RESEARCH INTERESTS

 
What We Do

Broadly speaking, the Bohn Group uses the tools of molecular nanotechnology to define the state-of-the-art in chemical analysis of mass-limited samples - generally speaking samples ranging from ~106 molecules (1.6 attomoles = 1.6 x 10-18 mole) down to a single molecule.

 

Our research currently focuses on problems in three thematic areas: low-dimensional analytical electronics and photonics, spectroelectrochemical sensors, and chemical imaging.  In all three areas we simultaneously develop new measurement tools and use these to accomplish new chemical analyses.  We specifically make heavy use of nanofabrication, high sensitivity molecular spectroscopy, and electrochemistry - usually in combination with each other.

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Low-Dimensional Analytical Electronics and Photonics

In the low-dimensional analytical electronics and photonics (AEP) area, we exploit the special properties of zero- and one-dimensional nanostructures to perform chemical measurements not possible in macroscopic structures. 

 

Tool Development. We have invented a new kind of structure - the electrochemical zero-mode waveguide (E-ZMW) - to explore single molecule spectroelectrochemistry, and we are developing capped zeptoliter volume electrochemical cells  (1 zL = 10-21 L) with ring-disk, dual-ring, and even triple-ring electrode architectures.  Also, following up on earlier work in the construction of atomic-scale junctions we have developed an addressable, reconfigurable, direct-write approach to the fabrication of nanofilaments for applications in non-classical optics.

 

New Measurements. To address sensitivity issues, we have developed a number of signal amplification strategies utilizing recessed electrode arrays for redox cycling as well as gated membranes for enhanced selectivity.  Furthermore, we exploit the joint electrochemical and spectroscopic properties of these nanostructures to carry out chemical dynamics studies of single electron transfer reaction events and single nanoparticle catalysts.

 

The Electrochemical Zero-Mode Waveguide

We have invented a special kind of nanophotonic platform – an electrochemical zero mode waveguide (E-ZMW) which, at the most basic level, is a small (d < 100 nm, typ.) cylindrical perforation in a thin metal film, capable of trapping optical radiation. The trapped radiation can interact with molecules contained in the zeptoliter-scale (1 zL = 10-21 L) volume bounded by the radiation field and enclosed within the nanopore.  The E-ZMW is a bifunctional electrochemical ZMW structure, in which the optical cladding layer serves (1) to confine radiation and (2) as an electrochemical working electrode.  The small volumes enclosed by the active region of the E-ZMW, their close proximity to the working electrode, and the small distances over which diffusive mass transport occurs combine to give the E-ZMW some powerful new properties, including the ability to observe single electron transfer events using simultaneous optical and electrochemical measurements.

 

For more information see:

 

Zhao, J., et al. “Potential-Dependent Single Molecule Blinking Dynamics for Flavin Adenine Dinucleotide Covalently Immobilized in Zero-Mode Waveguide Array of Working Electrodes,” Faraday Disc. 2013, 164, 57-69.

 

Zaino, L.P. III, et al. “Single Molecule Spectroelectrochemistry of Freely Diffusing Flavin Mononucleotide in Zero-Dimensional Nanophotonic Structures,” Faraday Disc. 2015, 184, 101-115.

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SEM images of a small portion of a recessed dual ring electrode ZMW array.  (A) Plan view image showing a small portion of a high density 30x30 nanopore array with ~250 nm pitch.  (B) Cross-sectional SEM image of 3 adjacent E-ZMW nanopores showing dual recessed Au ring electrodes separated by ~100 nm of SiNx.

 

Capped Zeptoliter Volume Electrochemical Cells

One issue which arises when using the nanopore-based structures in electrochemistry is that molecules may diffuse out of the pore, thereby changing its population.  At high concentrations this is a negligible effect.  However, when pore occupancies approach  ~ 1, it is important know and control the pore occupancy during an experiment.  To address this issue and also to make it possible to carry out long time integrations, we are elaborating the basic E-ZMW structure to include a capping layer.  Surprisingly, not only do we observe strict sequestration of ions, but also rectification and current enhancement - two very interesting effects that we are putting to use in electroanalytical experiments.

