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Bacteria generally interact with the environment via processes involving their cell-envelope. Thus, techniques that may shed light on their surface chemistry are attractive tools for providing an understanding of bacterial interactions. One of these tools is Al Kα-excited photoelectron spectroscopy (XPS) with its estimated information depth of <10 nm. XPS-analyses of bacteria have been performed for several decades on freeze-dried specimens in order to be compatible with the vacuum in the analysis chamber of the spectrometer. A limitation of these studies has been that the freeze-drying method may collapse cell structure as well as introduce surface contaminants. However, recent developments in XPS allow for analysis of biological samples at near ambient pressure (NAP-XPS) or as frozen hydrated specimens (cryo-XPS) in vacuum. In this work, we have analyzed bacterial samples from a reference strain of the Gram-negative bacterium Pseudomonas fluorescens using both techniques. We compare the results obtained and, in general, observe good agreement between the two techniques. Furthermore, we discuss advantages and disadvantages with the two analysis approaches and the output data they provide. XPS reference data from the bacterial strain are provided, and we propose that planktonic cells of this strain (DSM 50090) are used as a reference material for surface chemical analysis of bacterial systems.
Surface-supported metal-organic frameworks HKUST-1 (Hong Kong University of Science and Technology) were used as a model system for a development of a near ambient pressure (NAP) XPS based approach to investigate interaction with atmospheres of water, methanol or pyridine at pressures ranging from 1 to 4 mbar. The films were grown on a gold substrate functionalized with a COOH-terminated self-assembled monolayer using liquidphase epitaxy in a step-by-step fashion. Measurement protocols were developed and optimised for different gases in order to obtain spectra of similar quality in terms of signal intensity, noise and shape. Peak shapes were found to depend on the efficiency of charge compensation. Reference measurements in argon proved to be a useful strategy not only for the evaluation of the Cu(II)-fraction in pristine samples, but also to identify the contributions by the respective gas atmosphere to the C 1s and O 1s photoelectron spectra. Reduced copper was found during the exposition of HKUST-1 to water vapour and pyridine, but this effect was not observed in case of methanol. Additionally, it was established that there are no changes in relative Cu(II) percentage with increasing exposure time. This indicates that saturation was reached already at the lowest time of gas exposure. A detailed elucidation of the mechanism of Cu(II) reduction to Cu(I) in HKUST-1 mediated by water and pyridine is part of ongoing work and not in the scope of the present paper.
Metal-organic frameworks (MOFs) are suitable materials for gas storage of small molecules due to their nanoporous, crystalline structure. The extensively studied MOF HKUST-1 consist of Cu(II)-dimers in a paddlewheel structure, with 1,3,5-benzenetricarboxylic acid (BTC) as organic linker. Instability to humidity remains an issue for many types of MOFs, and for HKUST-1, it has been found that exposure to water vapor creates a surface barrier which reduces the gas uptake rate.
Near-ambient pressure XPS (NAP-XPS) is a promising method for investigations of the stability and interaction of HKUST-1 with various gas molecules. The oxidation state of copper can be monitored before, during and after exposure to various gases. This does not only provide information on the stability of the MOFs, but also on the interaction with the gas molecules.
NAP-XPS measurements of HKUST-1 exposed to methanol, pyridine and water vapor were performed with EnviroESCA, a laboratory NAP-XPS instrument developed by SPECS. Cu 2p, O 1s and C 1s core level spectra and Cu LMM Auger spectra were acquired at pressures ranging from high vacuum to 4 mbar to assess the oxidation state of copper and the stability of the organic linker. Reference measurements in argon atmosphere were conducted in order to have reference spectra with similar full width of half maxima (FHWM) as the spectra measured in reactive atmospheres. Peak fit analysis of the Cu 2p3/2 core level spectra shows that, as expected, the photoelectron signal mainly originates from Cu(II) species. The contribution from Cu(I) is negligible for samples exposed to argon and methanol, but approximately 4% for samples exposed to water vapor and 8% for samples exposed to pyridine. Within the error of the peak fit analysis, there are no changes in relative Cu(II) percentage with increasing water, methanol or pyridine exposure time pointing to saturation reached already at the lowest time of exposure.
