Novel Aptamer-based Enzyme-linked Assay for the Detection of ATP

doi:10.7503/cjcu20140129

Abstract

Abstract:

A novel aptamer-based enzyme-linked assay for the detection of adenosine triphosphate(ATP) was established based on the specific recognition of aptamer towards target and the efficient catalytic ability of horseradish peroxidase(HRP). Aptamer binding with targets caused that duplex DNA containing short chain DNA and aptamer dissociated and dissociated DNA was captured by another DNA immobilized on other plate through hybridization. A fluorescein isothiocyanate(FITC) was tagged on dissociated DNA, a HRP as signal transduction element was linked on microplate through specific interactions with anti-FITC-HRP and catalyzed methyenedianiline(TMB) substrate to produce color change. The detection of ATP was achieved by color change and absorbance change at 450 nm. This method has good selectivity for ATP recognition interfering from other proteins such as GTP, UTP and CTP and the detection can be carried in complex sample such as 10% and 50% serum. Experimental results show that there is a good linear relationship with ATP concentration ranged from 50 to 400 nmol/L, and the detection limit is 26 nmol/L.

Key words: Aptamer, Enzyme-linked assay, Adenosine triphosphate(ATP), Selectivity, Serum

CLC Number:

O657.1

TrendMD:

ZHAO Qiuling, LIU Lingling, YANG Lina. Novel Aptamer-based Enzyme-linked Assay for the Detection of ATP[J]. Chem. J. Chinese Universities, 2014, 35(6): 1161.

Figures/Tables 7

Table 1 DNA sequences used in this experiment

Name	DNA sequences(5'-3')
A1	Biotin-AGCT ACC TGG GGG AGT ATT GCG GAG GAA GGT
B	GTC ACC TTC CTC CGC AAT
B1	FITC-GTC ACC TTC CTC CGC AAT
C1	Biotin-ATT GCG GAG GAA GGT GAC

Fig.1 Sensing principle of aptamer based enzyme-linked assay

Fig.2 Fluorescence spectra of A1+B/SYBR Green I system upon analyzing different concentrations of ATPATP concentration/(nmol·L-1), a—g: 0, 50, 100, 150, 200, 250, 300. λex=497 nm.

Fig.3 Photograph of ATP concentration detection in Tris-HCl buffer

Fig.4 Corresponding calibration plot of A450 vs. ATP concentration(A) and linear part of the plot in a range of 0—400 nmol/L(B) The experiment was performed under optimal conditions. Error bars: SD, n=3.

Fig.5 Selectivity of the ELAA assay for ATP over CTP, GTP and UTP

Fig.6 Detection results of ATP in the complex sample systemsError bars: SD, n=3.

References 23

[1]	Tortorano A. M., Esposto M. C., Prigitano A., Grancini A., Ossi C., Cavanna C., Cascio G. L., J. Clin. Microbiol., 2012, 50, 1051—1053
[2]	Liao N. D., Lee W. Y., Cancer Cytopathology, 2012, 120(4), 269—275
[3]	Sapir A., Shalev A. H., Skalka N., Bronshtein A., Altstein M., Environmental Toxicology and Chemistry, 2013, 32(3), 585—593
[4]	Irengeand L. M., Gala J. L., Appl. Microbiol. Biotechnol., 2012, 93, 1411—1422
[5]	Fan Z. Z., Xu J. F., Shen P. P., J. Anal. Sci., 2011, 27(7), 113—118
	(范祚舟, 徐加发, 沈萍萍.分析科学学报, 2011,27(7), 113—118)
[6]	Fang X., Tan W., Acc. Chem. Res., 2010, 43(1), 48—57
[7]	Tan X., Chen T., Xiong X., Mao Y., Zhu G., Yasun E., Li C., Zhu Z., Tan W., Anal. Chem., 2012, 84(20), 8622—8627
[8]	Li J., Fu H., Wu L., Zheng A., Chen G., Yang H., Anal. Chem., 2012, 84(12), 5309—5315
[9]	Zuo X., Song S., Zhang J., Pan D., Wang L., Fan C., J. Am. Chem. Soc., 2007, 129(5), 1042—1043
[10]	Baldrich E., Acero J. L., Reekmans G., Laureyn W., O’Sullivan C. K., Anal. Chem., 2005, 77(15), 4774—4784
[11]	Higuchi A., Siao Y. D., Yang S.T., Hsieh P.V., Fukushima H., Chang Y., Ruaan R.C., Chen W.Y., Anal. Chem., 2008, 80(17), 6580—6586
[12]	Sharma A. K., Kent A. D., Heemstra J. M., Anal. Chem., 2012, 84(14), 6104—6109
[13]	Nie J., Deng Y., Deng Q., Zhang D., Zhou Y., Zhang X., Talanta, 2013, 106, 309—314
[14]	Park H., Paeng I. R., Anal. Chim. Acta, 2011, 685(1), 65—73
[15]	Barthelmebs L., Jonca J., Hayat A., Beatriz P. S., Marty J. L., Food Control, 2011, 22(5), 737—743
[16]	Tian J., Wang Y., Chen S., Jiang Y., Zhao Y., Zhao S., Analyst, 2012, 137, 978—982
[17]	Han K., L., Lin Z., Li G., Electrochem. Commun., 2009, 11(1), 157—160
[18]	Zhang K., Zhu X., Wang J., Xu L. , Li G., Anal. Chem., 2010, 82(8), 3207—3211
[19]	Zhu X., Sun L., Chen Y., Ye Z., Shen Z., Li G., Biosensors and Bioelectronics, 2013, 47, 32—37
[20]	Zeng X., Zhang X., Yang W., Jia H., Li Y., Anal. Chem., 2012, 424, 8—11
[21]	Wang J., Wang L., Liu X., Liang Z., Song S., Li W., Li G., Fan C., Adv. Mater., 2007, 19, 3943—3946
[22]	Zhang L., Wei H., Li J., Li T., Li D., Li Y., Wang E., Biosensors and Bioelectronics, 2010, 25, 1897—1901
[23]	Zuo X., Song S., Zhang J., Pan D., Wang L., Fan C., J. Am. Chem. Soc., 2007, 129, 1042—1043