 

For more information see:

 

Fu, K., et al. “Electrochemistry at Single Molecule Occupancy in Nanopore-Confined Recessed Ring-Disk Electrode Arrays,” Faraday Disc. 2016, 193, 51-64.

 

Han, D., et al. “Single-Molecule Spectroelectrochemical Cross-Correlation During Redox Cycling in Recessed Dual Ring Electrode Zero-Mode Waveguides,” Chem. Sci. 2017, 8, 5345-5355.

(Left) Schematic illustration of a capped zeptoliter electrochemical cell supporting redox cycling.  (Right) Successive cyclic voltammograms of 0.5 mM Fe(CN)63/4- showing both amplification (250x) of the current signal and rectification (R ~ 6.3).

 

Addressable Direct-Write Fabrication of Nanofilaments

Conductive nanofilaments can be grown by electrodeposition through polymer electrolytes as a potential approach to new devices, such as resistive random-access memory, electrochemical metallization memory (ECM), and chemical sensors. These structures  function by exhibiting a binary change in electrical resistance, with distinct “on” and “off” states typically separated by several orders of magnitude; mediated by the redox formation, and subsequent dissolution, of a conductive nanofilament between two fixed electrodes.  Motivated by the possibility of using nanoscale filaments for the addressable, reconfigurable, direct-write in situ wiring of nanoparticle arrays as a route to the creation of nanoelectronic and nanophotonic materials and devices requiring nanometer scale control of the dielectric response function, we are exploring the use of conductive AFM (C-AFM) preare nanofilaments under a variety of conditions, including where nanoparticles are used as nanoscale bipolar electrodes.

 

For more information see:

 

Crouch, G.M., et al. “Addressable Direct-Write Nanoscale Filament Formation and Dissolution by Nanoparticle-Mediated Bipolar Electrochemistry,” ACS Nano 2017, 11, 4976-4984.

 

Chao, Z., et al. “Direct-write formation and dissolution of silver nanofilaments in ionic liquid-polymer electrolyte composites,” Small 2018, in press.

(Left) Schematic illustration of the use of nanoparticles as nanoscale bipolar electrodes in the direct-write conductive AFM deposition of nanofilaments.  (Right) Histogram of the initial formation times under three different poly(ethylene oxide) loadings.

 

Recessed Electrode Arrays for Redox Cycling

Surface charge characteristics and the electric double layer (EDL) govern the transport of ions into and out of nanopores, producing a permselective concentration polarization, which dominates the electrochemical response of nanoelectrodes in solutions of low ionic strength, for example when electrochemistry is carried out in the absence of supporting electrolyte. We are using highly ordered, 0-D nanopore electrode arrays (NEAs), with each nanopore presenting a pair of recessed electrodes, in order to couple EDL effects with redox cycling, thereby achieving electrochemical detection with both improved sensitivity and selectivity.

 

For more information see:

 

Ma, C., et al. “Recessed Ring-Disk Nanoelectrode Arrays Integrated in Nanofluidic Structures for Selective Electrochemical Detection,” Analyt. Chem., 2013, 85, 9882-9888.[DOI 10.1021/ac403417w]

 

Ma, C., et al. “Redox Cycling in Nanoscale Recessed Ring-Disk Electrode Arrays for Enhanced Electrochemical Sensitivity,” ACS Nano 2013, 7, 5483-5490.

 

Fu, K., et al. “Ion Selective Redox Cycling in Zero-Dimensional Nanopore Electrode Arrays at Low Ionic Strength,” Nanoscale 2017, 9, 5164-5171.

(Left) Schematic illustration of the interaction and uptake of cations (blue) and anions (red) in a cation permselective nanopore.  Furthermore, the small spacing of the electrodes greatly facilitates redox cycling. (Center) Photo of electrochemical chip holding 8 pairs of dual-electrode sensor arrays.  (Right) Cyclic voltammograms of 1 mM Ru(NH3)63+ on the NEAs obtained by sweeping the bottom electrode while holding the top electrode at 0.1V either with (blue) or without (red) 0.1 M KCl, or floating the top electrode in 0.1 M KCl (black). Top electrode (solid) and bottom electrode currents (dashed) displayed.  (Inset) Magnified CV obtained with the top electrode floating.