Bacterial samples are typically freeze dried or cryo-prepared prior to XPS analysis to allow for measurements in ultra-high vacuum (UHV). The sample environment in the near-ambient pressure (NAP) XPS instrument EnviroESCA allows for measurements in up to 15 mbar water vapor, thus, sample preparation is no longer restricted to UHV-compatible techniques. For instance, biofilms grown in medium can be transferred directly from the medium to the measurements chamber, maintaining a humid environment throughout the measurements. Considering the complexity of bacterial samples, sample preparation must be carefully considered in order to obtain meaningful and reproducible results.
In this talk, various strategies for sample preparation of bacteria and biofilms for NAP-XPS measurements will be discussed. Model systems of planktonic bacteria, artificial biofilms resembling the exopolysaccharide matrix and biofilms have been characterised in various conditions. The stability and homogeneity of the samples was assessed by monitoring the C1s core level peak at different sample locations. The quality of the XPS-spectra is also influenced by the gas environment, which will be exemplified by core level spectra of P. Fluorescens acquired in air, water vapor and ultra-high vacuum.
Near-ambient pressure x-ray photoelectron spectroscopy (NAP-XPS) is a less traditional form of XPS that allows samples to be analyzed at relatively high pressures, i.e., at greater than 2500 Pa. With NAP-XPS, XPS can probe moderately volatile liquids, biological samples, porous materials, and/or polymeric materials that outgas significantly. In this submission, we show survey, C 1s, O 1s, and N
1s narrow scans from an aqueous solution of a common protein, bovine serum albumin. The C 1s peak envelope is well fit to four symmetric peaks of equal width that correspond to carbon bonded to carbon and hydrogen (C-1), carbon singly bonded to oxygen (C-2), carbonyl and/or amide carbon (C-3), and carboxyl carbon (C-4). Two possible peak fits are considered for the N 1s and O 1s peak envelopes. The N 1s signal is fit to four peaks that correspond to amine (—NH2), Amide (OvCZNH2), ammonium (—NH3 +), and N2(g) nitrogen, and alternatively to three peaks that correspond to amine, amide, and N2(g) nitrogen. The O 1s peak envelope is similarly fit to three and four components.
Metal-organic frameworks (MOFs) are suitable materials for gas storage of small molecules due to their nanoporous, crystalline structure. The extensively studied MOF HKUST-1 consist of Cu(II)-dimers in a paddlewheel structure, with 1,3,5-benzenetricarboxylic acid (BTC) as organic linker. Instability to humidity remains an issue for many types of MOFs, and for HKUST-1, it has been found that exposure to water vapor creates a surface barrier which reduces the gas uptake rate.
Near-ambient pressure XPS (NAP-XPS) is a promising method for investigations of the stability and interaction of HKUST-1 with various gas molecules. The oxidation state of copper can be monitored before, during and after exposure to various gases. This does not only provide information on the stability of the MOFs, but also on the interaction with the gas molecules and the reversibility of the processes.
NAP-XPS measurements of HKUST-1 exposed to methanol, pyridine and water vapor were performed with EnviroESCA, a laboratory NAP-XPS instrument developed by SPECS. Cu 2p, O 1s and C 1s core level spectra were acquired in pressure ranging from 10-5 to 8 mbar to assess the oxidation state of copper and the stability of the organic linker. HKUST-1 does not show interaction with pyridine, but there are indications of copper-reduction upon exposure to methanol and water vapor. Radiation induced damage is considered and two different approaches for determining the ratio of Cu(I) to Cu(II) will be discussed.
Near-ambient pressure XPS makes it possible to characterise samples not compatible to ultra-high vacuum, and enables the study of liquid-solid, gas-liquid and gas-solid interfaces. NAP-XPS meas-urements of biofilms, suspended nanoparticles and metal-organic frameworks were performed with EnviroESCA developed by SPECS.
An interesting application is surface characterisation of biofilms, which are bacterial communities embedded in a self-produced polysaccharide matrix. Various model systems ranging from pure polysaccharides of alginate to biofilms harvested directly from the growth medium have been char-acterised in humid conditions[1].
NAP-XPS also makes it possible to characterise nanoparticles in solution. Silver nanoparticles in aqueous solution were characterised and the Ag 3d-spectrum compared to spectra obtained of dried nanoparticles in UHV-conditions[2]. The binding energy of the Ag 3d-core level peak was shifted by 0,6 eV towards higher binding energy for suspended nanoparticles compared to the dried sample measured in UHV. This can be assigned to a change in surface potential at the water-nanoparticle interface.
