General Label-Free Fluorescent Aptamer Binding Assay Using Cationic Conjugated Polymers

Abstract

With more and more new aptamers being reported, a general, cost-effective yet reliable aptamer binding assay is still needed. Herein, we studied cationic conjugated polymer (CCP)-based binding assays taking advantage of the conformational change of aptamer after binding with a target, which is reflected by the fluorescence change of the CCP. Poly(3-(3′-N,N,N-triethylamino-1′-propyloxy)-4-methyl-2,5-thiophene hydrochloride) (PMNT) was used as a model CCP in this study, and the optimal buffer was close to physiological conditions with 100 mM NaCl and 10 mM MgCl2. We characterized four aptamers for K+, adenosine, cortisol, and caffeine. For cortisol and caffeine, the drop in the 580 nm peak intensity was used for quantification, whereas for K+ and adenosine, the fluorescence ratio at 580 over 530 nm was used. The longer stem of the stem-loop structured aptamer facilitated binding of the target and enlarged the detection signal. High specificity was achieved in differentiating targets with analogues. Compared with the SYBR Green I dye-based staining method, our method achieved equal or even higher sensitivity. Therefore, this assay is practicable as a general aptamer binding assay. The simple, label-free, quick response, and cost-effective features will make it a useful method to evaluate aptamer binding. At the same time, this system can also serve as label-free biosensors for target detection.

An RNA‐Cleaving DNAzyme That Requires an Organic Solvent to Function

“…To provide a proof of concept for the potential utility of E3 in sensing applications, we constructed a small-moleculeresponding aptazyme system from E3 and a well-studied DNA aptamer that binds adenine-containing molecules including adenosine, AMP, ADP and ATP. [19] E3 was first combined with the aptamer to create the aptazyme E3Apt; a regulatory oligonucleotide (RON) was then used to suppress the cleavage activity of E3Apt towards FRQ30 through the formation of a stable duplex (Figure 7A and Figure S22A), an aptazyme designing strategy previously reported by us. [20] Introduction of a cognate target for the aptamer should lead to the formation of target-aptamer complex, alleviating the suppression of RON and allowing the E3 element of the aptazyme to cleave its substrate.…”

Aptamers Targeting Membrane Proteins for Sensor and Diagnostic Applications

“…that effectively mediate interdisciplinary biological applications [175]. Optical reporting with aptamer-conjugated polymers is simple, cost-effective, can be fluorescence-or colorimetrybased, and is achievable in both a labeled and an unlabeled manner [176,177].…”

Surfactant-Assisted Label-Free Fluorescent Aptamer Biosensors and Binding Assays

“…Developing assays to study aptamer binding is of great importance to validate newly selected aptamers, characterize aptamer binding, and design biosensors [ 1 , 2 , 3 , 4 , 5 ]. Among the numerous aptamer binding assays [ 6 , 7 , 8 , 9 ], those based on DNA staining dyes such as SYBR Green I, thioflavin T, and thiazole orange have been very popular [ 10 , 11 , 12 , 13 , 14 , 15 ] due to their label-free nature and cost-effectiveness. In a typical assay, a dye and an aptamer are mixed, and their fluorescence is monitored as a function of the target analyte concentration.…”