PCR-Free Methodology for Detection of Single-Nucleotide Polymorphism with a Cationic Polythiophene Reporter

Abstract

This study presents a nonamplification-based nucleic acid assay for the detection of single-nucleotide polymorphism (SNP) associated with familial Mediterranean fever (FMF) besides polymerase chain reaction (PCR)-based methodologies. The major objective is to show the potential of the proposed assay for rapid screening of FMF in a Mediterranean region of 400 million population. The assay relies on binding difference of specially designed wild and mutant primers to the target genomic DNA, followed by determination of unbound primers by quick titration of a cationic polythiophene reporter. The fluorescent reporter exhibits signal transition from 525 to 580 nm in the presence of unbound primers, and it correlates the binding affinity of label-free primers to the homozygous wild and mutant genomes. As a proof of concept, 26 real samples are studied relying on the ON and OFF fluorescence signals of the cationic polythiophene reporter. The results are analyzed by principal component analysis (PCA), which provides clear separation of healthy and patient individuals. The further analysis by support vector machine (SVM) classification has revealed that our assay converges to 96% overall accuracy. These results support that the PCR-free nucleic acid assay has a significant potential for rapid and cost-effective screening of familial Mediterranean fever.

A Perspective on Polythiophenes as Conformation Dependent Optical Reporters for Label-Free Bioanalytics

“…In this study, the solution-based assay was translated to poly- 13). 88 This study described a solution based assay of polythiophene polyelectrolyte and target genes.…”

“…Monomeric thiophenes bearing different side chains have been mixed or copolymerized to display entirely novel properties and unique responses upon interactions with analyte molecules. The side chains with various functional groups and charge at the 3- and 4-positions of thiophene rings not only make the hydrophobic backbone soluble in polar solvents, but are also directly involved in both electrostatic and hydrophobic interactions with different analyte molecules. − …”

“…The diagnostic potential of PTs has been recently demonstrated by the detection of SNP associated with Familial Mediterranean Fever (FMF) with non-amplification-based nucleic acid assay (Figure ). This study described a solution based assay of polythiophene polyelectrolyte and target genes.…”

“…The fluorescent PTs are analyzed using principal component analysis (PCA), providing a clear separation of healthy and patient individuals. Reprinted with permission from ref . Copyright 2021 American Chemical Society.…”

The Soft Nanodots as Fluorescent Probes for Cell Imaging: Analysis of Cell and Spheroid Penetration Behavior of Single Chain Polymer Dots

“…The conjugated polymers and their nanoparticles have gained significant attention over the past decades due to their excellent photophysical and colloidal characteristics. [1][2][3][4][5][6][7][8][9] They underpin a vast range of implementation in biosensing, cell imaging, drug delivery, and photodynamic therapy. [10][11][12][13][14][15][16][17] The luminescence characteristic of organic nanoparticles relying on conjugated polymers enables tracking in inter-and intracellular milieu, by feature of enhanced photophysical phenomenon high brightness, extinction coefficients, quantum yield, and photostability.…”