Experimental Study On The Detection Of Porphyromonas Gingivalis With Multifunctional Nanospheres

ObjectivesNaYF₄: Er Yb and Fe₃O₄ nanoparticles were prepared by high-temperature oil-phase method and chemical coprecipitation method respectively,then these two nanoparticles were modified with the monoclonal antibody of Porphyromonas gingivalis so as to achieve the purpose of enriching and identifying P.gingivalis.To provide a new idea for the application of upconversion and magnetic nanomaterials in the field of oral examination and diagnosis and the basis for detection of multiple periodontal pathogens simultaneously.Methods1.NaYF₄: Er Yb nanoparticles were prepared by high-temperature oil-phase method.The morphology,spectroscopy and surface charge of nanoparticles were characterized by of Transmission Electron Microscope?TEM?,fluorescence spectroscopy and Zeta potential meter,respectively.2.Fe₃O₄ nanoparticles were prepared by chemical coprecipitation method.The morphology and the magnetic properties of Fe₃O₄ were evaluated by TEM and vibrational sample magnetometer?VSM?,respectively.3.NaYF₄: Er@SiO₂ and Fe₃O₄@SiO₂ were prepared by encapsulating these nanoparticles with silica.The morphology,spectra and the magnetic intensity of nanospheres were characterized by TEM,fluorescence spectroscopy and VSM.4.NaYF₄:Er@SiO₂ and Fe₃O₄@SiO₂ were activated by N-hydroxysuccinimide and 1-?3-dimethylaminopropyl?-3-ethylcarbodiimide hydrochloride.Then these nanospheres were combined with antibodies,after that,incubated with P.gingivalis;the empty nanospheres were incubated with P.gingivalis as the control group to investigate the nonspecific adsorption of these nanospheres.5.After activated,NaYF₄:Er@SiO₂ and Fe₃O₄@SiO₂ were reacted with monoclonal antibodies?NaYF₄:Er@SiO₂-Ab,Fe₃O₄@SiO₂-Ab?and then incubated with P.gingivalis,Fusobacterium nucleatum?F.nucleatum?,Streptococcus mutans?S.mutans?and phosphate solution,respectively.6.NaYF₄:Er@SiO₂-Ab and Fe₃O₄@SiO₂-Ab were added to a bacteria solution of P.gingivalis,F.nucleatum and S.mutans to detect P.gingivalis.7.NaYF₄:Er@SiO₂-Ab and Fe₃O₄@SiO₂-Ab were added to district concentration of the P.gingivalis solutions?105,104,103,102,10 and 0 CFU/mL?to evaluate the sensitivity of this detection system.Results1.The average size of NaYF₄: Er Yb nanoparticles and NaYF₄:Er@SiO₂,with good dispersion,were about 30 nm and 100 nm,respectively.Fluorescence spectra showed that three peaks were observed at 519 nm,542 nm and 655 nm,and the peak intensity of NaYF₄: Er@SiO₂ was similar to NaYF₄: Er Yb nanoparticles.2.The average size of Fe₃O₄ and Fe₃O₄@SiO₂ were about 7 nm and 50 nm,respectively.The magnetic hysteresis curve at room temperature showed that the magnetic intensity of Fe₃O₄@SiO₂ was similar to Fe₃O₄ nanoparticles.3.Fluorescence microscopy showed that green fluorescence can be observed in conjugated antibody group alone.4.When excited by 980 nm NIR,there was no green fluorescence in the blank,F.nucleatum and S.mutans group,except the P.gingivalis group.5.After adding NaYF₄:Er@SiO₂-Ab and Fe₃O₄@SiO₂-Ab,green fluorescence could be observed under the fluorescence microscope after 980 nm NIR excitation.6.In addition to 0 CFU/m L group,other groups can be found green fluorescence under 980 nm NIR excitation after adding NaYF₄:Er@SiO₂-Ab and Fe₃O₄@SiO₂-Ab.Conclusions1.NaYF₄: Er Yb and Fe₃O₄ nanoparticles had good physical and chemical properties.After encapsulation with silica,the fluorescence intensity of NaYF₄: Er Yb and the magnetic properties of Fe₃O₄ were almost unchanged.2.Without conjugating with antibodies,the nanospheres would not capture any bacteria that means,the nanospheres have no nonspecific adsorption.3.This detection system can detect P.gingivalis in 10 CFU/mL bacteria solution,indicating that the sensitivity of this detection system is excellent.