Analysis of Bacteriophage-Host Interaction by Raman Tweezers.

Bacteriophages, or "phages" for short, are viruses that replicate in bacteria. The therapeutic and biotechnological potential of phages and their lytic enzymes is of interest for their ability to selectively destroy pathogenic bacteria, including antibiotic-resistant strains. Introduction of phage preparations into medicine, biotechnology, and food industry requires a thorough characterization of phage-host interaction on a molecular level. We employed Raman tweezers to analyze the phage-host interaction of Staphylococcus aureus strain FS159 with a virulent phage JK2 (=812K1/420) of the Myoviridae family and a temperate phage 80α of the Siphoviridae family. We analyzed the timeline of phage-induced molecular changes in infected host cells. We reliably detected the presence of replicating phages in bacterial cells within 5 min after infection. Our results lay the foundations for building a Raman-based diagnostic instrument capable of real-time, in vivo, in situ, nondestructive characterization of the phage-host relationship on the level of individual cells, which has the potential of importantly contributing to the development of phage therapy and enzybiotics.

Detection of Chloroalkanes by Surface-Enhanced Raman Spectroscopy in Microfluidic Chips.

Optofluidics, a research discipline combining optics with microfluidics, currently aspires to revolutionize the analysis of biological and chemical samples, e.g., for medicine, pharmacology, or molecular biology. In order to detect low concentrations of analytes in water, we have developed an optofluidic device containing a nanostructured substrate for surface enhanced Raman spectroscopy (SERS). The geometry of the gold surface allows localized plasmon oscillations to give rise to the SERS effect, in which the Raman spectral lines are intensified by the interaction of the plasmonic field with the electrons in the molecular bonds. The SERS substrate was enclosed in a microfluidic system, which allowed transport and precise mixing of the analyzed fluids, while preventing contamination or abrasion of the highly sensitive substrate. To illustrate its practical use, we employed the device for quantitative detection of persistent environmental pollutant 1,2,3-trichloropropane in water in submillimolar concentrations. The developed sensor allows fast and simple quantification of halogenated compounds and it will contribute towards the environmental monitoring and enzymology experiments with engineered haloalkane dehalogenase enzymes.

Raman microspectroscopy and laser-induced breakdown spectroscopy for the analysis of polyethylene microplastics in human soft tissues.

People are exposed to microplastics (MPs) on a large scale in everyday life. However, it is not clear whether MPs can also be distributed and retained in certain tissues. Therefore, the development of analytical methods capable of detecting MPs in specific human organs/tissues is of utmost importance. In this study, the use and combination of spectroscopic techniques, namely Raman microspectroscopy and laser-induced breakdown spectroscopy (LIBS), was tested for the detection of polyethylene (PE) MPs in human tonsils. Preliminary results showed that Raman microspectroscopy was able to detect MPs down to 1 µm in size and LIBS down to 10 µm. In the next step, human tonsils were spiked with PE MPs, and digested. The filtered particles were analyzed using Raman microspectroscopy and LIBS, and complemented by X-ray fluorescence (XRF). The results showed that Raman microspectroscopy could reliably detect PE MPs in spiked human tonsils, while LIBS and XRF served as a reference analytical method to characterize particles that could not be classified by Raman microspectroscopy for their non-organic origin. The results of this study, supported by a current feasibility study conducted on clinical samples, demonstrated the reliability and feasibility of this approach for monitoring MPs in biotic samples.

Laser-based techniques: Novel tools for the identification and characterization of aged microplastics with developed biofilm.

Microplastics found in the environment are often covered with a biofilm, which makes their analysis difficult. Therefore, the biofilm is usually removed before analysis, which may affect the microplastic particles or lead to their loss during the procedure. In this work, we used laser-based analytical techniques and evaluated their performance in detecting, characterizing, and classifying pristine and aged microplastics with a developed biofilm. Five types of microplastics from different polymers were selected (polyamide, polyethylene, polyethylene terephthalate, polypropylene, and polyvinyl chloride) and aged under controlled conditions in freshwater and wastewater. The development of biofilm and the changes in the properties of the microplastic were evaluated. The pristine and aged microplastics were characterized by standard methods (e.g., optical and scanning electron microscopy, and Raman spectroscopy), and then laser-induced breakdown spectroscopy (LIBS) and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) were used. The results show that LIBS could identify different types of plastics regardless of the ageing and major biotic elements of the biofilm layer. LA-ICP-MS showed a high sensitivity to metals, which can be used as markers for various plastics. In addition, LA-ICP-MS can be employed in studies to monitor the adsorption and desorption (leaching) of metals during the ageing of microplastics. The use of these laser-based analytical techniques was found to be beneficial in the study of environmentally relevant microplastics.

