Binding Studies of Cationic Conjugated Polymers and DNA for Label-Free Fluorescent Biosensors

Abstract

Cationic conjugated polymers (CCPs), especially polythiophene, have been extensively used as probes for developing DNA and aptamer-based biosensors. Although many interesting applications have been achieved, a fundamental understanding of this system remains quite limited. In this work, we performed systematic binding assays to understand the interactions between poly(3-(3′-N,N,N-triethylamino-1′-propyloxy)-4-methyl-2,5-thiophene) (PMNT) and DNA. The fluorescence of PMNT at 530 nm initially decreased and then a peak at 580 nm emerged after binding with single-stranded DNA (ssDNA). The binding force between PMNT and DNA was dominated by electrostatic interactions at first and then DNA base-mediated interactions also became important. Since the bases in double-stranded DNA (dsDNA) were shielded, their fluorescence changes were quite different. To best differentiate ssDNA and dsDNA, the optimal pH was between 6 and 8, and the optimal NaCl concentration was around 0.3 M. Moreover, by changing the sequence and length of ssDNA, poly-T had the largest fluorescence shift and poly-A had the smallest change. Under the optimized conditions, the PMNT-based biosensor had a detection limit of 1 nM DNA, which was similar to the SYBR Green I-based assay.

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

“…In this work, we chose a cationic polythiophene derivative, PMNT (Figure A), as a model CCP since it showed obvious fluorescence changes in response to the conformational change of DNA. , The average molecular weight of our PMNT was 38 kDa (132 thiophene monomer units) with a molecular weight distribution of 1.87 as characterized by gel permeation chromatography (Figure S1). We chose this length since it is much longer than most aptamers (typically between 20 and 60 nucleotides), ensuring more comparable results for different aptamers.…”

“…38,39 DNA hybridization can also be probed by CCP. 40,41 However, this method has only been applied in limited cases, such as the detection of metal ions or proteins. No work was performed on small-molecule binding aptamers, which are a major aptamer type.…”

“…Thymine-rich aptamers can fold DNA into a duplex through thymine–Hg 2+ –thymine interactions. Both reactions led to a conformational change of a CCP, resulting in a color or fluorescence change. , DNA hybridization can also be probed by CCP. , However, this method has only been applied in limited cases, such as the detection of metal ions or proteins. No work was performed on small-molecule binding aptamers, which are a major aptamer type.…”

Water-soluble polythiophene-based colorimetry for the quick and accurate detection of SARS-CoV-2 RNA

“…Cationic water-soluble poly [3-(3′-N,N,N-triethylamino-1′-propyloxy)-4-methyl-2,5-thiophene hydrochloride] (PMNT, Figure S1 ) has been reported as a DNA chromogenic reagent through electrostatic interaction [ 18 ]. The mechanism of the interactions between PMNT and DNA has been further explored recently [ 19 ]. The PMNT in the aqueous solution alone exhibits yellow color, denoting a less conjugated and random nonplanar coil shape.…”

Sequence-Defined Conjugated Oligomers in Donor–Acceptor Dyads

“…Conjugated polymers form the basis of numerous aspects of chemical and materials research including, but not limited to, organic electronic devices, − energy storage, − fluorescent biosensors and therapeutics, − and visible-light-driven photocatalysis. , The intense interest in these materials is due, in part, to their large degree of broadly tunable electronic and physical properties. While these properties are typically dictated by the identity of the initial monomer, they may also be influenced by the chain length, monomer sequence, and side chain composition. − However, the inherent disperse nature of polymers often prevents the development of clear structure–property relationships that are needed to further elucidate fundamental processes and advance the application of these materials.…”