锌掺杂碳点联合蓝光照射对金黄色葡萄球菌生长及其生物膜形成的抑制作用

Inhibitory effects of zinc-doped carbon dots combined with blue light radiation on growth and biofilm formation of Staphylococcus aureus

目的:通过水热法合成锌掺杂碳点(CDs),观察锌掺杂CDs联合蓝光对金黄色葡萄球菌及其生物膜形成的抑制作用,并初步探讨相关机制。方法:采用水热法合成锌掺杂CDs,采用透射电子显微镜(TEM)、荧光光谱和傅立叶红外光谱仪(FT-IR)观察其表征。实验分为空白对照组、单独CDs溶液组、单独蓝光照射组和CDs+蓝光照射组。单独CDs溶液组细胞采用不同浓度(50、75和100 mg·L-1)CDs溶液处理,单独蓝光照射组采用蓝光照射10、20和40 min,CDs+蓝光照射组采用上述浓度CDs溶液处理后,按上述时间进行蓝光照射,空白对照组仅采用培养基处理后避光培养。CCK-8法检测各组L929和MC3T3-E1细胞增殖率。选取抑菌效果最佳组(100 mg·L-1 CDs组)采用N-乙酰半胱氨酸(NAC)处理清除活性氧,实验分为对照组、100 mg·L-1 CDs组、0.5 mmol·L-1 NAC组和0.5 mmol·L-1NAC+100 mg·L-1 CDs组,采用分光光度法测定各组菌液浓度,采用结晶紫法检测各组金黄色葡萄球菌生物膜形成量,采用平板菌落计数法记录各组菌落数。结果:TEM检测,成功合成粒径约1.8 nm的锌掺杂CDs。荧光光谱检测,CDs最佳激发波长为342 nm,最佳发射波长为440 nm,且具有激发依赖性。FT-IR检测,CDs具有羟基、羧基和氨基等官能团。CCK-8法检测,共培养24 h时,100 mg·L-1 CDs组L929和MC3T3-E1细胞增殖率均达80%。光照20和40 min时,与空白对照组比较,单独蓝光照射组菌液浓度降低(P<0.05或P<0.01),光照40 min时金黄色葡萄球菌生物膜形成量减少(P<0.01)。光照10 min时,与单独蓝光照射组比较,CDs+蓝光照射组菌液浓度和生物膜形成量均降低(P<0.01);与100 mg·L-1 CDs组比较,0.5 mmol·L-1NAC+100 mg·L-1 CDs组菌液浓度和生物膜形成量明显增加(P<0.01)。结论:锌掺杂CDs联合蓝光后通过光催化作用能有效抑制金黄色葡萄球菌的生长和生物膜形成。

Objective:To synthesize the zinc-doped carbon dots(CDs)by hydrothermal method and to observe the inhibitory effects of zinc-doped CDs combined with blue light on the formation of Staphylococcus aureus(S.aureus),and to explore the related mechanism.Methods:The zinc-doped(CDs)were synthesized by hydrothermal method,and the characteristics were observed by transmission electron microscope(TEM),fluorescence spectrometer and Fourier transform-infrared spectrometer(FT-IR).The experiment was divided into blank control group,CDs group,blue light radiation group,and CDs+blue light radiation group.The cells in CDs group were treated with different concentrations(50,75,100 mg·L-1)of CDs,the cells in blue light radiation group were irradiated with blue light for 10,20,and 40 min,the cells in CDs+blue light radiation group were treated with CDs combined with blue light,and the cells in blank control group were only treated by culture medium in the dark.CCK-8 assay was used to determine the proliferation rates of the L929 and MC3T3-E1 cells.The reactive oxygen species were scavenged by adding N-acetylcysteine(NAC)in the best bacteriostatic effect group(100 mg·L-1 CDs group),and the experiment was divided into contro group,100 mg·L-1 CDs group,0.5 mmol·L-1NAC group,and 0.5 mmol·L-1 NAC+100 mg·L-1 CDs group.The concentrations of the bacteria suspension in various groups were detected by spectrophotometer,the bacterial biofilm formation amounts of S.aureus in various groups were detected by crystal violet staining,and the plate count method was used to record the colony counts in various groups.Results:The TEM results showed that the particle size of the zinc-doped CDs constructed successfully was about 1.8 nm.The fluorescence spectra showed that the optimum excitation wavelength of CDs was 342 nm and the optimum emission wavelentgh was 450 nm.The FT-IR spectrum showed that CDs had hydroxyl,carboxy,amino and other functional groups.The CCK-8 assay results showed that after co-culture for 24 h,the proliferation rates of L929 and MC3T3-E1 cells in 100 mg·L-1 CDs group were up to 80%.Compared with blank control group,the concentrations of bacteria solution in blue light radiation for 20 and 40 min groups were decreased(P<0.05 or P<0.01),the biofilm formation amount of S.aureus was decreased after blue light radiation for 40 min(P<0.01).Compared with blue light radiation group,the concentration of bacteria solution and the biofilm formation amount of S.aureus in CDs+blue light radiation(10 min)group were decreased after blue light radition for 10 min(P<0.01).Compared with 100 mg·L-1 CDs group,the concentration of bacteria solution and the biofilm formation amount of bacteria in 0.5 mmol·L-1 NAC+100 mg·L-1 CDs group were increased(P<0.01).Conclusion:Zinc-doped CDs combined with blue light can inhibit the growth of S.aureus and the biofilm formation by photocatalysis effectly.