Solvent-sensitive nanoparticle-enhanced PCR assay for the detection of enterotoxigenic Escherichia coli

Stimulus-responsive nanoparticles are among the most utilized nanoscale materials in biomedical applications. As these nanoparticles exhibit a manipulable response to a particular stimulus, such as pH, heat, and organic solvent, they are potential signalling units in diagnostic assays. This study aims to enhance the limit of detection and reduce the turnaround time of magnetic nanoparticle polymerase chain reaction (PCR) enzyme-linked gene assay (MELGA), an advanced PCR-based technique termed the solvent-sensitive nanoparticle (SSNP)-enhanced PCR assay. This technique was proposed to detect pathogenic enterotoxigenic Escherichia coli (ETEC) through applying stimulus-responsive nanoparticles. The SSNPs were elaborated with three main components, including mesoporous silica nanoparticles as a structural unit, organic dye (Nile red) as a payload, and the corresponding organic solvent-sensitive polymer shell as "gatekeeper" (poly(maleic anhydride-alt-methyl vinyl ether, PMAMVE). A suitable organic solvent capable of inducing polymer swelling and dye dissolution was investigated by considering a solubility parameter. Using ethanol, the encapsulated Nile red can diffuse out of the SSNPs faster than other solvents and reach a constant concentration within 15 min. For the PCR inhibition study, various SSNPs concentrations (10-30 μg/reaction) were mixed with the ETEC gene and PCR reagent. The results showed that the particles in this concentration range did not inhibit PCR. By comparing the efficacy of conventional PCR, MELGA, and SSNP-enhanced PCR assay, the proposed technique showed a better detection limit than that of PCR, whereas that of MELGA was the lowest. Moreover, compared to MELGA or conventional PCR, this technique provided remarkably faster results in the postamplification process.