Comparison of turn-on and ratiometric fluorescent G-quadruplex aptasensor approaches for the detection of ATP
Two fluorescent aptasensor methods were developed for the detection of ATP in biochemical systems. The first method consisted of a label-free fluorescent “turn-on” approach using a guanine-rich ATP aptamer sequence and the DNA-binding agent berberine complex. In the presence of ATP, the ATP preferentially binds with its aptamer and conformationally changes into a G-quadruplex structure. The association of berberine with the G-quadruplex results in the enhancement of the fluorescence signal of the former. The detection limit of ATP was found to be 3.5 μM. Fluorescence, circular dichroism and melting temperature (Tm) experiments were carried out to confirm the binding specificity and structural changes. The second method employs the ratiometric fluorescent approach based on the Forster resonance energy transfer (FRET) for the detection of ATP using berberine along with a quencher (AuNRs, AgNPs) and a fluorophore (red quantum dots (RQDs), carbon dots (CDs)) labeled at 5′ and 3′ termini of the ATP-binding aptamer sequence. Upon addition of ATP and berberine, ATP specifically binds with its aptamer leading to the formation of G-quadruplex, and similarly, berberine also binds to the G-quadruplex. This leads to an enhancement of fluorescence of berberine while that of RQD and CDs were significantly quenched via FRET. The respective detection limits calculated were 3.6 μM and 3.8 μM, indicating these fluorescent aptasensor methods may be used for a wide variety of small molecules.
KeywordsAdenosine-5′-triphosphate FRET Gold nanorods Fluorescence Berberine Aptasensor
This work was financially supported by the Natural Sciences and Engineering Research Council of Canada.
Compliance with ethical standards
The human serum samples used were pooled human samples obtained under informed consent (Millipore). All experiments were performed in accordance with the policies of Carleton University.
Conflict of interest
The authors declare that they have no conflict of interest.
- 2.Zheng D, Seferos DS, Giljohann D a, Patel PC, Mirkin C a. Aptamer nano-flares for molecular detection in living cells. Nano Lett 2009;9(9):3258–3261.Google Scholar
- 9.Ellington A, Szostak JW. In vitro selection of RNA molecules that bind specific ligands. Nature. 1990;346:818–22.Google Scholar
- 16.Wang Y, Wang Y, Liu B. Fluorescent detection of ATP based on signaling DNA aptamer attached silica nanoparticles. Nanotechnology. 2008;19(41).Google Scholar
- 32.Ma Y, Ou TM, Tan JH, Hou JQ, Huang SL, Gu LQ, et al. Quinolino-benzo-[5, 6]-dihydroisoquindolium compounds derived from berberine: a new class of highly selective ligands for G-quadruplex DNA in c-myc oncogene. Eur J Med Chem. 2011;46(5):1906–13. https://doi.org/10.1016/j.ejmech.2011.02.020.CrossRefPubMedGoogle Scholar
- 35.Liu Y, Li B, Cheng D, Duan X. Simple and sensitive fluorescence sensor for detection of potassium ion in the presence of high concentration of sodium ion using berberine-G-quadruplex complex as sensing element. Microchem J. 2011;99(2):503–7. https://doi.org/10.1016/j.microc.2011.07.001.CrossRefGoogle Scholar
- 51.Leung KH, Lu L, Wang M, Mak TY, Chan DSH, Tang FK, et al. A label-free luminescent switch-on assay for ATP using a G-quadruplex-selective iridium(III) complex. PLoS One. 2013;8(10):1–7.Google Scholar