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This application note presents how EnviroESCA can be used to analyze bacterial samples under near ambient pressure conditions in various states of hydration using different levels of humidity. Such investigations of bacterial cell wall surfaces in their hydrated state are essential for studying biological interfaces at work.
The use of innovative near-ambient pressure (NAP-)XPS instrumentation allows the detailed analysis of irregularly-surfaced biofilms. NAP-XPS enables the surface analysis of bacterial samples in their natural hydrated state without complex sample preparation techniques such as freeze-drying or fast-freezing, which are needed for XPS analysis in ultrahigh vacuum.
Here, we present a study on agarose thin-film samples that represent a model system for the exopolysaccharide matrix of biofilms. Povidone-iodide (PVP-I) was selected as an antibacterial agent to evaluate our x-ray photoelectron spectroscopy (XPS)-based methodology to trace specific marker elements, here iodine, commonly found in organic matrices of antibiotics. The in-depth distribution of iodine was determined by XPS analyses with variable excitation energies and in combination with argon gas cluster ion beam sputter cycles. On mixed agarose/PVP-I nanometer-thin films, both methods were found to solve the analytical task and deliver independently comparable results. In the mixed agarose/PVP-I thin film, we found the outermost surface layer depleted in iodine, whereas the iodine is homogeneously distributed in the depth region between this outermost surface layer and the interface between the thin film and the substrate. Depletion of iodine from the uppermost surface in the thin-film samples is assumed to be caused by ultrahigh vacuum exposure resulting in a loss of molecular iodine (I2) as reported earlier for other iodine-doped polymers.
Water and aqueous reagents are essential in any biological process or system. But apart from a few special low vapor-pressure cases, liquids have not been accessible to any technique requiring UHV conditions. EnviroESCA opens up this exciting field of applications.
In this paper first results from water based samples are presented as a proof of concept to demonstrate the special capabilities of EnviroESCA analyzing liquid samples. The following solutions were investigated under near ambient pressure conditions: i.) water, ii.) brine, iii.) an oil in water dispersion, iv.) aqueous iron(II) sulfate heptahydrate, and v.) a suspension of nano silver particles in water.
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.
Multi-resistant bacteria embedded in infectious biofilms are one of the largest future threats in human healthcare. The mechanism of drug uptake and the origin of resistancy is quantitatively not well characterized and understood. Modern X-ray and synchrotron excited spectroscopic methods like XRF and NAP-XPS under ambient pressure conditions and liquid media will add new insights into this field.
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 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.
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.