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.

pH-responsive nanoparticle (PRNP) is one of the most thoroughly studied encapsulation systems in the field of nanomedicine especially therapeutic approaches, due to their controllable reaction in the particular range of pH. Herein, we aim at utilizing PRNPs to enhance efficacy of Magnetic Nanoparticles PCR Enzyme-Linked Gene Assay (MELGA). MELGA is originally based on polymerase chain reaction (PCR) and has been reported as the advanced technique developed for the detection of target gene at femtogram level. With the coupling magnetic nanoparticles (MNPs) as product enriching unit with biotin as a signal reporting unit, MELGA technique showed significantly improved sensitivity and specificity when compared to the conventional PCR in the study of detection enterotoxigenic Escherichia coli (ETEC), the major cause of traveler’s diarrhea and diarrhea in children. Nevertheless, it is challenging to further modify MELGA for faster turnaround time by reducing steps in procedure. Therefore, this proposed technique called PRNP-modified MELGA has been invented by producing indicator loaded PRNPs to replace biotin in order to cut enzyme-substrate reaction off. In the present study, synthesized PRNPs are composed of mesoporous silica nanomaterials (MSNs) loaded with indicator throughout their mesoporous structure and coated with the pH-responsive polymer as “Gate Keeper” or outer shell molecule which is supposed to swell at pH of response and release indicator for reporting. The comparison of candidates for outer shell molecules between poly dopamine (PDA) and chitosan (CH) was carried out by characterizing the pH-responsive behaviors. Results show that at pH of response (pH = 1.2 and 2.2), PRNPs coated with CH exhibited shaper releasing behavior than that that of the one coated with PDA. It is highly anticipated that this developing technique will be a tool towards a successful molecular assay for various diagnosis.