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  • 1. Johansson, Alexandra C.
    et al.
    Landström, Lars
    Öberg, Rasmus
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Andersson, Per Ola
    Monitoring deactivation processes of bacterial spores using fluorescence spectroscopy2022In: Proceedings Volume 12116, Chemical, Biological, Radiological, Nuclear, and Explosives (CBRNE) Sensing XXIII, 2022 / [ed] Jason A. Guicheteau, Chris R. Howle, SPIE - International Society for Optical Engineering, 2022, Vol. 12116Conference paper (Refereed)
    Abstract [en]

    There is a need for time efficient evaluation methods to discriminate between viable and dead bacterial spores. In this work, the potential to use the autofluorescence from spore suspensions for evaluation of spore deactivation processes is investigated. Bacillus thuringiensis and Bacillus anthracis ATCC 4229 spores were exposed to UV-radiation for deactivation and the fluorescence response was monitored at different radiation doses and the deactivation was evaluated via traditional bacterial incubation on agar culture plates. For excitation wavelengths of, e.g., 280 m and 330 nm, differences in the fluorescence response could be observed for different live:dead ratios.

  • 2.
    Jonsmoen, Unni Lise
    et al.
    Department of Paraclinical Sciences, Faculty of Veterinary Medicine, Norwegian University of Life Sciences (NMBU), Ås, Norway.
    Malyshev, Dmitry
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Öberg, Rasmus
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Dahlberg, Tobias
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Aspholm, Marina E.
    Department of Paraclinical Sciences, Faculty of Veterinary Medicine, Norwegian University of Life Sciences (NMBU), Ås, Norway.
    Andersson, Magnus
    Umeå University, Faculty of Science and Technology, Department of Physics. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Endospore pili - flexible, stiff and sticky nanofibers2023In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 122, no 13, p. 2696-2706Article in journal (Refereed)
    Abstract [en]

    Species belonging to the Bacillus cereus group form endospores (spores) whose surface is decorated with micrometers-long and nanometers-wide endospore appendages (Enas). The Enas have recently been shown to represent a completely novel class of Gram-positive pili. They exhibit remarkable structural properties making them extremely resilient to proteolytic digestion and solubilization. However, little is known about their functional and biophysical properties. In this work, we apply optical tweezers to manipulate and assess how wild type and Ena-depleted mutant spores immobilize on a glass surface. Further, we utilize optical tweezers to extend S-Ena fibers to measure their flexibility and tensile stiffness. Finally, by oscillating single spores, we examine how the exosporium and Enas affect spores’ hydrodynamic properties. Our results show that S-Enas (μm long pili) are not as effective as L-Enas in immobilizing spores to glass surfaces but are involved in forming spore to spore connections, holding the spores together in a gel-like state. The measurements also show that S-Enas are flexible but tensile stiff fibers, which support structural data suggesting that the quaternary structure is composed of subunits arranged in a complex to produce a bendable fiber (helical turns can tilt against each other) with limited axial fiber extensibility. Lastly, the results show that the hydrodynamic drag is 1.5-times higher for wild type spores expressing S- and L-Enas compared to mutant spores expressing only L-Enas or ”bald spores” lacking Ena, and 2-times higher compared to spores of the exosporium deficient strain. This study unveils novel findings on the biophysics of S- and L-Enas, their role in spore aggregation, binding of spores to glass, and their mechanical behavior upon exposure to drag forces.

