Assessment of lectin-binding analysis for in situ detection of glycoconjugates in biofilm systems
Assessment of lectin-binding analysis for in situ detection of glycoconjugates in biofilm systems
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DOI：
10.1099/00221287-147-2-299
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发表时间：
2001-02-01
期刊：
影响因子：
2.8
通讯作者：
Lawrence, JR
中科院分区：
文献类型：
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作者：
Neu, TR;Swerhone, GDW;Lawrence, JR
利用完全水化的河流生物膜对作为环境微生物群落中胞外聚合物的一部分的胞外糖缀合物进行了凝集素结合分析的表征的评估。用一组荧光缀合凝集素对该方法的适用性进行了评价，结果表明，凝集素结合分析能够对生物膜群落中的糖缀合物进行染色。凝集素染色也证明了生物膜基质内的空间异质性。此外，如果选择合适的组合，两种甚至三种凝集素的应用是可能的。凝集素结合分析可以与一般核酸染色相结合，以收集核酸和糖缀合物信号。研究了孵育时间、凝集素浓度、荧光标记、碳水化合物抑制、添加顺序和凝集素相互作用的影响。发现20分钟的孵育时间足以完成凝集素结合。不可能确定单个凝集素的饱和浓度，因此使用标准浓度进行测定。碳水化合物抑制试验表明，异硫氰酸荧光素（FITC）标记的凝集素比异硫氰酸四甲基罗丹明（TRITC）或花青染料（CY 5）标记的凝集素具有更特异的结合特性。添加的顺序和性质的荧光缀合物也被发现影响凝集素的结合模式，因此，选择一组凝集素的研究EPS基质必须基于其在生物膜系统中的行为进行研究的全面评估。尽管有这种必要性，凝集素结合分析代表了检查完全水合生物膜中的糖缀合物分布的有价值的工具。因此，可以鉴定细胞外生物膜位置内的化学异质性，以检查作用（例如，吸附性质、微环境）。细胞-细胞外聚合物质相互作用）。
An assessment of lectin-binding analysis for the characterization of extracellular glycoconjugates as part of the extracellular polymeric substances in environmental microbial communities was performed using fully hydrated river biofilms, The applicability of the method was evaluated for single, dual and triple staining with a panel of fluor-conjugated lectins, It was shown that lectin-binding analysis was able to stain glycoconjugates within biofilm communities. Lectin staining also demonstrated spatial heterogeneity within the biofilm matrix. Furthermore, the application of two or even three lectins was possible if suitable combinations were selected, The lectin-binding analysis can be combined with general nucleic acid stains to collect both nucleic acid and glycoconjugate signals. The effects of incubation time, lectin concentration, fluor labelling, carbohydrate inhibition, order of addition and lectin interactions were studied. An incubation time of 20 min was found to be sufficient for completion of lectin binding. It was not possible to ascertain saturating concentration for individual lectins, therefore a standard concentration was used for the assay, Carbohydrate inhibition tests indicated that fluorescein isothiocyanate (FITC)-conjugated lectins had more specific binding characteristics than tetramethyl rhodamine isothiocyanate (TRITC)- or cyanine dye (CY5)-labelled lectins. The order of addition and the nature of the fluor conjugate were also found to influence the binding pattern of the lectins, Therefore the selection of a panel of lectins for investigating the EPS matrix must be based on a full evaluation of their behaviour in the biofilm system to be studied. Despite this necessity, lectin-binding analysis represents a valuable tool to examine the glycoconjugate distribution in fully hydrated biofilms, Thereby, chemical heterogeneities within extracellular biofilm locations can be identified in order to examine the role (e.g, sorption properties, microenvironments. cell-extracellular polymeric substance interactions) of the extracellular polymeric substances in environmental biofilm systems.