基于胶体金检测核酸适配子与蛋白相互作用的新方法

doi:10.7503/cjcu20140951

摘要/Abstract

摘要：

基于核酸适配子对靶蛋白的高特异性及胶体金比色法的高度灵敏性, 建立了一种分析核酸适配子与靶蛋白亲和力以及简便快速检测蛋白质的新方法. 核酸适配子保护的胶体金在高盐条件下可保持稳定, 靶蛋白与胶体金竞争结合适配子, 使胶体金发生聚沉, 呈现颜色变化, 可通过检测A₅₂₀值分析适配子与靶蛋白的亲和力以及蛋白浓度. 通过对适配子浓度、靶蛋白浓度、共孵育时间、竞争反应时间和氯化钠浓度等关键因素进行优化, 确定了最佳检测条件.

关键词: 核酸适配子, 靶蛋白, 胶体金, 亲和力

Abstract:

A novel method was developed for assaying affinity of aptamer with target protein and detecting the protein, which was based on the specific recognition of aptamers toward targets and high sensitivity of colloidal gold colorimetric. Oligonucleotide aptamer can protect colloid gold from coagulation in high salt conditions. When the aptamer bound to the target protein, the naked gold nanoparticles aggregated and the color of the solution changed. Then the affinity of target protein with aptamer and the concentration of target protein could be detected by monitoring A₅₂₀ value. The best detection system was established, while the principal influence factors, such as the amount of aptamer and target protein, incubation time, competitive reaction time and the concentration of sodium chloride, were optimized.

Key words: Oligonucleotide aptamer, Target protein, Colloidal gold, Affinity

中图分类号:

TrendMD:

刘中成, 赵丽健, 刘利鹏, 张艳芬, 王南南, 陈瑶, 王向欢. 基于胶体金检测核酸适配子与蛋白相互作用的新方法. 高等学校化学学报, 2015, 36(4): 638.

LIU Zhongcheng, ZHAO Lijian, LIU Lipeng, ZHANG Yanfen, WANG Nannan, CHEN Yao, WANG Xianghuan. Novel Method Based on Colloidal Gold for Detection of Oligonucleotide Aptamer with Protein Interacting^†. Chem. J. Chinese Universities, 2015, 36(4): 638.

图/表 18

Table 1 Factors and levels for L16(45) orthogonal array design

Level	Factor
Level	A1 concentration/ (μmol·L^-1)	Cε3-Cε4 concentration/ (μg·μL^-1)	Incubation time/min	Competitive time/min	NaCl concentration/ (mmol·L^-1)
1	0.8	0.01	80	40	300
2	0.9	0.02	90	50	350
3	1.0	0.03	100	60	400
4	1.2	0.04	110	70	450

Scheme 1 Schematic illustration of the AuNP-based colorimetric assay of target protein using the ssDNA aptamer

Fig.1 Different states of colloidal gold a. Steady state(wine red); b. coagulation state.

Fig.2 TEM images of AuNPs in steady state(A) and coagulation state(B)

Fig.3 UV-Vis spectra of AuNPs in different state of aggregation a. Steady state; b. coagulation state.

Fig.4 Optimization for the concentration of aptamer A1 Concentration of A1/(μmol·L-1), a—j: 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8.

Fig.5 Influence of the concentration of aptamer A1

Fig.6 Influence of the concentration of target protein Cε3-Cε4

Fig.7 Optimization for the concentration of Cε3-Cε4 Concentration of Cε3-Cε4/(μg·μL-1), a—i: 0, 0.02, 0.04, 0.06, 0.08, 0.10, 0.12, 0.14, 0.16.

Fig.8 Optimization for the NaCl concentration Concentration of NaCl/(mmol·L-1), a—l: 0, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550.

Fig.9 Influence of NaCl concentration

Fig.10 UV-Vis spectra of AuNPs after coagulation reaction in different NaCl concentrations

Fig.11 Influence of time a. Incubation time of aptamer A1 with colloidal gold;b. competition time of target protein with colloidal gold.

Fig.12 Optimization for the incubation time of aptamer A1 with colloidal gold Incubation time/min, a—g: 0, 30, 60, 90, 120, 150, 180.

Fig.13 Optimization for the time for target protein competition with colloidal gold Competition time/min, a—f: 0, 30, 60, 90, 120, 150.

Fig.14 Colour change of AuNPs in the orthogonal design experiment A520 value, a—p: 0.260, 0.408, 0.291, 0.199, 0.312, 0.194, 0.200, 0.322, 0.313, 0.288,0.379, 0.241, 0.216, 0.448, 0.243, 0.202.

Fig.15 Analysis of Cε3-Cε4(a) and thrombin(b) protein detection(A) and absorption spectra for Cε3-Cε4(B) (B) Control group was added NaCl directly.

Fig.16 Specificity of different proteins detection a. Cε3-Cε4; b. OVA; c. BSA; d. skim milk;e. negative control; f. no protein.

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