Stable Isotope Raman Microspectroscopy (SIRM) for Quantitative and Nondestructive 2D & 3D Analysis of Biofilms: Microbial Degradation of Microplastic

基于稳定同位素的分析方法在不同的科学领域越来越重要。尽管基于质谱（MS）的方法能够灵敏地分析大量样品（例如，同位素比质谱，IRMS）或提供低至50 nm的空间分辨率（例如，纳米级二次离子质谱（NanoSIMS），这些方法是破坏性的并且需要耗时的样品制备。在这里，拉曼显微光谱（RM）与稳定同位素方法的组合-稳定同位素拉曼显微光谱（SIRM）-可以扩展具有非破坏性，定量和空间分辨分析的成熟技术的能力。SIRM提供具有共聚焦光学显微镜的空间分辨率的样品特征指纹谱，包含关于稳定同位素标记物质和标记量的信息（基于标记物质的带的红移）。同时，这些光谱提供了样品的化学成分和结构信息。此外，该方法不需要或只需要有限的样品制备，并且可以在原位和体内进行，没有水的光谱干扰。腐殖物质、微生物和生物膜，已在申请人项目的第一阶段得到证明。SIRM还成功地与共振和表面增强拉曼散射（Sers）效应相结合，以提高其灵敏度。所取得的成果以及其他研究人员目前的研究表明，SIRM在单细胞水平上表征微生物群落的潜力很大。该方法提供了关于碳代谢/流动和细胞活性的信息，并且已经被测试用于分析环境污染物的降解。然而，到目前为止，还没有进行微生物降解的一个最突出的新兴污染物-微塑料（MP）的研究。这个后续项目的目标是开发和评估一种基于SIRM的方法，定量和无损的2D和3D分析的生物膜参与微塑料的生物降解。SIRM与Sers相结合，有可能提供新的信息碳同化和一般代谢活动的微生物从塑料降解生物膜。除了使用昂贵或甚至不可用的稳定同位素标记的聚合物之外，还将应用替代方法-D2 O-和反向标记SIRM。将使用IRMS和NanoSIMS数据验证获得的SIRM结果。总而言之，这应该有助于我们建立可靠的方法来分析水环境中（微）塑料的生物降解，从而为我们提供单细胞水平的（直接）信息。

Stable isotope-based analytical methods gain increasing relevance in different scientific fields. Although mass spectrometry-based (MS) methods enable sensitive analysis of bulk samples (e.g., isotope ratio mass spectrometry, IRMS) or provide a spatial resolution down to 50 nm (e.g., nanoscale secondary ion mass spectrometry, NanoSIMS), these methods are destructive and require time-consuming sample preparation. Here, a combination of Raman microspectroscopy (RM) with the stable isotope approach – stable isotope Raman microspectroscopy (SIRM) – can extend the capabilities of the well-established techniques with a nondestructive, quantitative and spatially-resolved analysis. SIRM provides characteristic fingerprint spectra of samples with the spatial resolution of a confocal optical microscope, containing information on stable isotope-labeled substances and the amount of a label (based on red shift of bands of the labeled substances). Simultaneously, these spectra deliver information on the chemical composition and structure of samples. Furthermore, this method requires no or limited sample preparation, and can be performed in situ and in vivo without spectral interference of water.The feasibility of SIRM for the quantitative 2D & 3D studies of different samples, incl. humic substances, microorganisms and biofilms, has been demonstrated during the first phase of the applicant project. SIRM has been also successfully combined with resonance and surface-enhanced Raman scattering (SERS) effects, to improve its sensitivity. The achieved results as well as current studies of other researchers indicate a high potential of SIRM for the characterization of microbial communities at the single cell level. This method provides information on the carbon metabolism / flow and the cell activity, and has already been tested for the analysis of the degradation of environmental pollutants. However, no research has been performed so far on the microbial degradation of a most prominent emerging pollutant in the aquatic environment – microplastic (MP).The goal of this follow-up project is to develop and evaluate a SIRM-based method for quantitative and nondestructive 2D & 3D analysis of biofilms involved in the biodegradation of MP. SIRM in combination with SERS has a potential to provide novel information on the carbon assimilation and general metabolic activity of microorganisms from plastic-degrading biofilms. Besides the use of stable isotope-labeled polymers which are expensive or even unavailable, alternative approaches – D2O- and reverse-labeling SIRM – will be applied. The obtained SIRM results will be validated with IRMS and NanoSIMS data. Altogether, this should help us to establish reliable method for the analysis of biodegradation of (micro)plastic in the aquatic environment, thus providing us with (direct) information at the single cell level.