  • 3.
    Malyshev, Dmitry
    et al.
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Dahlberg, Tobias
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Öberg, Rasmus
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Landström, Lars
    Swedish Defence Research Agency (FOI), Umeå, Sweden.
    Andersson, Magnus
    Umeå University, Faculty of Science and Technology, Department of Physics. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Reference Raman Spectrum and Mapping of Cryptosporidium parvum Oocysts2022In: Journal of Raman Spectroscopy, ISSN 0377-0486, E-ISSN 1097-4555, Vol. 53, no 7, p. 1293-1301Article in journal (Refereed)
    Abstract [en]

    Cryptosporidium parvum is a protozoan parasite and among the most infectious diarrhea-causing pathogens, leading to severe health problems for malnourished children and immunocompromised individuals. Outbreaks are common even in developed countries, originating from water or food contamination and resulting in suffering and large costs for society. Therefore, robust, fast and highly specific detection strategies of Cryptosporidium are needed. Label-free detection techniques such as Raman spectroscopy have been suggested, however high-resolution reported spectra in the literature are limited. In this work, we report reference Raman spectra at 3 cm-1 resolution for viable and inactivated Cryptosporidium oocysts of the species C. parvum, gathered at a single oocyst level using a laser tweezers Raman spectroscopy system. We furthermore provide tentative Raman peak assignments for the Cryptosporidium oocysts, along with Raman mapping of the oocysts’ heterogeneous internal structure. Finally, we compare the C. parvum Raman spectrum with other common enterotoxigenic pathogens: Escherichia coli, Vibrio cholerae, Bacillus cereus and Clostridium difficile. Our results show a significant difference between C. parvum Raman spectra and the other pathogens.

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  • 4.
    Malyshev, Dmitry
    et al.
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Jones, Imogen Anne
    Faculty of Health, University of Plymouth, Plymouth, UK.
    McKracken, Matthew
    Faculty of Health, University of Plymouth, Plymouth, UK.
    Öberg, Rasmus
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Harper, Glenn M.
    Faculty of Health, University of Plymouth, Plymouth, UK.
    Joshi, Lovleen Tina
    Faculty of Health, University of Plymouth, Plymouth, UK.
    Andersson, Magnus
    Umeå University, Faculty of Science and Technology, Department of Physics. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Hypervirulent R20291 Clostridioides difficile spores show disinfection resilience to sodium hypochlorite despite structural changes2023In: BMC Microbiology, E-ISSN 1471-2180, Vol. 23, no 1, article id 59Article in journal (Refereed)
    Abstract [en]

    Clostridioides difficile is a spore forming bacterial species and the major causative agent of nosocomial gastrointestinal infections. C. difficile spores are highly resilient to disinfection methods and to prevent infection, common cleaning protocols use sodium hypochlorite solutions to decontaminate hospital surfaces and equipment. However, there is a balance between minimising the use of harmful chemicals to the environment and patients as well as the need to eliminate spores, which can have varying resistance properties between strains. In this work, we employ TEM imaging and Raman spectroscopy to analyse changes in spore physiology in response to sodium hypochlorite. We characterize different C. difficile clinical isolates and assess the chemical’s impact on spores’ biochemical composition. Changes in the biochemical composition can, in turn, change spores’ vibrational spectroscopic fingerprints, which can impact the possibility of detecting spores in a hospital using Raman based methods.

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  • 5.
    Malyshev, Dmitry
    et al.
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Robinson, Nicholas Finlay
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Öberg, Rasmus
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Dahlberg, Tobias
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Andersson, Magnus
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Reactive oxygen species generated by infrared laser light in optical tweezers inhibits the germination of bacterial spores2022In: Journal of Biophotonics, ISSN 1864-063X, E-ISSN 1864-0648, Vol. 15, no 8, article id e202200081Article in journal (Refereed)
    Abstract [en]

    Bacterial spores are highly resistant to heat, radiation and various disinfection chemicals. The impact of these on the biophysical and physicochemical properties of spores can be studied on the single-cell level using optical tweezers. However, the effect of the trapping laser on spores' germination rate is not fully understood. In this work, we assess the impact of 1064 nm laser light on the germination of Bacillus thuringiensis spores. The results show that the germination rate of spores after laser exposure follows a sigmoid dose-response relationship, with only 15% of spores germinating after 20 J of laser light. Under anaerobic growth conditions, the percentage of germinating spores at 20 J increased to 65%. The results thereby indicate that molecular oxygen is a major contributor to the germination-inhibiting effect observed. Thus, our study highlights the risk for optical trapping of spores and ways to mitigate it.

