Competitive fluorescence anisotropy/polarization assay for ATP using aptamer as affinity ligand and dye-labeled ATP as fluorescence tracer

Abstract

A B S T R A C TWe developed an aptamer-based competitive fluorescence anisotropy (FA)/fluorescence polarization (FP) assay for adenosine triphosphate (ATP). Different from the traditional fluorescence polarization immunoassays for small molecules, here DNA aptamer against ATP was used as affinity ligand, and tetramethylrhodamine (TMR) labeled ATP served as fluorescent tracer. The binding between TMR-labeled ATP and aptamer gave large FA due to molecular volume increase and restricted rotation of the dye-labeled ATP. When ATP was added in solution, ATP competitively displaced the TMR-labeled ATP from aptamer affinity complex, causing decrease of FA of TMR-labeled ATP. The buffer containing MgCl 2 and incubation at low temperature were preferred for large FA change in the FA assay. The FA change was further enhanced in this competitive FA assay by increasing the molecular weight of aptamer through extension of aptamer sequences or conjugating streptavidin protein on aptamer. This method allowed for the detection of ATP in the range from 0.5 μM to 1 mM, generating the maximum FA change about 0.187 (corresponding maximum FP change about 0.242). The detection of ATP spiked in diluted urine or serum sample was achieved, showing capability for analysis in complex sample matrix. This assay also enabled the detection of the analogues of ATP, e.g. adenosine, adenosine monophosphate (AMP), and adenosine diphosphate (ADP) with similar sensitivity. This aptamer-based competitive FA assay takes advantages of aptamer in ease of synthesis, good thermal stability, and facile modulating the molecular mass of aptamer.

Recent advances in fluorescence anisotropy/polarization signal amplification

“…In addition, small molecules and metal ions also have been analyzed by a direct FA/FP format. 22,24,47–49 Likewise, the FA/FP variation experimentally observed is somewhat negligible. Through changing the structure of aptamer and the coupling mode of aptamer and fluorescent dye, some studies have been developed to overcome this constraint.…”

“…Significantly, streptavidin as a large-sized protein has been employed as an effective signal amplifier to detect small molecules 49,53 and analyze the DNA–protein interactions. 54 Unlike other FA/FP signal amplification strategies that use dual binding DNA probes, these methods implement signal amplification through the binding of streptavidin with biotinylated DNA.…”

Aptamer fluorescence anisotropy assays for detection of aflatoxin B1 and adenosine triphosphate using antibody to amplify signal change

“…The detection limit of ATP was 1 mM, which is comparable to that in some previous aptamer-based FP/FA assays for ATP (Table S2 †). [8][9][10]18,[30][31][32][33] The higher detection limits of our FA assays for ATP (1 mM) than that of our FA assay for AFB1 (25 pM) can be attributed to the lower affinity of aptamer against ATP 31 and high affinity of aptamer against AFB1. 24 This method was selective for ATP detection (Fig.…”

Liquid-to-gel transition for visual and tactile detection of biological analytes

“…A) Sensor design: DNA strands PxD1 and PxD2 bind two ATP molecules, 6 which stabilize a G-quadruplex structure. The G-quadruplex binds hemin (brown oval) and catalyzes H 2 O 2 -dependent oxidation of acetylacetone to acetylacetone radical, which then triggers polymerization of acrylamaide and bisacrylamide.…”

“…The sequence for ATP aptamer was isolated by Huizenga and Szostak. 6a The aptamer is known to bind two ATP molecules and was used in a number of assays for ATP detection. 6 In our design, the ATP aptamer sequence was split into two fragments, PxD1 and PxD2 (Figure 1A).…”