Institute of Biochemistry and Physiology of Plants and Microorganisms, Saratov Scientific Centre of the Russian Academy of Sciences (IBPPM RAS), 410049 13 Prospekt Entuziastov, Saratov, 410049, Russia.
Institute of Biochemistry and Physiology of Plants and Microorganisms, Saratov Scientific Centre of the Russian Academy of Sciences (IBPPM RAS), 410049 13 Prospekt Entuziastov, Saratov, 410049, Russia.
Talanta. 2023 Nov 1;264:124773. doi: 10.1016/j.talanta.2023.124773. Epub 2023 Jun 9.
Bacterial biofilms colonize chronic wounds and surfaces of medical devices, thus making the development of reliable methods for imaging and detection of biofilms crucial. Although fluorescent identification of bacteria is sensitive and non-destructive, the lack of biofilm-specific fluorescent dyes limits the application of this technique to biofilm detection. Here, we demonstrate, for the first time, that fluorescent glutathione-stabilized gold nanoclusters (GSH-AuNCs) without targeting ligands can specifically interact with extracellular matrix components of Gram-negative and Gram-positive bacterial biofilms resulting in fluorescent staining of bacterial biofilms. By contrast, fluorescent bovine serum albumin-stabilized gold nanoclusters and 11-mercaptoundecanoic acid - stabilized gold nanoclusters do not stain the extracellular matrix of biofilms. According to molecular docking studies, GSH-AuNCs show affinity to several targets in extracellular matrix, including amyloid-anchoring proteins, matrix proteins and polysaccharides. Some experimental evidence was obtained for the interaction of GSH-AuNCs with the lipopolysaccharide (LPS) that was isolated from the matrix of Azospirillum baldaniorum biofilms. Based on GSH-AuNCs properties, we propose a new fluorescent method for the measurement of biofilm biomass with a limit of detection 1.7 × 10 CFU/mL. The sensitivity of the method is 10-fold higher than the standard biofilm quantification with the crystal violet assay. There is a good linear relationship between the fluorescence intensity from the biofilms and the number of CFU from the biofilms in the range from 2.6 × 10 to 6.7 × 10 CFU/mL. The developed nanocluster-mediated method of biofilm staining was successfully applied for quantitative detection of biofilm formation on urinary catheter surface. The presented data suggest that fluorescent GSH-AuNCs can be used to diagnose medical device-associated infections.
细菌生物膜定植于慢性创面和医疗器械表面，因此开发可靠的成像和检测生物膜的方法至关重要。虽然荧光鉴定细菌具有灵敏性和非破坏性，但缺乏生物膜特异性荧光染料限制了该技术在生物膜检测中的应用。在这里，我们首次证明，无靶向配体的荧光谷胱甘肽稳定的金纳米簇（GSH-AuNCs）可以与革兰氏阴性和革兰氏阳性细菌生物膜的细胞外基质成分特异性相互作用，从而导致细菌生物膜的荧光染色。相比之下，荧光牛血清白蛋白稳定的金纳米簇和 11-巯基十一酸稳定的金纳米簇不会对生物膜的细胞外基质进行染色。根据分子对接研究，GSH-AuNCs 显示出与细胞外基质中的多个靶标（包括淀粉样蛋白锚定蛋白、基质蛋白和多糖）的亲和力。我们获得了一些实验证据，证明 GSH-AuNCs 与从 Azospirillum baldaniorum 生物膜基质中分离出的脂多糖（LPS）相互作用。基于 GSH-AuNCs 的特性，我们提出了一种新的荧光方法来测量生物膜生物量，检测限为 1.7×10 CFU/mL。该方法的灵敏度比结晶紫法标准生物膜定量法高 10 倍。生物膜的荧光强度与生物膜的 CFU 数量之间存在良好的线性关系，范围为 2.6×10 至 6.7×10 CFU/mL。开发的基于纳米簇的生物膜染色方法成功地应用于定量检测尿导管表面的生物膜形成。所提出的数据表明，荧光 GSH-AuNCs 可用于诊断与医疗器械相关的感染。
