Aptamer-Conjugated Magnetic Nanoparticles For Targeting And Bioluminescent Detection Of Staphylococcus Aureus

Staphylococcus aureus(S.aureus),a common pathogen,is popular in food.Under certain conditions,S.aureus can produce high temperature resistance toxin,causing food poisoning and huge economic losses,thus posts great threats on human food security and economic development.However,regular methods for detection of S.aureus,such as the standard method,ELISA and PCR method,have shortcomings in complicated operation and low detection sensitivity,which can not meet the needs of modern rapid quantitative detection. Therefore, it is urgent to establish a rapid and effective method for adsorption and detection of S.aureus.In this paper,we proposed a sensitive ATP bioluminescence and ATP amplification assay for detecting S.aureus,which was captured by aptamer modified magnetic nanoparticles (Apt-MNPs).First,Fe3O4 magnetic nanoparticles (about 500 nm in diameter) were prepared by solvothermal method;then silica was coated on the surface followed by modifying amino groups to form amine-functionalized nanoparticles.After that, Apt-MNPs were fabricated by immobilizing a specific aptamer on the surface of amine-functionalized magnetic nanoparticles by biotin-avidin reaction. Apt-MNPs were applied to capture S.aureus and the capture rate was optimized.The results showed that:when 100 μL of Apt-MNPs (1 mg/mL) and 50 μL of S.aureus suspension (≤5×10 CFU/mL) were mixed and incubated at room temperature for 10 min, the capture rate of Apt-MNPs for S.aureus reached 99%.Then we used ATP bioluminescence assay to detect S.aureus enriched by Apt-MNPs and the ATP bioluminescence assay was optimized.The optimization results of ATP bioluminescence showed that:CTAB solution (0.02%) was used to release bacteria ATP;extraction time was 2 min;the volume ratio of bacteria to CTAB was 5:l;the pH value of Tris-HCl was 7.4;β-CD(0.008%) was used to embed the excess CTAB;ATP bioluminescence reaction system was consist of firefly luciferin solution (0.25 mg/mL) and luciferase solution(0.07 mg/mL).When S.aureus was captured by Apt-MNPs with 10-fold enrichment, and the above ATP bioluminescence assay showed a wide dynamic range from 2.5×102 CFU/mL to 2.5×106 CFU/mL (R2=0.953) and the detection limit was 70 cfu/mL.Finally, in order to further reduce the detection limit,we used ATP amplification technology for amplifying the ATP in low concentration of S.aureus captured by Apt-MNPs and made a reaction black box device connected to the mobile phone camera for ATP bioluminescence reaction.The mobile phone camera successfully captured the luminescence of ATP bioluminescence reaction with optimized conditions.The results showed that:when the reaction volume ratio of ATP solution (≥10-13M),firefly luciferase solution (1 mg/mL) and firefly luciferin solution (0.25 mg/mL) was 1:1:1,the mobile phone camera can captured the strongest luminescence;amplifying the ATP in low concentration of S.aureus captured by Apt-MNPs,when the ATP amplification time was 15 min,the mobile phone camera can captured the luminescence and the detection limit was 32 CFU/mL.