Depth-Resolved Nanospray Desorption Electrospray Ionization Mass Spectroscopy in Electrochemically Active Biofilms
Authors: Atci Erhan , Ewing Timothy , Beyenal Haluk 
Published: February 2020

Recently, some bacterial biofilms have been shown to colonize and transfer their extracellular electrons to solid electrodes in microbial fuel cells and in bioelectrochemical systems [1, 2]. This class of microorganisms is described as electrochemically active biofilms (EABs) that are able to transfer electrons outside the cell to insoluble electron acceptors (iron or metal oxides) or to solid electrodes via extracellular electron transfer [3, 4]. The need to understand the distribution of proteins involved in electron transfer in EABs is of critical importance. These proteins can be identified using mass spectroscopy (MS). It is known that there is a wide spectrum of applications in which mass spectroscopy (MS) can provide information from a sample of EABs, and these applications can easily translate to all areas of research in EABs. However, the spatial diversity of microbial functions within EABs cannot be resolved because all of the current MS analysis techniques are conducted in the bulk liquid, which provides no spatial resolution with respect to the structure of EABs. Separation techniques can be combined with MS to present a more focused data set with regard to composition; however, MS analysis still lacks the capacity to investigate variation with depth of EABs. Depth profiles will have the potential to elucidate the mechanisms of the surrounding matrix and the roles of the microorganism with respect to the layers of EABs and the growth interface. To date, this MS technique has not been used for depth profiling in EABs. This is mostly because there was no tool available to extract samples from different depths in EABs. Microcapillaries with a several-micrometer tip diameter can be used in EABs without damaging its structure for the depth profiling of selected chemicals. The goal of this work was to develop a microcapillary system which can extract samples at desired depth inside the EABs.
Ambient pressure surface ionization mass spectrometry is used to obtain a chemical analyte for sampling from interfaces without special sample preparation [5]. Desorption electrospray ionization (DESI) is an ambient ionization technique in which charged droplets from an electrosonic spray ionization source are aimed towards a surface with a proximal atmospheric pressure mass spectrometer inlet. In this technique, analyte molecules are collected from flat surfaces followed by ionization using a self-aspirating nanoelectrospray. This technique directly transports and ionizes an analyte that is desorbed from a surface into a liquid and it is called as nanospray DESI (nano-DESI). The nanospray capillary transports the charged liquid to the mass spectrometer inlet directly, eliminating splashing while minimizing analyte transport distance. The target application of the developed microcapillary system is to interface it with a nano-DESI sensor which can be used to characterize in situ, depth-resolved analyses of metabolites and possibly proteins.
The developed nano-DESI sensor is composed of two microcapillaries placed in an outer case made of glass. Figure 1 shows an image of a developed nano-DESI sensor. In this system, the solvent delivery and its collection were made from the same microcapillaries. We managed to operate the sensor between 10 nL/min and 100 mL/min flow rates. After optimization of the flow rates, we tested it in EABs. EABs were grown according to our previously published paper and book [6, 7]. While we had succeeded in developing a nano-DESI sensor, we found unexpected challenges using it with EABs. Finally, the microcapillary system developed for this work enabled us to use it for quantifying electron transfer processes in EABs [7].

References:

1. Hamelers, H.M., et al., New applications and performance of bioelectrochemical systems. Applied Microbiology and Biotechnology, 2010. 85(6): p. 1673-1685.
2. Logan, B.E., Exoelectrogenic bacteria that power microbial fuel cells. Nat Rev Micro, 2009. 7(5): p. 375-381.
3. Lovley, D.R., Microbial fuel cells: novel microbial physiologies and engineering approaches. Current Opinion in Biotechnology, 2006. 17(3): p. 327-332.
4. Logan, B.E., et al., Microbial Fuel Cells:  Methodology and Technology†. Environmental Science & Technology, 2006. 40(17): p. 5181-5192.
5. Roach, P.J., J. Laskin, and A. Laskin, Nanospray desorption electrospray ionization: an ambient method for liquid-extraction surface sampling in mass spectrometry. Analyst, 2010. 135(9): p. 2233-2236.
6. Lewandowski, Z. and H. Beyenal, Fundamentals of Biofilm Research, Second Edition. 2014: Taylor & Francis.
7. Babauta, J.T. and H. Beyenal, Local Current Variation by Depth in Geobacter Sulfurreducens Biofilms. Journal of The Electrochemical Society, 2014. 161(13): p. H3070-H3075.

Figure 1