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  • 6.
    Malyshev, Dmitry
    et al.
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Öberg, Rasmus
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Dahlberg, Tobias
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Wiklund, Krister
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Landström, Lars
    Andersson, Per Ola
    Andersson, Magnus
    Umeå University, Faculty of Science and Technology, Department of Physics. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Laser induced degradation of bacterial spores during micro-Raman spectroscopy2022In: Spectrochimica Acta Part A - Molecular and Biomolecular Spectroscopy, ISSN 1386-1425, E-ISSN 1873-3557, Vol. 265, article id 120381Article in journal (Refereed)
    Abstract [en]

    Micro-Raman spectroscopy combined with optical tweezers is a powerful method to analyze how the biochemical composition and molecular structures of individual biological objects change with time. In this work we investigate laser induced effects in the trapped object. Bacillus thuringiensis spores, which are robust organisms known for their resilience to light, heat, and chemicals are used for this study. We trap spores and monitor the Raman peak from CaDPA (calcium dipicolinic acid), which is a chemical protecting the spore core. We see a correlation between the amount of laser power used in the trap and the release of CaDPA from the spore. At a laser power of 5 mW, the CaDPA from spores in water suspension remain intact over the 90 min experiment, however, at higher laser powers an induced effect could be observed. SEM images of laser exposed spores (after loss of CaDPA Raman peak was confirmed) show a notable alteration of the spores' structure. Our Raman data indicates that the median dose exposure to lose the CaDPA peak was ∼60 J at 808 nm. For decontaminated/deactivated spores, i.e., treated in sodium hypochlorite or peracetic acid solutions, the sensitivity on laser power is even more pronounced and different behavior could be observed on spores treated by the two chemicals. Importantly, the observed effect is most likely photochemical since the increase of the spore temperature is in the order of 0.1 K as suggested by our numerical multiphysics model. Our results show that care must be taken when using micro-Raman spectroscopy on biological objects since photoinduced effects may substantially affect the results.

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  • 7.
    Malyshev, Dmitry
    et al.
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Öberg, Rasmus
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Landström, Lars
    Andersson, Per Ola
    Dahlberg, Tobias
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Andersson, Magnus
    Umeå University, Faculty of Science and Technology, Department of Physics. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    pH induced changes in Raman, UV-Vis absorbance, and fluorescence spectra of dipicolinic acid (DPA)2022In: Spectrochimica Acta Part A - Molecular and Biomolecular Spectroscopy, ISSN 1386-1425, E-ISSN 1873-3557, Vol. 271, article id 120869Article in journal (Refereed)
    Abstract [en]

    Dipicolinic acid (DPA) is an essential component for the protection of DNA in bacterial endospores and is often used as a biomarker for spore detection. Depending upon the pH of the solution, DPA exists in different ionic forms. Therefore, it is important to understand how these ionic forms influence spectroscopic response. In this work, we characterize Raman and absorption spectra of DPA in a pH range of 2.0–10.5. We show that the ring breathing mode Raman peak of DPA shifts from 1003 cm−1 to 1017 cm−1 and then to 1000 cm−1 as pH increases from 2 to 5. The relative peak intensities related to the different ionic forms of DPA are used to experimentally derive the pKa values (2.3 and 4.8). We observe using UV–vis spectroscopy that the changes in the absorption spectrum of DPA as a function of pH correlate with the changes observed in Raman spectroscopy, and the same pKa values are verified. Lastly, using fluorescence spectroscopy and exciting a DPA solution at between 210–330 nm, we observe a shift in fluorescence emission from 375 nm to 425 nm between pH 2 and pH 6 when exciting at 320 nm. Our work shows that the different spectral responses from the three ionic forms of DPA may have to be taken into account in, e.g., spectral analysis and for detection applications.

