Preparation of pH-Responsive Nanoparticles (PRNPs) for Detection of Pathogenic Escherichia coli from Stool Sample of Diarrheagenic Patients

摘要

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.