Metal-organic frameworks (MOFs) are suitable materials for gas storage of small molecules due to their nanoporous, crystalline structure. However, instability in humidity remains an issue for many types of MOFs. XPS-measurements of the MOF-structure HKUST-1 were performed in various NAP-conditions to assess the stability of the sample and its interaction with the gas molecules as water, methanol and pyridine.
Synchrotron XPS in the soft-X-ray regime is suitable for the detection of light elements commonly found in biological samples. Various model systems of biofilms have been developed and characterised at synchrotron- and lab-based facilities. By obtaining the chemical composition at various information depths, the vertical distribution of iodine in an artificial biofilm have been determined.
The proposed ISO Technical Report provides a description of a variety of physical methods of analytical chemistry by which bacteria and biofilms can be analysed. The state of the art, sample requirements and strengths associated with each method are identified. Presented at the DIN-meeting for NA 062-08-16 AA „Chemische Oberflächenanalyse und Rastersondenmikroskopie“
X-ray photoelectron spectroscopy (XPS) provides elemental and chemical information from the outermost ~10 nm of the sample surface. This is in the same order of magnitude as the thickness of the outer bacterial membrane of gram-negative bacteria, as well as outer membrane molecules as exopolysaccharides and lipopolysaccharides, commonly attached to the cell surface. With the development of near-ambient pressure (NAP)-XPS, bacteria can be analysed with minimal sample preparation.
EnviroESCA is a laboratory based NAP-XPS instrument, equipped with a monochromated Al Kα radiation source and a differentially pumped energy analyser connected to an exchangeable sample environment. It allows for measurements in various gas-atmospheres, including water vapor, which makes it possible to characterise bacteria and other biological samples close to their natural, hydrated state. Artificial model-biofilms of exopolysaccharides, planktonic Pseudomonas Fluorescens and biofilms of Escherichia Coli have been characterised in hydrated and dried state.
High-resolution XPS-spectra from carbon, oxygen, nitrogen and phosphorous can be assigned to carbohydrates, lipids and proteins in general agreement with literature. Especially the carbon 1s peak is of interest. A series of measurements of an E. coli biofilm from 11 mbar in humid environment to 1 mbar air reveal changes in the C1s peak, which suggests that the bacterial surface undergo substantial Change.
This application note presents how EnviroESCA can be used to analyze E. coli biofilms on silicon under near ambient pressure conditions in various states of hydration. Such investigations of the outer bacterial cell surface in their hydrated state are essential for studying biological interfaces at work.
INTRODUCTION: X-ray photoelectron spectroscopy (XPS) provides elemental and chemical information from the outermost ~10 nm of the sample surface. This is in the same order of magnitude as the thickness of the outer bacterial membrane of gram-negative bacteria, as well as outer membrane molecules as exopolysaccharides and lipopolysaccharides, commonly attached to the cell surface. With the development of near-ambient pressure (NAP)-XPS, bacteria can be analysed with minimal sample preparation. This is in contrast to ultra-high vacuum XPS, where complex sample preparation including freeze-drying or fast-freezing is required, a treatment that may introduce artefacts or degrade the biological constituents.
METHODS: EnviroESCA is a laboratory based NAP-XPS instrument, equipped with a monochromated Al Kα radiation source and a differentially pumped energy analyser connected to an exchangeable sample environment, a basic build-up is displayed in Figure 1c. It allows for measurements in various gas-atmospheres, including water vapor, which makes it possible to characterise bacteria and other biological samples close to their natural, hydrated state. Artificial model-biofilms of exopolysaccharides, planktonic Pseudomonas Fluorescens and biofilms of Escherichia Coli have been characterised in hydrated and dried state.
RESULTS: High-resolution XPS-spectra from carbon, oxygen, nitrogen and phosphorous can be assigned to carbohydrates, lipids and proteins in general agreement with litterature2,3. Especially the carbon 1s peak is of interest (see Figure 1a and b), which reveal components originating from aliphatic carbon, single bounded carbon and double bounded/acetal carbon. A series of measurements of an E. coli biofilm from 11 mbar in humid environment to 1 mbar air reveal changes in the C1s peak, which suggests that the bacterial surface undergo substantial change.
DISCUSSION & CONCLUSIONS:
Bacterial samples can be analysed under near-ambient pressure conditions in various states of hydration with minimal sample preparation. Such investigations of bacterial cell wall surfaces are essential for studying biological interfaces at work.