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  • 8.
    Nilsson, Daniel P.G.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Jonsmoen, Unni Lise
    Department of Paraclinical Sciences, Faculty of Veterinary Medicine, Norwegian University of Life Sciences, Norway.
    Malyshev, Dmitry
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Öberg, Rasmus
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Wiklund, Krister
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Andersson, Magnus
    Umeå University, Faculty of Science and Technology, Department of Physics. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Physico-chemical characterization of single bacteria and spores using optical tweezers2023In: Research in Microbiology, ISSN 0923-2508, E-ISSN 1769-7123, Vol. 174, no 6, article id 104060Article in journal (Refereed)
    Abstract [en]

    Spore-forming pathogenic bacteria are adapted for adhering to surfaces, and their endospores can tolerate strong chemicals making decontamination difficult. Understanding the physico-chemical properties of bacteria and spores is therefore essential in developing antiadhesive surfaces and disinfection techniques. However, measuring physico-chemical properties in bulk does not show the heterogeneity between cells. Characterizing bacteria on a single-cell level can thereby provide mechanistic clues usually hidden in bulk measurements. This paper shows how optical tweezers can be applied to characterize single bacteria and spores, and how physico-chemical properties related to adhesion, fluid dynamics, biochemistry, and metabolic activity can be assessed.

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  • 9.
    Qamar, Saqib
    et al.
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Öberg, Rasmus
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Malyshev, Dmitry
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Andersson, Magnus
    Umeå University, Faculty of Science and Technology, Department of Physics. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    A hybrid CNN-Random Forest algorithm for bacterial spore segmentation and classification in TEM images2023In: Scientific Reports, E-ISSN 2045-2322, Vol. 13, no 1, article id 18758Article in journal (Refereed)
    Abstract [en]

    We present a new approach to segment and classify bacterial spore layers from Transmission Electron Microscopy (TEM) images using a hybrid Convolutional Neural Network (CNN) and Random Forest (RF) classifier algorithm. This approach utilizes deep learning, with the CNN extracting features from images, and the RF classifier using those features for classification. The proposed model achieved 73% accuracy, 64% precision, 46% sensitivity, and 47% F1-score with test data. Compared to other classifiers such as AdaBoost, XGBoost, and SVM, our proposed model demonstrates greater robustness and higher generalization ability for non-linear segmentation. Our model is also able to identify spores with a damaged core as verified using TEMs of chemically exposed spores. Therefore, the proposed method will be valuable for identifying and characterizing spore features in TEM images, reducing labor-intensive work as well as human bias.

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  • 10.
    Valijam, Shayan
    et al.
    Umeå University, Faculty of Science and Technology, Department of Physics. Faculty of Electrical Engineering, K. N. Toosi University of Technology, Tehran, Iran.
    Nilsson, Daniel P. G.
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Malyshev, Dmitry
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Öberg, Rasmus
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Salehi, Alireza
    Faculty of Electrical Engineering, K. N. Toosi University of Technology, Tehran, Iran.
    Andersson, Magnus
    Umeå University, Faculty of Science and Technology, Department of Physics. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Fabricating a dielectrophoretic microfluidic device using 3D-printed moulds and silver conductive paint2023In: Scientific Reports, E-ISSN 2045-2322, Vol. 13, no 1, article id 9560Article in journal (Refereed)
    Abstract [en]

