Rolling Circle Amplification-based Polyvalent Molecular Beacon Probe for Signal-amplifying and Sensitive-Detection of Thrombin †

doi:10.7503/cjcu20190450

Abstract

Abstract:

A long chain structure of DNA synthesized by rolling circle amplification(RCA) was used to turn on aptamer-based molecular beacons(MBs). Owing to the multiple complementary repeat units, the RCA scaffold can hybridize tens or hundreds of MBs to form polyvalent aptamer probes. The unfold ability and the fluorescence intensity of MBs were both improved by RCA compared to short single chains. Experimental results demonstrated that the system is significantly more effective than monovalent molecular beacon in targeting thrombin and signal sensitivity because of multivalent effects. Once the poly valent molecular beacon binding to the thrombin, there was a linear relationship between fluorescence signal and thrombin concentration, and the detection limit of thrombin concentration was 0.2 nmol/L. The establishment of this system is conducive to the realization of highly sensitive and specific detection of thrombin.

Key words: Rolling circle amplification, Polyvalent molecular beacon, Aptamer, Thrombin

CLC Number:

O657.3
O648

TrendMD:

Zhiqing ZHANG,Shanshan WANG,Zichen ZHANG,Jie MA,Xiufeng WANG,Ting ZHOU,Fang WANG,Guodong ZHANG. Rolling Circle Amplification-based Polyvalent Molecular Beacon Probe for Signal-amplifying and Sensitive-Detection of Thrombin ^†[J]. Chem. J. Chinese Universities, 2019, 40(12): 2465.

Figures/Tables 7

Name	DNA sequence(5'-3')	Source
Circle DNA1	TGTCTTCGCCTTCTTGTTTCCTTTCCTTGAAACTTCTTCCTTTCT-	TaKaRa Biotechnology(Dalian) Co., Ltd.
	TTCTTTCGACTAAGCACC
Primer DNA2	GGCGAAGACAGGTGCTTAGTC	TaKaRa Biotechnology(Dalian) Co., Ltd.
Repeated sequence of	GGTGCTTAGTCGAAAGAAAGAAAGGAAGAAGTTTCAAGGAA-	TaKaRa Biotechnology(Dalian) Co., Ltd.
RCA product	AGGAAACAAGAAGGCGAAGACA
MB	(FAM)GGAAGAAGTGGTTGGTGTGGTTGGCCCCTGTCTTCGC-	Sangon Biotechnology(Shanghai) Co., Ltd.
	CTTCTTGTTTCCTTTCCTTGAAACTTCTTCC(DABCYL)
FC short chain	TTTCTTTCTTTCGACTAAGCACC	Sangon Biotechnology(Shanghai) Co., Ltd.
63-cDNA	GGTGCTTAGTCGAAAGAAAGAAAGGAAGAAGTTTCAAGGAA-	Sangon Biotechnology(Shanghai) Co., Ltd.
	AGGAAACAAGAAGGCGAAGACA

Fig.1 Electrophoresis gel(A) and UV-Vis spectrum(B) of RCA products

Fig.2 Illustration of the signal-amplifying of molecular beacon(A) and poly(MB) detection of thrombin(B)

Fig.3 Fluorescence spectra(A) of different concentrations of FC and the signal-to-background ratio(S/B)(B) cMB=30 nmol/L. a. control; cFC:cRCA:cMB : b. 0:1:3; c. 1:1:3; d.10:1:3; e. 30:1:3; f. 100:1:3; g. 300:1:3; h.1000:1:3.

Fig.4 Fluorescence spectra(A, B) and relative fluorescence intensity ratios(C, D) under different conditions (A) a. control; cRCA:cMB : b. 1:100; c.1:30; d.1:10; e.1:5; f. 1:3; g. 1:1. cMB=30 nmol/L. (B) a. Control; c63-cDNA:cMB: b. 1:100; c.1:30; d.1:10; e.1:3; f. 1:1; g. 3:1; h. 10:1; i. 30:1; j. 100:1. cMB=30 nmol/L. (C) RCA-MB assembly and 63-cDNA-MB assembly with 30 nmol/L MB; (D) RCA-MB assembly with different concentrations of MB.

Fig.5 Detection sensitivity of MB-RCA for thrombin (A) Fluorescence spectra. cMB =100 nmol/L. cThrombin/(μmol·L-1): a. 0; b. 0.0001; c. 0.0003; d. 0.001; e. 0.003;f. 0.01; g. 0.03; h. 0.1; i. 0.3; j. 1. (B) Relative fluorescence intensity ratio vs. the concentration of thrombin.

Method	Linear range/(nmol·L^-1)	Detection limit/(nmol·L^-1)	Ref.
Electrochemical sensor based on aptamers	40—100	10	[26]
Aptamer biosensor based on guanine-quenching		0.81	[27]
Fluorescence detection based on molecular beacon	0—30	0.83	[23]
Fluorescence detection based on dual quenching molecular beacon	0.4—40	0.18	[28]
Polyvalent molecular beacon probe base on RCA	0.1—30	0.20	This work

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