 

Gated Membranes for Enhanced Selectivity

Given the strong nature of the permselectivity effect, we are seeking to extend the idea by introducing additional gating concepts to control the composition inside a nanopore. In one set of experiments, we address the properties and behavior of single nanoparticles, which can reveal how particle-to-particle heterogeneity affects ensemble properties derived from traditional bulk measurements.  High-bandwidth, low noise electrochemical measurements are needed to examine the fast heterogeneous electron-transfer behavior of single nanoparticles with sufficient fidelity to resolve the behavior of individual nanoparticles, and the top ring electrode serves to “gate” the transport of silver nanoparticles (AgNPs) into individual attoliter-volume nanopores.  In another set of experiments, we are developing the natural permselectivity of ionomer membranes, like Nafion, to produce high-performance redox cycling-based electrochemical diodes, which exhibit large rectification ratio, fast response times, and simplified circuitry without the need for external electrodes.

 

For more information see:

Fu, K., et al. “Voltage-Gated Nanoparticle Transport and Collisions in Attoliter-Volume Nanopore Electrode Arrays,” Small 2018, 14, 1703248(1-10).

Fu, K., et al. “Asymmetric Nafion-Coated Nanopore Electrode Arrays as Redox Cycling-Based Electrochemical Diodes,” ACS Nano 2018, in press.

(Top) Representative amperometric traces from the bottom electrode (EBE = +0.3 V) obtained by applying different voltages to the top electrode. (Bottom) Simulated electric field and particle trajectories of AgNPs in nanopores with ETE = 0.0 V (gate closed, (left)) or +0.7 V (gate open, (right)), while EBE was fixed at +0.3 V.

Schemes and SEM images of asymmetric Nafion-coated nanopore electrode arrays, Nafion@NEA. (Top, left) Schematic illustration of Nafion@NEA, where Nafion (light blue) acts as a cation exchange membrane to allow cations (red spheres) to pass through, while rejecting anions (green spheres), for subsequent redox cycling inside the NEA. (Top, right) The Nafion@NEA acts as a redox cycling-based diode when using the top and bottom electrodes are operated in a two terminal configuration. (Bottom, left) Tilted SEM image near the edge of the Nafion film, indicating that the Nafion conformally coats the NEA. Scale bar is 2 mm. (Bottom, right) Cross-sectional SEM image showing the stacked metal (disk)-insulator-metal (ring) (MIM) structure in the vertical direction, as well the well-sealed Nafion at the top. Scale bar is 400 nm.

 

Single Electron Transfer Reaction Events

Despite their fundamental importance in life processes, direct electrochemical observation of single electron transfer events in biological systems is still quite challenging. The principal problem is that the generated currents are at, or below, the noise floor of electrical measurements that can be made at accessible gain-bandwidth product. We are utilizing the electrochemical zero-mode waveguide (E-ZMW) to couple biological electron transfer reactions to changes in luminescence.  The defining characteristic of these architectures is that single molecule spectroscopic and electrochemical data can be acquired simultaneously.  For example, flavoenzymes are fluorigenic molecules, because flavin adenine dinucleotide (FAD) cofactors are strongly fluorescent in the oxidized state, while they are non-emissive in the reduced state (FADH2). We are exploiting this property to study complex reaction assemblies, in which one electrode of the E-ZMW is used to control the production of reactive oxygen species (superoxide, H2O2, etc.) status of the zeptoliter-volume nanopore which

 

For more information see:

 

Han, D., et al. “Single-Molecule Spectroelectrochemical Cross-Correlation During Redox Cycling in Recessed Dual Ring Electrode Zero-Mode Waveguides,” Chem. Sci. 2017, 8, 5345-5355.

 

Fu, K., et al. “Electrochemistry at Single Molecule Occupancy in Nanopore-Confined Recessed Ring-Disk Electrode Arrays,” Faraday Disc. 2016, 193, 51-64.