A presentation held for the seminar "Ausgewählte analytische Methoden der Physik" hosted by Prof. Birgit Kanngießer at TU Berlin. The first part focus on depth-dependent XPS-measurements (XPS, synchrotron HAXPES) to obtain a concentration profile of iodine in an artificial biofilm. In the second part, NAP-XPS measurements of various bacterial samples are presented.
Surface characterisation of Escherichia coli under various conditions by near-ambient pressure XPS
(2018)
Bacteria are inherently in a hydrated state and therefore not compatible to ultra-high vacuum techniques such as XPS without prior sample preparation involving freeze drying or fast freezing.
This has changed with the development of near-ambient pressure (NAP)-XPS, which makes it possible to characterise the bacterial surface with minimal sample preparation. This paper presents NAP-XPS measurements of Escherichia coli under various NAP conditions: at 11 mbar in a humid environment, at 2 mbar after drying in the chamber, pre-dried at 4 mbar, and at 1 mbar after overnight pumping at 10^−4 mbar. The high-resolution spectra of carbon, nitrogen, and oxygen are presented and found to be in general agreement with XPS measurements from freeze-dried and fast-frozen bacteria. However, it was found that the amount of carbon components associated with polysaccharides increases relative to aliphatic carbon during drying and increases further after overnight pumping. This implies that drying has an impact on the bacterial surface.
The XPS information depth of approximately 10 nm is in the same size range as the outer membrane of the gram-negative bacteria, which makes XPS a suitable tool for determining the elemental composition of the bacterial surface and monitor changes caused by outer stress like dehydration or exposure to antimicrobials. However, bacteria are inherently in a hydrated state, and therefore only compatible to ultra-high vacuum after extensive sample preparation, which may degrade the sample constituents. This issue is addressed by the development of near-ambient pressure (NAP)-XPS, which enables bacteria and biofilms to be characterised in their native wet state. Artificial biofilms, bacteria and biofilms of Escherichia coli have been characterised with the laboratory NAP-XPS instrument EnviroESCA from SPECS GmbH, at pressures ranging from high vacuum to 12 mbar, and in both humid and dry environment. By studying biological samples in their native wet state, new insight about composition and transport of drugs through cell membranes and the extracellular polymeric substance (EPS) of biofilms can be obtained. In this contribution, the latest progress on biofilm characterisation by NAP-XPS will be presented, and measurement capabilities and limitations will be discussed.
Surface characterisation of biofilms by nearambient pressure X-ray photoelectron spectroscopy
(2018)
The XPS information depth of approximately 10 nm is in the same size range as the outer membrane of the gram-negative bacteria, which makes XPS a suitable tool for determining the elemental composition of the bacterial surface and monitor changes caused by outer stress like dehydration or exposure to antimicrobials. However, bacteria are inherently in a hydrated state, and therefore only compatible to ultra-high vacuum after extensive sample preparation, which may degrade the sample constituents. This issue is addressed by the development of near-ambient pressure (NAP)-XPS, which enables bacteria and biofilms to be characterised in their native wet state. Artificial biofilms, bacteria and biofilms of Escherichia coli have been characterised with the laboratory NAP-XPS instrument EnviroESCA from SPECS GmbH, at pressures ranging from high vacuum to 12 mbar, and in both humid and dry environment. By studying biological samples in their native wet state, new insight about composition and transport of drugs through cell membranes and the extracellular polymeric substance (EPS) of biofilms can be obtained.
The degradation of the chemotherapy drug 5-fluorouracil by a non-pristine metal surfaces is studied.Using density functional theory, X-ray photoelectron spectroscopy and X-ray absorption spectroscopy we show that the drug is entirely degraded by medical-grade silver surfaces, already at body temperature,and that all of the fluorine has left the molecule, presumably as HF. Remarkably, this degradation is even more severe than that reported previously for 5-fluorouracil on a pristine monocrystalline silver surface(in which case 80% of the drug reacted at body temperature) [1]. We conclude that the observed reaction is due to a reaction pathway, driven by H to F attraction between molecules on the surface, which results in the direct formation of HF; a pathway which is favoured when competing pathways involving reactive Ag surface sites are made unavailable by environmental contamination. Our measurements indicate that realistically cleaned, non-pristine silver alloys, which are typically used in medical applications, can result in severe degradation of 5-fluorouracil, with the release of HF – a finding which may have important implications for the handling of chemotherapy drugs.