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Monitoring intracellular metal ion complexation with an acetylene-tagged ligand by Raman spectroscopy.

ABSTRACT: We propose to monitor molecular vibrations to identify metal ion-ligand complexation by means of Raman spectroscopy, which has been applied to track vibrational modes of molecules and to obtain a structural fingerprint. We prepared ligand molecules for Zn²⁺ ion complexation with a dipycolylaminoethyl aniline (DPEA) skeleton and phenylacetylene unit as the Raman tag which showed a typical band around 2200 cm^-1. Among the labeled ligands synthesized in this study, A-DPEA showed a strong band attributed to the acetylene unit at 2212 cm^-1, while the addition of Zn²⁺ ion resulted in a band shift to 2220 cm^-1 due to complex formation. The addition of other metal ions and titration experiments showed that A-DPEA bound with Zn²⁺ selectively with a dissociation constant (K _d) that was estimated to be 0.22 μM. We also conducted cellular experiments and found that complexation between A-DPEA and Zn²⁺ also occurred in cells, with a shift in the Raman signal of the ligand from 2212 to 2215 cm^-1. Thus, complex formation of the metal ion was identified by monitoring the Raman band shift.

SUBMITTER: Takemura S

PROVIDER: S-EPMC9056985 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Monitoring intracellular metal ion complexation with an acetylene-tagged ligand by Raman spectroscopy.

Takemura Seiya S Watanabe Hikaru H Nishihara Tatsuya T Okamoto Akimitsu A Tanabe Kazuhito K

RSC advances 20200901 59

We propose to monitor molecular vibrations to identify metal ion-ligand complexation by means of Raman spectroscopy, which has been applied to track vibrational modes of molecules and to obtain a structural fingerprint. We prepared ligand molecules for Zn<sup>2+</sup> ion complexation with a dipycolylaminoethyl aniline (DPEA) skeleton and phenylacetylene unit as the Raman tag which showed a typical band around 2200 cm<sup>-1</sup>. Among the labeled ligands synthesized in this study, A-DPEA show ...[more]

PMID: 35517095

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Project description:Antibiotics resistance developed by biofilms has posed a clinical challenge in the effective treatment of bacterial infections. However, the resistance mechanisms have not been well understood due to a lack of suitable tools for dynamic observation of the interplay between antibiotics and biofilm. In this work, with the use of rapid hyperspectral stimulated Raman scattering microscopy associated with an aryl-alkyne-based Raman tag synthesized, we investigate dynamic interactions between vancomycin and Staphylococcus aureus (S. aureus) biofilm to gain new insights into the resistance mechanisms of the biofilm. Methods: We utilize spectral focusing hyperspectral stimulated Raman scattering microscopy ensued with multivariate curve resolution analysis to spectrally decompose S. aureus biofilm into its major components (i.e., bacteria and extracellular polymeric substances). Concurrently, vancomycin is conjugated with aryl-alkyne Raman tag (Raman peak at 2218 cm-1) for in vivo tracking of its uptake into biofilm without tissue interference. Results: We find that vancomycin penetration is a non-uniform diffusion process with penetration depths limited by the preferential affinity to the cell clusters. Semi-quantitative analysis shows that the majority of vancomycin binds to the bacteria, achieving intracellular concentrations of up to 4- to 10- fold higher than the administered dosage. The diffusion constant of ~3.16 µm2/min based on the diffusion and antibiotic binding equations is obtained that well accounts for the antibiotic penetration into the biofilm. SRS longitudinal monitoring of antibiotic effect on the growth of biofilms shows that the antibiotics can eradicate the upper layer of the biofilm exposed to sufficient dosages, while the lower layer of the biofilm at a sub-inhibitory dose remains viable, eventually re-growing to significant bio-volume. Conclusion: The Raman-tagged hyperspectral SRS microscopy developed is a powerful imaging tool for dynamic monitoring of inhibitory effects of antibiotics on the growing biofilm in vivo, which would facilitate the formulation of new antibiotics for more effective treatments of bacterial infections in near future.

Project description:BackgroundMonitoring and control of both growth media and microbial biomass is extremely important for the development of economical bioprocesses. Unfortunately, process monitoring is still dependent on a limited number of standard parameters (pH, temperature, gasses etc.), while the critical process parameters, such as biomass, product and substrate concentrations, are rarely assessable in-line. Bioprocess optimization and monitoring will greatly benefit from advanced spectroscopy-based sensors that enable real-time monitoring and control. Here, Fourier transform (FT) Raman spectroscopy measurement via flow cell in a recirculatory loop, in combination with predictive data modeling, was assessed as a fast, low-cost, and highly sensitive process analytical technology (PAT) system for online monitoring of critical process parameters. To show the general applicability of the method, submerged fermentation was monitored using two different oleaginous and carotenogenic microorganisms grown on two different carbon substrates: glucose fermentation by yeast Rhodotorula toruloides and glycerol fermentation by marine thraustochytrid Schizochytrium sp. Additionally, the online FT-Raman spectroscopy approach was compared with two at-line spectroscopic methods, namely FT-Raman and FT-infrared spectroscopies in high throughput screening (HTS) setups.ResultsThe system can provide real-time concentration data on carbon substrate (glucose and glycerol) utilization, and production of biomass, carotenoid pigments, and lipids (triglycerides and free fatty acids). Robust multivariate regression models were developed and showed high level of correlation between the online FT-Raman spectral data and reference measurements, with coefficients of determination (R2) in the 0.94-0.99 and 0.89-0.99 range for all concentration parameters of Rhodotorula and Schizochytrium fermentation, respectively. The online FT-Raman spectroscopy approach was superior to the at-line methods since the obtained information was more comprehensive, timely and provided more precise concentration profiles.ConclusionsThe FT-Raman spectroscopy system with a flow measurement cell in a recirculatory loop, in combination with prediction models, can simultaneously provide real-time concentration data on carbon substrate utilization, and production of biomass, carotenoid pigments, and lipids. This data enables monitoring of dynamic behaviour of oleaginous and carotenogenic microorganisms, and thus can provide critical process parameters for process optimization and control. Overall, this study demonstrated the feasibility of using FT-Raman spectroscopy for online monitoring of fermentation processes.

Dataset Information

Monitoring intracellular metal ion complexation with an acetylene-tagged ligand by Raman spectroscopy.

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Monitoring intracellular metal ion complexation with an acetylene-tagged ligand by Raman spectroscopy.

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