(Left) Schematic diagram of a single enzyme in an E-ZMW.  The ZMW confines the optical radiation near the bottom Au working electrode, where a single GR enzyme molecule is immobilized. The top Pt working electrode is poised at a potential to generate ROS, e.g. H2O2. Single GR enzyme turnover is indicated by the observation of an emission discontinuity, characteristic of FAD « FADH2. (Right) Single enzyme luminescence time traces. (top) EB (= potential of the bottom Au OC/WE) floats. Asterisks (*) denote events exceeding the 99% confidence level for photon emission. (bottom) EB = -0.6 V, a potential at which GR is expected to be pinned in the reduced, non-emissive state, FADH2.

 
Spectroelectrochemical Sensors

The coupling of electron transfer events to spectroscopic phenomena is not only useful for fundamental studies - it can also be used for enhanced chemical sensing.  We are using the novel electrochemical construct of bipolar electrodes, particularly closed bipolar electrodes (CBEs), in order to produce chemical sensors where the measurement and the readout are conveniently separated into two physically separated regions.

 

Tool Development. In the CBE sensor architecture, there are two electrochemical cells - an analytical cell, in which the analyte is recognized and oxidized/reduced, and a reporter cell, in which a convenient electrochemical reporter reaction is chosen to convert the electrochemical signal into a light-driven readout. The key to CBE is that one electrode in each cell is at open circuit potential, and these are electrically connected to each other.  We are examining the use of electrochromism, electrofluorigenic reactions, and electrochemically-driven changes to localized surface plasmon resonance (LSPR) readouts.

 

New Measurements. The sensor efforts are currently targeting two specific biomedical problems - the detection of pathogenic bacteria by whole-cell recognition and detection, and multiplex detection of a panel of biomarkers for the deadly sepsis syndrome. 

 

Electron-Optical Conversion for Sensor Readout

Detecting and identifying infectious agents and potential pathogens in complex environments and characterizing their mode of action is a critical need. While traditional diagnostics target a single characteristic, advances in detection technologies are enabling approaches in which multiple modes of action are combined to obtain enhanced performance characteristics. Particularly appealing in this regard, electrophotonic devices capable of coupling light to electron translocation.

 

Electrofluorigenic Reactions. Our first efforts in this area coupled nanopore electrode arrays supporting redox cycling in an analytical cell to electrofluorigenic reactions (dark ® emissive). A nanopore electrode array is used to support self-induced redox cycling, amplifying the current amplification.  This electron transfer reaction is connected to a remote (mm-distance) planar bipolar electrode, which couples the electrochemical reactions of the analyte with that of a reporter, such as a potential-switchable fluorescent indicator, in the cell at the distal end of the bipolar electrode.  The inherent sensitivity of the fluorescence readout has led to LODs in the pM concentration range.

 

Electrochromic Reactions. A CBE-enabled method for electrochemical sensing based on the electrochromic response of a methylviologen (MV) reporter is developed, characterized, and rendered in a field-deployable format. CBE-enabled devices based on two thin-layer-cells of ITO and Pt are used to couple the analytical reaction in one cell with an MV reporter reaction, producing a color change in the complementary cell. Then, smartphone-based detection and RGB analysis are employed to further simplify the sensing scheme. The method produces linear relationship between the analyte concentration, the quantity of MV generated, and the colorimetric response, yielding LODs in the low µM, well-sufficient for routine clinical monitoring of a wide range of diagnostic targets.

 

Electrochemical LSPR. Given the success of the electrofluorigenic and electrochromic approaches, we are exploring the use of electric chemically modified LSPR architectures in the reporter cell of CBE-enabled sensor architectures. This approach Will likely fill in a middle ground between the high sensitivity, but complex, electrofluorigenic measurements and the simpler, but less sensitive, electrochromic measurements.

 

For more information see:

 

Ma, C., et al. “Self-Induced Redox Cycling Coupled Luminescence on Nanopore Recessed Disk-Multiscale Bipolar Electrodes,” Chem. Sci. 2015, 6, 3173-3179.

 

Xu, W., et al. “Closed Bipolar Electrode-Enabled Dual-Cell Electrochromic Detectors for Chemical Sensing,” Analyst 2016, 141, 6018-6024.

