A Label-Free, Mix-and-Detect ssDNA-Binding Assay Based on Cationic Conjugated Polymers

Abstract

The accurate, simple, and efficient measurement of the concentration of single-stranded DNA (ssDNA) is important for many analytical applications, such as DNA adsorption, biosensor design, and disease diagnosis, but it is still a challenge. Herein, we studied a cationic conjugated polymer (CCP)-based ssDNA assay taking advantage of the obvious fluorescence change of CCPs upon binding ssDNA. Poly(3-(3′-N,N,N-triethylamino-1′-propyloxy)-4-methyl-2,5-thiophene hydrochloride) (PMNT) achieved an apparent dissociation constant (Kd) of 57 ± 4 nM for ssDNA, indicating a very high binding affinity between PMNT and ssDNA. This allowed us to develop a CCP-based ssDNA biosensor with a detection limit of 0.6 nM, similar to the fluorescence-dye-based method using SYBR Green I and SYBR Gold. Our CCP-based biosensor produced smaller differences among ssDNA samples with different base compositions. In addition, the existence of double-stranded DNA (dsDNA) at different concentrations did not interfere with the fluorescence of PMNT, indicating that our CCP-based biosensor was more suitable for the measurement of ssDNA. Compared with fluorescence-intensity-based quantification, our CCP system allowed ratiometric quantification, which made the calibration easier and more robust. We then applied our method to the quantification of ssDNA on AuNPs using both unmodified and thiolated ssDNA, and the accurate quantification of ssDNA was achieved without any fluorophore modification. This method provides an alternative approach for the measurement of ssDNA.

Sensing, Imaging, and Therapeutic Strategies Endowing by Conjugate Polymers for Precision Medicine

“…[ 82 ] The increase in conjugated length on the polymer backbone further causes absorbance to shift toward higher wavelengths, resulting in a color change. [ 83 ] Besides, conjugated polymer bind to dsDNA with negligible conformational changes, preserving the optical properties. [ 84 ] Typically, conjugated polymer‐assisted colorimetric detection is mainly applied to ssDNA, dsDNA, aptamers, SNPs, and pathogens.…”

π-Conjugated Polymer Nanoparticles from Design, Synthesis to Biomedical Applications: Sensing, Imaging, and Therapy

“…This has stimulated enormous research and development of highly fluorescent π-CP as an active material in light-emitting devices and as a sensing layer in biosensor devices [ 74 ]. Thus, various fluorescent biosensing materials have been developed based on fluorescent π-CP for sensing metal ions, small molecules to microorganisms and cells [ 75 , 76 , 77 ]. The detection mechanism could be achieved by two approaches, by quenching the fluorescence of the polymers after the reaction with the target or by the emission of fluorescence.…”

Catalytic Hairpin Assembly-Based Self-Ratiometric Gel Electrophoresis Detection Platform for Reliable Nucleic Acid Analysis

“…Recently, gel electrophoresis was employed in bioanalysis research to detect the reconfiguration of large DNA nanostructures, termed DNA nanoswitches [12][13][14][15]. The target-induced conformational change of DNA nanoswitches was applied in the detecting of miRNA [16,17], Zika virus [18], DNA [19][20][21], and more [22,23]. More recently, by combining the signal amplification feature of HCR with the CRISPR-Cas12a system, the Yigit group reported a reprogrammable gel electrophoresis-based approach for DNA virus detection [24].…”