In Situ Detection of Amino Acids from Bacterial Biofilms and Plant Root Exudates by Liquid Microjunction Surface-Sampling Probe Mass Spectrometry

Abstract

The plant rhizosphere is a complex and dynamic chemical environment where the exchange of molecular signals between plants, microbes, and fungi drives the development of the entire biological system. Exogenous compounds in the rhizosphere are known to affect plant-microbe organization, interactions between organisms, and ultimately, growth and survivability. The function of exogenous compounds in the rhizosphere is still under much investigation, specifically with respect to their roles in plant growth and development, the assembly of the associated microbial community, and the spatiotemporal distribution of molecular components. A major challenge for spatiotemporal measurements is developing a nondisruptive and nondestructive technique capable of analyzing the exogenous compounds contained within the environment. A methodology using liquid microjunction-surface sampling probe-mass spectrometry (LMJ-SSP-MS) and microfluidic devices with attached microporous membranes was developed for in situ, spatiotemporal measurement of amino acids (AAs) from bacterial biofilms and plant roots. Exuded arginine was measured from a living Pantoea YR343 biofilm, which resulted in a chemical image indicative of biofilm growth within the device. Spot sampling along the roots of Populus trichocarpa with the LMJ-SSP-MS resulted in the detection of 15 AAs. Variation in AA concentrations across the root system was observed, indicating that exudation is not homogeneous and may be linked to local rhizosphere architecture and different biological processes along the root.

Structure-Driven Liquid Microjunction Surface-Sampling Probe Mass Spectrometry

“…When imaging plant root systems, a 4× reduction in analysis time significantly reduces stress on the plant. In our experience, after about 2 h of analysis, the Populus plant begins to exhibit flaccid leaves, areas of leaves turning brown, and roots losing their healthy pinkish hue . SD sampling would require 1816 sampling locations over 2 h to image the entire microfluidic device, while the 0.85 and 0.5 mm GD samplings would involve collection of 3008 and 8692 sampling locations over 3.1 and 9 h, respectively.…”

“…MS imaging can provide spatially resolved chemical information and has been used to chemically profile leaves, roots, and biofilms. − Unfortunately, many of these reports require flash-freezing, sectioning, or other preparation protocols that are difficult to implement for the plant rhizosphere and destroy the plant. Recently, our group developed the capability to spatially resolve the chemical composition of fluids inside of microfluidic devices nondestructively using a porous membrane microfluidic framework coupled with liquid microjunction surface-sampling probe (LMJ-SSP)-MS. , The membrane acts as a barrier to the atmosphere while enabling liquid extraction anywhere along its surface by the LMJ-SSP, which is chemically characterized by MS. , Using this methodology, the spatial distribution of amino acids released from Populus trichocarpa roots grown in a soil-analogue microfluidic habitat were measured in situ for the first time . However, image acquisition times over several hours can negatively affect plant heath and limit temporal studies.…”

Applications of ambient ionization mass spectrometry in 2022: An annual review

“…By applying the MasSpec Pen directly to the skin of the live frog, the authors achieved the measurement of target alkaloids throughout a month-long feeding study, in addition to performing untargeted analysis of skin excretions. Other techniques have also been demonstrated in the analysis of plant metabolites, with Walton et al using the liquid micro junction-surface sampling probe (LMJ-SSP) to study the spatial distribution of amino acids in plant material and bacterial biofilms 148 and Nakata et al using picolitre pressure-probe electrospray-ionization MS to study the effects of salt stress in single plant cells. 149 The early identification of bacterial species is of crucial importance for rapid diagnostics in order to ensure appropriate and timely treatment of infections.…”

“…Other techniques have also been demonstrated in the analysis of plant metabolites, with Walton et al. using the liquid micro junction‐surface sampling probe (LMJ‐SSP) to study the spatial distribution of amino acids in plant material and bacterial biofilms 148 and Nakata et al. using picolitre pressure‐probe electrospray‐ionization MS to study the effects of salt stress in single plant cells 149 …”

Monitoring Growth and Removal of Pseudomonas Biofilms on Cellulose-Based Fabrics

“…Catto and Capitelli [36] summarized approaches to study the EPS matrix, such as fluorescence microscopy, spectroscopic techniques, protein stains, or polysaccharide quantification. Recent studies [37][38][39][40] reported on novel methods to evaluate biofilms and their metabolites without destroying the EPS matrix, such as scanning Kelvin probe (SKP), Fourier transform infrared microscopy (FTIR), digital holographic tomography (DHT), Raman spectroscopy, surface-enhanced Raman scattering (SERS) spectroscopy, and liquid microjunction surface sampling probe accoupled to mass spectrometry. To identify the spatial distribution of bacteria residing in biofilm, FISH (fluorescence in situ hybridization)-based techniques and immunofluorescence are used [34].…”