    Dielectrophoresis is an electric field-based technique for moving neutral particles through a fluid. When used for particle separation, dielectrophoresis has many advantages compared to other methods, like providing label-free operation with greater control of the separation forces. In this paper, we design, build, and test a low-voltage dielectrophoretic device using a 3D printing approach. This lab-on-a-chip device fits on a microscope glass slide and incorporates microfluidic channels for particle separation. First, we use multiphysics simulations to evaluate the separation efficiency of the prospective device and guide the design process. Second, we fabricate the device in PDMS (polydimethylsiloxane) by using 3D-printed moulds that contain patterns of the channels and electrodes. The imprint of the electrodes is then filled with silver conductive paint, making a 9-pole comb electrode. Lastly, we evaluate the separation efficiency of our device by introducing a mixture of 3 μm and 10 μm polystyrene particles and tracking their progression. Our device is able to efficiently separate these particles when the electrodes are energized with ±12 V at 75 kHz. Overall, our method allows the fabrication of cheap and effective dielectrophoretic microfluidic devices using commercial off-the-shelf equipment.

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  • 11.
    Valijam, Shayan
    et al.
    Umeå University, Faculty of Science and Technology, Department of Physics. Faculty of Electrical Engineering, K. N. Toosi University of Technology, Tehran, Iran.
    Nilsson, Daniel
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Öberg, Rasmus
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Albertsdóttir Jonsmoen, Unni Lise
    Department of Paraclinical Sciences, Faculty of Veterinary Medicine, Norwegian University of Life Sciences, Ås, Norway.
    Porch, Adrian
    School of Engineering, Cardiff University, Cardiff, United Kingdom.
    Andersson, Magnus
    Umeå University, Faculty of Science and Technology, Department of Physics. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Malyshev, Dmitry
    Umeå University, Faculty of Science and Technology, Department of Physics.
    A lab-on-a-chip utilizing microwaves for bacterial spore disruption and detection2023In: Biosensors & bioelectronics, ISSN 0956-5663, E-ISSN 1873-4235, Vol. 231, article id 115284Article in journal (Refereed)
    Abstract [en]

    Bacterial spores are problematic in agriculture, the food industry, and healthcare, with the fallout costs from spore-related contamination being very high. Spores are difficult to detect since they are resistant to many of the bacterial disruption techniques used to bring out the biomarkers necessary for detection. Because of this, effective and practical spore disruption methods are desirable. In this study, we demonstrate the efficiency of a compact microfluidic lab-on-chip built around a coplanar waveguide (CPW) operating at 2.45 GHz. We show that the CPW generates an electric field hotspot of ∼10 kV/m, comparable to that of a commercial microwave oven, while using only 1.2 W of input power and thus resulting in negligible sample heating. Spores passing through the microfluidic channel are disrupted by the electric field and release calcium dipicolic acid (CaDPA), a biomarker molecule present alongside DNA in the spore core. We show that it is possible to detect this disruption in a bulk spore suspension using fluorescence spectroscopy. We then use laser tweezers Raman spectroscopy (LTRS) to show the loss of CaDPA on an individual spore level and that the loss increases with irradiation power. Only 22% of the spores contain CaDPA after exposure to 1.2 W input power, compared to 71% of the untreated control spores. Additionally, spores exposed to microwaves appear visibly disrupted when imaged using scanning electron microscopy (SEM). Overall, this study shows the advantages of using a CPW for disrupting spores for biomarker release and detection.

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  • 12.
    Öberg, Rasmus
    et al.
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Dahlberg, Tobias
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Malyshev, Dmitry
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Andersson, Magnus
    Umeå University, Faculty of Science and Technology, Department of Physics. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Monitoring bacterial spore metabolic activity using heavy water-induced Raman peak evolution2023In: The Analyst, ISSN 0003-2654, E-ISSN 1364-5528, Vol. 148, no 9, p. 2141-2148Article in journal (Refereed)
    Abstract [en]

