MAGNETIC NANOPARTICLES STABILIZED WITH ENZYME-CONJUGATED POLYMER FOR SEPARATION AND DETECTION OF MUTANS STREPTOCOCCI

Abstract

This research aims to develop a simple, yet effective assay for mutans streptococci (Streptococcus mutans and Streptococcus sobrinus) detection based on a combination of magnetic separation and selective filtration. Magnetic nanoparticles (MNPs) synthesized by solvothermal process were first grafted with poly(acrylic acid) (PAA) bearing active carboxyl groups, readily available for conjugation with cell wall binding domain (CWBD) of automutanolysin that is specific to mutans streptococci. The functionalized MNPs were characterized by XRD, FT-IR, TEM, DLS, and TGA. To determine the bacteria binding, the CWBD-conjugated MNPs were mixed with bacteria solution and then isolated by a magnet. The isolated bacteria bound CWBD-conjugated MNPs were re-suspended and then passed through a membrane (pore size 0.8 µm) by vacuum filtration. The intensity of the colored spots on the membrane was found to increase linearly with concentration of both targeted bacteria, Mutans streptococci in a concentration range of 1x102 - 1x107 CFU/mL. The CWBD-conjugated MNPs were more specific to S.mutans and S.sobrinus with capture efficiency of 77 and 69%, respectively than non-targeted bacteria, S. salivarius and S. sanguinis with capture efficiency of 15 and 38%, respectively. Taking a benefit of peroxidase-like activity of MNPs, an addition of TMB and H2O2 on the spots on the membrane could amplify the signal and thus improve the detection limit of the assay for S. mutans detection from 20 to 8 CFU/mL. With the ease of operation, high sensitivity and specificity as well as the quick response time, the colorimetric assay employing CWBD-conjugated MNPs is a promising detection tool for mutans streptococci detection that may be implemented in clinical setting.