Novel Method Based on Colloidal Gold for Detection of Oligonucleotide Aptamer with Protein Interacting†

doi:10.7503/cjcu20140951

Abstract

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

CLC Number:

TrendMD:

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^†[J]. Chem. J. Chinese Universities, 2015, 36(4): 638.

Figures/Tables 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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