Ratiometric Imaging of the in Situ pH Distribution of Biofilms by Use of Fluorescent Mesoporous Silica Nanosensors
Ratiometric Imaging of the in Situ pH Distribution of Biofilms by Use of Fluorescent Mesoporous Silica Nanosensors
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DOI：
10.1021/acsami.9b09978
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发表时间：
2019-09-11
影响因子：
9.5
通讯作者：
Casey, Eoin
中科院分区：
文献类型：
--
作者：
Fulaz, Stephanie;Hiebner, Dishon;Casey, Eoin
生物膜是由微生物群落组成的，这些微生物被包裹在自身产生的细胞外聚合物基质中。虽然生物膜的顽固性和持久性是由多种因素造成的，但抗菌敏感性的降低与生物膜结构内pH梯度的产生密切相关。嵌入生物膜内的细胞会形成局部酸性微环境，该环境不受外部pH的影响。因此，pH监测是理解三维异质生物膜复杂性的一种有前景的方法。通过合成介孔二氧化硅纳米粒子（直径为47±5纳米），并将其与对pH敏感的染料（荧光素）和对pH不敏感的染料（罗丹明B）作为内标（染料 - 介孔二氧化硅纳米粒子）结合，设计了一种荧光pH纳米传感器。当pH从8.5降至3.5时，荧光素的荧光强度（I - F）显著降低。相反，罗丹明B的荧光强度（I - R）在任何pH下都保持恒定。在4.5到7.5的工作pH范围内，I - F / I - R的比值相对于pH产生了一条S形曲线。染料 - 介孔二氧化硅纳米粒子能够测量荧光假单胞菌WCS 365生物膜微菌落内的pH梯度。生物膜显示出空间上不同的低pH区域，这些区域被封闭在与高细胞密度区域相对应的大簇中。还存在一些由于传质效应而在整个微菌落中不明显扩散的小的低pH区域。在微菌落内核中检测到的最低pH为5.1，朝着微菌落外部逐渐增加到中性pH。染料 - 介孔二氧化硅纳米粒子能够完全穿透生物膜基质，并能够对整个生物膜内的pH梯度和分布进行定量比率分析，且该分析与纳米粒子浓度无关。
Biofilms are communities of microorganisms enclosed in a self-generated matrix of extracellular polymeric substances. While biofilm recalcitrance and persistence are caused by several factors, a reduction in antimicrobial susceptibility has been closely associated with the generation of pH gradients within the biofilm structure. Cells embedded within the biofilm create a localized acidic microenvironment, which is unaffected by the external pH. Therefore, pH monitoring is a promising approach for understanding the complexities of a three-dimensional heterogeneous biofilm. A fluorescent pH nanosensor was designed through the synthesis of mesoporous silica nanoparticles (47 +/- 5 nm diameter) conjugated to a pH-sensitive dye (fluorescein) and a pH-insensitive dye (rhodamine B) as an internal standard (dye-MSNs). The fluorescence intensity of fluorescein (I-F) reduced significantly as the pH was decreased from 8.5 to 3.5. In contrast, the fluorescence intensity of rhodamine B (I-R) remained constant at any pH. The ratio of I-F/I-R produced a sigmoidal curve with respect to the pH, in a working pH range between 4.5 and 7.5. Dye-MSNs enabled the measurement of pH gradients within Pseudomonas fluorescens WCS 365 biofilm microcolonies. The biofilms showed spatially distinct low-pH regions that were enclosed into large clusters corresponding to high-cell-density areas. Also present were small low-pH areas that spread indistinctly throughout the microcolony caused by the mass transfer effect. The lowest detected pH within the inner core of the microcolonies was 5.1, gradually increasing to a neutral pH toward the exterior of the microcolonies. The dye-MSNs were able to fully penetrate the biofilm matrix and allowed a quantitative ratiometric analysis of pH gradients and distribution throughout the biofilm, which was independent of the nanoparticle concentration.