    Endospore-forming bacteria are associated with food spoilage, food poisoning, and infection in hospitals. Therefore, methods to monitor spore metabolic activity and verify sterilization are of great interest. However, current methods for tracking metabolic activity are time-consuming and resource intensive. This work investigates isotope labeling and Raman microscopy as a low-cost rapid alternative. Specifically, we monitor the Raman spectrum of enterotoxic \textit{B. cereus} spores undergoing germination and cell division in D2O-infused broth. During germination and cell division, water is metabolized and deuterium from the broth is incorporated into proteins and lipids, resulting in the appearance of a Raman peak related to C-D bonds at 2190 cm-1. We find that a significant C-D peak appears after 2 h of incubation at 37◦C. Further, we found that the peak appearance coincides with the observed first cell division indicating little metabolic activity during germination. Lastly, the germination and cell growth rate of spores were not affected by adding 30 % heavy water to the broth. This shows the potential for real-time monitoring of metabolic activity from a bacterial spore to a dividing cell. In conclusion, our work proposes tracking the evolution of the C-D Raman peak in spores incubated with D2O-infused broth as an effective and time-, and cost-efficient method to monitor the outgrowth of a spore population, simultaneously allowing us to track for how long the bacteria have grown and divided.

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  • 13.
    Öberg, Rasmus
    et al.
    Umeå University, Faculty of Science and Technology, Department of Physics. Division of CBRN Defence and Security, Swedish Defence Research Agency (FOI), Umeå, Sweden.
    Landström, Lars
    Division of Weapons, Protection and Security, Swedish Defence Research Agency (FOI), Norra Sorunda, Sweden.
    Gracia-Espino, Eduardo
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Larsson, Andreas
    Division of CBRN Defence and Security, Swedish Defence Research Agency (FOI), Umeå, Sweden.
    Andersson, Magnus
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Andersson, Per Ola
    Division of CBRN Defence and Security, Swedish Defence Research Agency (FOI), Umeå, Sweden.
    Characterization of carfentanil and thiofentanil using surface-enhanced raman spectroscopy and density functional theory2024In: Journal of Raman Spectroscopy, ISSN 0377-0486, E-ISSN 1097-4555Article in journal (Refereed)
    Abstract [en]

    Fentanyls are synthetic opioids up to 10,000 times more potent than morphine. Although initially developed for medical applications, fentanyl and its analogues have recently grown synonymous with the ongoing opioid epidemic. To combat the continued spread of these substances, there is a need for rapid and sensitive techniques for chemical detection. Surface-enhanced Raman spectroscopy (SERS) has the potential for trace detection of harmful chemical substances. However, vibrational spectra obtained by SERS often differ between SERS substrates, as well as compared with spectra from normal Raman (NR) spectroscopy. Herein, SERS and NR responses from two fentanyl analogues, carfentanil (CF) and thiofentanil (TF), were measured and analysed with support from density functional theory (DFT) modelling. Using commercially available silver nanopillar SERS substrates, the SERS signatures of samples diluted in acetonitrile between 0.01 and 1000 µg/mL were studied. Relative SERS peak intensities measured in the range of 220–1800 cm−1 vary with concentration, while SERS and NR spectra largely agree for CF at higher concentrations ((Formula presented.) 100 µg/mL). For TF, three distinct NR peaks at 262, 366 and 667 cm−1 are absent or strongly suppressed in the SERS spectrum, attributed to the lone-pair electrons of the thiophene's sulphur atom binding to the Ag surface. The concentration dependence of the Raman peak at (Formula presented.) 1000 cm−1, assigned to trigonal bending of the phenyl ring, approximately follows a Langmuir adsorption isotherm. This work elucidates similarities and differences between SERS and NR in fentanyl detection and discusses the chemical rationale behind these differences.

