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

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 & PRIME;- N,N,N-triethylamino-1 & PRIME;-propyloxy) -4-methyl-2, 5-thiop hene ) (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.

Automated summary

In this study, systematic binding assays were conducted to understand the interactions between PMNT and DNA, revealing that the fluorescence changes were driven by electrostatic interactions and DNA base-mediated interactions. The optimal conditions for distinguishing ssDNA and dsDNA were determined to be pH 6 to 8 and a NaCl concentration of approximately 0.3 M. By altering the sequence and length of ssDNA, different levels of fluorescence shift were observed, with poly-T showing the largest change and poly-A the smallest. The PMNT-based biosensor had a detection limit of 1 nM DNA under optimized conditions, comparable to the SYBR Green I-based assay.