Rapid Identification of Pathogens Causing Bloodstream Infections by Raman Spectroscopy and Raman Tweezers.

The search for the "Holy Grail" in clinical diagnostic microbiology-a reliable, accurate, low-cost, real-time, easy-to-use method-has brought up several methods with the potential to meet these criteria. One is Raman spectroscopy, an optical, nondestructive method based on the inelastic scattering of monochromatic light. The current study focuses on the possible use of Raman spectroscopy for identifying microbes causing severe, often life-threatening bloodstream infections. We included 305 microbial strains of 28 species acting as causative agents of bloodstream infections. Raman spectroscopy identified the strains from grown colonies, with 2.8% and 7% incorrectly identified strains using the support vector machine algorithm based on centered and uncentred principal-component analyses, respectively. We combined Raman spectroscopy with optical tweezers to speed up the process and captured and analyzed microbes directly from spiked human serum. The pilot study suggests that it is possible to capture individual microbial cells from human serum and characterize them by Raman spectroscopy with notable differences among different species. IMPORTANCE Bloodstream infections are among the most common causes of hospitalizations and are often life-threatening. To establish an effective therapy for a patient, the timely identification of the causative agent and characterization of its antimicrobial susceptibility and resistance profiles are essential. Therefore, our multidisciplinary team of microbiologists and physicists presents a method that reliably, rapidly, and inexpensively identifies pathogens causing bloodstream infections-Raman spectroscopy. We believe that it might become a valuable diagnostic tool in the future. Combined with optical trapping, it offers a new approach where the microorganisms are individually trapped in a noncontact way by optical tweezers and investigated by Raman spectroscopy directly in a liquid sample. Together with the automatic processing of measured Raman spectra and comparison with a database of microorganisms, it makes the whole identification process almost real time.

Raman Spectroscopy-A Novel Method for Identification and Characterization of Microbes on a Single-Cell Level in Clinical Settings.

Rapid and accurate identification of pathogens causing infections is one of the biggest challenges in medicine. Timely identification of causative agents and their antimicrobial resistance profile can significantly improve the management of infection, lower costs for healthcare, mitigate ever-growing antimicrobial resistance and in many cases, save lives. Raman spectroscopy was shown to be a useful-quick, non-invasive, and non-destructive -tool for identifying microbes from solid and liquid media. Modifications of Raman spectroscopy and/or pretreatment of samples allow single-cell analyses and identification of microbes from various samples. It was shown that those non-culture-based approaches could also detect antimicrobial resistance. Moreover, recent studies suggest that a combination of Raman spectroscopy with optical tweezers has the potential to identify microbes directly from human body fluids. This review aims to summarize recent advances in non-culture-based approaches of identification of microbes and their virulence factors, including antimicrobial resistance, using methods based on Raman spectroscopy in the context of possible use in the future point-of-care diagnostic process.

Raman Microspectroscopic Analysis of Selenium Bioaccumulation by Green Alga Chlorella vulgaris.

Selenium (Se) is an element with many commercial applications as well as an essential micronutrient. Dietary Se has antioxidant properties and it is known to play a role in cancer prevention. However, the general population often suffers from Se deficiency. Green algae, such as Chlorella vulgaris, cultivated in Se-enriched environment may be used as a food supplement to provide adequate levels of Se. We used Raman microspectroscopy (RS) for fast, reliable, and non-destructive measurement of Se concentration in living algal cells. We employed inductively coupled plasma-mass spectrometry as a reference method to RS and we found a substantial correlation between the Raman signal intensity at 252 cm-1 and total Se concentration in the studied cells. We used RS to assess the uptake of Se by living and inactivated algae and demonstrated the necessity of active cellular transport for Se accumulation. Additionally, we observed the intracellular Se being transformed into an insoluble elemental form, which we further supported by the energy-dispersive X-ray spectroscopy imaging.