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  • 14.
    Öberg, Rasmus
    et al.
    Umeå University, Faculty of Science and Technology, Department of Physics. Swedish Defence Research Agency (FOI), Umeå, Sweden.
    Sil, Timir B.
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Johansson, Alexandra C.
    Swedish Defence Research Agency (FOI), Umeå, Sweden.
    Malyshev, Dmitry
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Landström, Lars
    Swedish Defence Research Agency (FOI), Norra Sorunda, Sweden.
    Johansson, Susanne
    Swedish Defence Research Agency (FOI), Umeå, Sweden.
    Andersson, Magnus
    Umeå University, Faculty of Science and Technology, Department of Physics. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Andersson, Per Ola
    Swedish Defence Research Agency (FOI), Umeå, Sweden.
    UV-induced spectral and morphological changes in bacterial spores for inactivation assessment2024In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 128, no 7, p. 1638-1646Article in journal (Refereed)
    Abstract [en]

    The ability to detect and inactivate spore-forming bacteria is of significance within, for example, industrial, healthcare, and defense sectors. Not only are stringent protocols necessary for the inactivation of spores but robust procedures are also required to detect viable spores after an inactivation assay to evaluate the procedure’s success. UV radiation is a standard procedure to inactivate spores. However, there is limited understanding regarding its impact on spores’ spectral and morphological characteristics. A further insight into these UV-induced changes can significantly improve the design of spore decontamination procedures and verification assays. This work investigates the spectral and morphological changes to Bacillus thuringiensis spores after UV exposure. Using absorbance and fluorescence spectroscopy, we observe an exponential decay in the spectral intensity of amino acids and protein structures, as well as a logistic increase in dimerized DPA with increased UV exposure on bulk spore suspensions. Additionally, using micro-Raman spectroscopy, we observe DPA release and protein degradation with increased UV exposure. More specifically, the protein backbone’s 1600–1700 cm–1 amide I band decays slower than other amino acid-based structures. Last, using electron microscopy and light scattering measurements, we observe shriveling of the spore bodies with increased UV radiation, alongside the leaking of core content and disruption of proteinaceous coat and exosporium layers. Overall, this work utilized spectroscopy and electron microscopy techniques to gain new understanding of UV-induced spore inactivation relating to spore degradation and CaDPA release. The study also identified spectroscopic indicators that can be used to determine spore viability after inactivation. These findings have practical applications in the development of new spore decontamination and inactivation validation methods.

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  • 15.
    Öberg, Rasmus
    et al.
    Umeå University, Faculty of Science and Technology, Department of Physics. Swedish Defence Research Agency (FOI), Umeå, Sweden.
    Sil, Timir Baran
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Ohlin, C. André
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Andersson, Magnus
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Malyshev, Dmitry
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Assessing CaDPA levels, metabolic activity, and spore detection through deuterium labeling2024In: The Analyst, ISSN 0003-2654, E-ISSN 1364-5528, Vol. 149, no 6, p. 1861-1871Article in journal (Refereed)
    Abstract [en]

    Many strains among spore-forming bacteria species are associated with food spoilage, foodborne disease, and hospital-acquired infections. Understanding the impact of environmental conditions and decontamination techniques on the metabolic activity, viability, and biomarkers of these spores is crucial for combatting them. To distinguish and track spores and to understand metabolic mechanisms, spores must be labeled. Staining or genetic modification are current methods for this, however, these methods can be time-consuming, and affect the viability and function of spore samples. In this work, we investigate the use of heavy water for permanent isotope labeling of spores and Raman spectroscopy for tracking sporulation/germination mechanisms. We also discuss the potential of this method in observing decontamination. We find that steady-state deuterium levels in the spore are achieved after only ∼48 h of incubation with 30% D2O-infused broth and sporulation, generating Raman peaks at cell silent region of 2200 and 2300 cm−1. These deuterium levels then decrease rapidly upon spore germination in non-deuterated media. We further find that unlike live spores, spores inactivated using various methods do not lose these Raman peaks upon incubation in growth media, suggesting these peaks may be used to indicate the viability of a spore sample. We further observe several Raman peaks exclusive to deuterated DPA, a spore-specific chemical biomarker, at e.g. 988 and 2300 cm−1, which can be used to track underlying changes in spores involving DPA. In conclusion, permanent spore labeling using deuterium offers a robust and non-invasive way of labeling bacterial spores for marking, viability determination, and characterising spore activity.

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