 

Xu, W., et al. “Electrochromic Sensor for Multiplex Detection of Metabolites Enabled by Closed Bipolar Electrode Coupling,” ACS Sens. 2017, 2, 1010-1026.

 

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Schematic side view of the electrofluorigenic array and illustration of the mechanism of self-induced redox cycling for Ru(NH3)62/3+  (abbreviated as Ru2/3+).  SIRC in the analytical cell is monitored by a reporter redox couple (Repox/red) at a remote location (reporter cell).

Battery operation of a closed-BPE dual cell with colorimetric readout by smartphone camera yields a simple, inexpensive, field-deployable electrochemical sensor.

 

Pathogenic Bacteria

Bacterial infections have posed a persistent health threat to humans, livestock, and crops throughout human history.  Consequently, there is an urgent need to develop both efficient bacterial diagnostics as well as effective treatments. Current gold standard microbiology methods for bacterial detection have excellent specificity and sensitivity, allowing for bacterial strain identification and single cell detection; however, these protocols require culturing bacteria in a strictly controlled laboratory environment and often take days for an accurate reading.  To address these challenges we are developing efficient schemes for whole-cell bacterial sensing based on the combination of a molecular recognition motif (aptamer for Pseudomonas aeruginosa, siderophore for Acinetobacter baumannii) bound to an LSPR substrate.  Surprisingly, the approach is extraordinarily sensitive, producing reliable detection of a single bacteria cell in the case of P. aeruginosa.  We are exploring the extension fo this platform technology to the whole cell detection of other pathogens, in particular Mycobacterium tuberculosis.

 

For more information see:

 

Hu, J. and Bohn, P.W. “Optical Biosensing of Bacteria and Bacterial Communities,” J. Anal. Test. 2017, 1, 1-17.

 

Hu, J., et al. “Whole-Cell Pseudomonas aeruginosa Localized Surface Plasmon Resonance Aptasensor,” Analyt. Chem. 2018, 90, 2326-2332.


LSPR wavelength shift as a function of bacterial concentration for the whole-cell detection of P. aeruginosa. The detections of 10 cfu mL-1 (statistically significant at the 99.9% confidence level) corresponds to detecting a single bacterial cell.

 

CBE-Enabled Multiplex Sensor for Sepsis Biomarkers

Recently, our laboratory has begun to develop integrated CBE-based biosensing platforms aimed at the early and accurate diagnosis of sepsis, a potentially life-threatening syndrome caused by infection. Several biomarkers are used in clinical tests to diagnose and manage the severity of patient’s illness, e.g. lactate, C-reactive protein, the liver enzymes ALT and AST, creatinine, procalcitonin, and bilirubin. There is hope that a multiplex assay ported to a PoC device, by reducing the time to accurate diagnosis, can have a substantive impact on patient outcomes. It is a challenging task detect multiple analytes simultaneously in a portable device, but our proof-of-principle experiment with the enzyme-mediated, CBE-enabled multiplex detection of analytes that do not themselves have to be redox-active opens the door to such a potential life-saving diagnostic tool.

 

For more information see:

 

Xu, W., et al. “Electrochromic Sensor for Multiplex Detection of Metabolites Enabled by Closed Bipolar Electrode Coupling,” ACS Sens. 2017, 2, 1010-1026.

 

The use of enzyme-mediated redox reactions coupled to electrochromic readout allows the CBE-enabled scheme to be extended to multiplex detection of important biomarkers for the rapid diagnosis of sepsis.

Chemical Imaging
 

Most bacteria in natural and clinical settings grow as surface-attached biofilms. Biofilms contain bacteria organized into a highly structured community encased by a matrix composed of extracellular polymeric substances (EPS). Furthermore, much of what we know regarding the chemical profile of bacterial communities arises from some combination of homogenized samples and extrapolation of nucleic acid profile data—yet, most organisms heterogeneously occupy 2D and 3D space. We seek to advance an understanding of spatial variations in bacterial behavior that are currently underestimated or entirely missed when assessing the proteomic, genomic, or phenotypes using homogeneous methods.

