Effect Of Zinc-doped Carbon Dots Combined With Blue Light Bacteria And Biofilm Formation

Background:Bacterial infection often leads to the occurrence of many serious diseases,which brings a huge public health burden.In particular,the increase of antibiotic resistance of pathogenic bacteria makes its effective prevention and treatment more difficult.Among them,the formation of bacterial biofilm and the difficulty of removing it are the main causes of bacterial resistance and persistent infection,which promotes the exploration of antibacterial strategies,such as: nanotechnology,photodynamic antimicrobial therapy and micro-electric technology.Photodynamic therapy is one of the ways to treat cancer,especially skin cancer.In recent years,this therapy has been applied to treat a variety of infectious diseases,including bacteria,also has been named photodynamic antimicrobial therapy.It is believed that the mechanism is that reactive oxygen species produced by laser-inspired photosensitizers act on the cell membrane,which destroys the biofilm structure and other functional units and causes microbial death or apoptosis.This unique antibacterial mechanism makes it difficult for bacteria to develop drug resistance.Recent studies have found that carbon dots,also known as carbon nanodots,are quantum dot-like nanomaterials.their optical properties and photocatalytic functions are similar to those of traditional nano-semiconductors.For example,effective photoinduced charge separation to form free radicals and anions and their radiative recombination can not only produce fluorescence emission,but also drive various catalytic processes with relatively strong photodynamic effects.These properties make carbon dots promising photodynamic antimicrobial therapy photosensitizers.furthermore,in recent years,research on the antibacterial effect of carbon dots has become more and more extensive.Some scholars have synthesized hybrid hydrogels composed of carbon dots,protoporphyrin and DNA,and found that it can kill gram-positive bacteria Staphylococcus aureus(S.aureus)by carbon dots energy transfer.Also some scholars have synthesized the composite of carbon dots,which can inhibit the growth of S.aureus and Escherichia coli(E.coli)when the blue light time reaches 60 min.Although the above studies have clarified the antibacterial properties of carbon dots,they also show shortcomings,such as high cytotoxicity,complex preparation methods,difficult access of raw materials and long illumination time.Therefore,for the first time,zinc-doped carbon dots with good water solubility,non-toxicity and photostability were prepared by simple and green hydrothermal method with citric acid,ethylenediamine and zinc acetate as carbon sources.Under the condition of blue-light-inspired zinc-doped carbon dots,the growth of S.aureus and the biofilm formation of S.aureus and Streptococcus mutans(S.mutans)were clearly defined,and the related mechanism was preliminarily discussed in order to provide theoretical basis for the application of zinc-doped carbon dots in this field.Methods:1.Preparation of zinc-doped carbon dotsZinc-doped carbon dots were prepared by one-step hydrothermal method and characterized by transmission electron microscope,fluorescence spectrum and Fourier infrared spectroscopy.2.The cell cytotoxicity of zinc-doped carbon dotsThe L929,MC3T3-E1 cell cultured in 96-well plates were divided into blank control group,and different concentrations of zinc-doped carbon dots solution(50?g.ml-1,75?g.ml-1,100?g.ml-1).After 24 h culture,the cytotoxicity of zinc-doped carbon dots was detected by CCK8 assay.3.Detection of antibacterial ability of zinc-doped carbon dotsStaphylococcus aureus(ATCC25923)and Streptococcus mutans(UA159)were selected as experimental strains.The bacteria which concentration were 1×106 cfu.ml-1 were co-cultured under the condition of carbon dots,blue light and combination,the number of bacteria in each group was detected by rapid spectrophotometry.4.Detection of biofilm formationS.aureus and S.mutans were cultured with zinc-doped carbon dots,blue light and combined application,the bacterial biofilm was detected by crystal violet staining after 48 h.5.The observation of Scanning electron microscopeS.aureus and S.mutans were co-cultured with zinc-doped carbon dots combined with blue light for 40 min.The untreated group set as blank control group.Then fixed with 2.5% glutaraldehyde solution after 4h and overnight at 4? condition.The samples were dehydrated by ethanol gradient,then treated with gold spray.The biofilm structure and morphology of bacteria were observed by Scanning electron microscope.6.The mechanism of antibacterial activityTo investigate whether to produce reactive oxygen species and its role in the bacteriostatic process by observing the effect of 100?g.ml-1 zinc-doped carbon dots on the growth and biofilm formation of S.aureus under 40 min of blue light after adding reactive oxygen scavenger acetylcysteine(N-acetyl-L-cysteine,NAC).Rapid spectrophotometry and plate colony count were used to detect the number of bacteria,and the amount of biofilm formation was detected by crystal violet staining.Results:1.TEM showed that the particle size of zinc-doped CDs was about 1.8 nm.Fluorescence spectra showed that the CDs had 342 nm excited light and 450 nm emission light.FT-IR showed that CDs had hydroxyl,carboxy,amino and other functional groups.2.CCK8 resultsThe results of CCK8 showed that when the concentration of carbon dots was 50?g.ml-1,75?g.ml-1,100?g.ml-1,the cytotoxicity are garde 1,showing low cytotoxicity of carbon dots.3.Antibacterial ability of zinc-doped carbon dots resultsZinc-doped carbon dots had no antibacterial effect on the two bacteria selected in the experiment,the growth of S.aureus was inhibited by blue light for 20 min,but it had no effect on S.mutans,the antibacterial effect of zinc-doped carbon dots was obvious combined with blue light,and the growth of S.aureus was inhibited by blue light for 10 min,but there was no obvious inhibition on the growth of S.mutans.4.Bacterial biofilm resultsZinc-doped carbon dots did not affect the formation of the two bacterial biofilms selected in the experiment,and inhibited the formation of the above two bacterial biofilms at 40 min of blue light,but there was obvious inhibition of biofilm formation when zinc-doped carbon dots combined blue light.5.Scanning electron microscope resultsThe combination of zinc-doped carbon dots and blue light can reduce the number of S.aureus,shrink the morphology of bacteria,and had no obvious biofilm structure,the number of S.mutans were reduced,also no complete biofilm structure.6.Antibacterial mechanism resultsAfter NAC treatment with reactive oxygen scavenger,the bacterial concentration,colony number and biofilm formation increased significantly compared with carbon dots group.It suggested that zinc-doped carbon dots may produce reactive oxygen species by photocatalytic reaction under blue light,thus inhibiting bacterial growth and biofilm formation.Conclusions:1.Zinc-doped carbon dots were prepared by hydrothermal method,which were high water solubility,good stability,excellent fluorescence properties and low toxicity;2.Zinc-doped carbon dots combined with blue light can inhibit the growth of S.aureus,and had no antibacterial effect on S.mutans,also can inhibit the biofilm formation of S.aureus and S.mutans;3.Zinc-doped carbon dots maybe produce reactive oxygen species through photocatalytic reaction under blue light,thus producing irreversible damage to bacteria.