probes were constructed: one was modified with detection antibody and DNA probe 1(detection probe), and the other was labeled with DNA probe 2 which was complementary to DNA probe 1(signal probe). When target antigen was present, a sandwich structure(capture antibody-target antigen-detection probe-signal probe) was formed through the interaction of the antibody-antigen and the hybridization of complemenatry DNAs. Then the chip was dyed by the gold deposition solution, and the signal was amplified. The results were observed with a microscope linked with an image analysis software. This system could simultaneously detect multiple markers in 40 min. The detection limit of cTnⅠ was 10 pg/mL, which was equivalent to ECLIA in clinical. For HFABP and MYO, the detection limit could be as low as 640 and 10 pg/mL, respectively, and the sensitivity was improved greatly when compared with ECLIA and ELISA.

Key words: Gold nanoparticles probe, Protein chip, Hybridization, Myocardial injury marker

中图分类号:

O657.39

TrendMD:

黄丽君, 张丽华, 毛红菊, 王萍, 蒋友旭, 李盼盼, 金庆辉, 赵建龙. 基于纳米金复合探针-蛋白芯片检测心肌损伤标志物. 高等学校化学学报, 2015, 36(9): 1687.

HUANG Lijun, ZHANG Lihua, MAO Hongju, WANG Ping, JIANG Youxu, LI Panpan, JIN Qinghui, ZHAO Jianlong. Detection of Myocardial Injury Markers Based on Double Gold Nanoparticles Probes and Protein Chip^†. Chem. J. Chinese Universities, 2015, 36(9): 1687.

图/表 10

Scheme 1 Schematic of the detection of myocardial injury markers based on double gold nanoparticle probes and protein chip

Fig.1 TEM images of bare AuNPs(A) and AuNPs modified with detection antibodies and DNA probes(B)

Fig.2 UV-Vis spectra of AuNPs and AuNPs probesa. AuNPs; b. signal probe; c. cTnⅠ detection probe;d. HFABP detection probe; e. MYO detection probe.

Fig.3 Electrophoresis results of AuNPs before and after modificationLane 1: HFABP detection probe; lane 2: cTnⅠ detection probe; lane 3: MYO detection probe; lane 4: AuNPs; lane 5: signal probe; lane 6: DNA probe(25 nt); lane 7: DNA ladder(20 bp).

Fig.4 Optimization results for the amount of detection probe(A) and signal probe(B)

Fig.5 Detection results of protein chips before and after amplification(A1—A3) Detection results of signal AuNPs probe; (B1—B3) detection results of double AuNPs probes hybridization.(A1), (B1) blank control; (A2), (B2) 1 ng/mL; (A3), (B3) 32 ng/mL.

Fig.6 Specificity of myocardial injury markers detected by the developed protein chip(A) Blank control; (B) specific test of HFABP; (C) specific test of cTnⅠ; (D) specific test of MYO.

Fig.7 Standard curves of HFABP(a),cTnⅠ(b) and MYO(c)R2: a. 0.995; b. 0.994; c. 0.990.

Table 1 Comparision between ECLIA and protein chip for the detection of cTnⅠ and MYO in serum

Method	AMI(n=42)				UA(n=30)				Normal control(n=30)
	cTnⅠ		MYO		cTnⅠ		MYO		cTnⅠ		MYO
	+	-	+	-	+	-	+	-	+	-	+	-
Protein chip	31	11	37	5	6	24	15	15	1	29	7	23
ECLIA kit	30	12	35	7	6	24	16	14	1	29	6	24
χ²	0.060		0.389		0		0.067		0		0.098
P	0.807		0.533		1.000		0.796		1.000		0.754

Table 2 Comparision between protein chip and ELISA for the detection of HFABP in serum

Method	AMI(n=42)		UA(n=30)		Normal control(n=30)
	HFABP		HFABP		HFABP
	+	-	+	-	+	-
Protein chip	39	3	9	21	3	27
ELISA kit	36	6	11	19	4	28
χ²	0.498		0.300		0.097
P	0.480		0.584		0.756

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基于纳米金复合探针-蛋白芯片检测心肌损伤标志物

Detection of Myocardial Injury Markers Based on Double Gold Nanoparticles Probes and Protein Chip^†

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基于纳米金复合探针-蛋白芯片检测心肌损伤标志物

Detection of Myocardial Injury Markers Based on Double Gold Nanoparticles Probes and Protein Chip†

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Detection of Myocardial Injury Markers Based on Double Gold Nanoparticles Probes and Protein Chip^†