 

Tool Development. Because these problems simply could not be addressed with the previously existing tools, in the last four years we have created new technology to identify and map constituents of host-associated communities. We developed a correlated chemical imaging approach, in which SIMS-based mass spectrometric imaging (MSI) - carried out by Jonathan Sweedler’s group at UIUC - and confocal Raman microscopy (CRM) are combined to analyze the chemical composition of microbial communities. Precise spatial correlation between SIMS and CRM images – carried out on different instruments hundreds of miles apart – is achieved using a chemical microdroplet array, allowing us to relocate regions of interest, and align image data with µm-scale precision. We also developed novel approaches to differences in sensitivity, dynamic range, and noise susceptibility between the platforms.

 

New Measurements. These new tools are being applied to two broad classes of problems: spatiotemporal mapping of chemical messengers and spatially- and temporally-organized antibiotic responses. Dramatic molecular differences in the spatiotemporal distributions of quinolone metabolites and signaling molecules are observed between planktonic and biofilm modes and between community compo­nents exhibiting distinct mobility modes, e.g. twitching vs. swarming vs. sessile biofilms. In addition, the distribution of alkyl quinolones can vary by several orders of magnitude within the same community upon exposure to antibiotics, suggesting that multiple intercellular signals can be triggered by one common cue. 

 

Correlated Chemical Imaging 

Vibrational imaging, in particular CRM, is already established as a powerful tool for investigating both com­position and function of bacteria. Coupling the high spatial resolution (Δx,y ~ 0.5 µm) chemical imaging capabilities and functional group specificity of CRM to multivariate statistical tools, yields a platform capable of discerning inherent variations in morphology, protein expression, and inter-cellular chemical signaling that can be used to identify changes in bacterial behavior, even in cases involving multiple species. Prior to our collaborative experiments, previous work in correlated imaging from optical and mass spectrometric experiments was sparse, empirical, and mostly focused on model systems. The technological motivations for implementing CRM/MSI are clear. Unlike many conventional chemical imaging modalities, such as fluorescence microscopy and positron emission tomogra­phy, MSI and CRM require no a priori knowledge about chemical composition, and sample perturbations from staining or genetic label incorporation are avoided. Thus, multimodal imaging approaches that combine MSI and CRM can generate information that is difficult or impossible to obtain with a single technique.

 

For more information see:

 

Masyuko, R., et al.  “Correlated Imaging – A Grand Challenge in Chemical Analysis,” Analyst, 2013, 138, 1924-1939.

 

Masyuko, R.N., et al. “Spatial Organization of Pseudomonas Aeruginosa Biofilms Probed by Correlated Laser Desorption Ionization Mass Spectrometry and Confocal Raman Microscopy,” Analyst 2014, 139, 5700-5708.

 

Lanni, E.J., et al. “Correlated Imaging with C60-SIMS and Confocal Raman Microscopy: Visualization of Cell-Scale Molecular Distributions in a Bacterial Biofilm,” Analyt. Chem.  2014, 86, 10885-10891.

CRM/SIMS correlated imaging workflow. (a) A microdroplet array is applied to the dried biofilm. (b) CRM is performed to locate ROIs, and array coordinates are recorded. (c) The sample is transferred to the SIMS instrument, and the array is used to navigate back to the ROIs. (d) The CRM and SIMS data are correlated, using the array for alignment.

 

Spatiotemporal Mapping of Chemical Messengers

CRM imaging is being used to characterize the tran­sition from planktonic cells to biofilms in P. aeruginosa grown under static conditions. Furthermore, the success of pathogens depends partly on the production and secretion of an arsenal of virulence factors, e.g. phenazines – a class of redox-active heterocyclic small-molecule metabolites85, such as pyocyanin (5-N-methyl-1-hydroxy-phenazine) which has been associated with lung damage in CF patients, and indeed is one of the hallmarks of the disease. As an example, we are using in situ SERS spatial and temporal mapping of P. aeruginosa bacterial communities to classify the major factors influencing community heterogeneity, thus circumventing problems inherent in the more common approach of averaging single spectra. It is clear that the combination of CRM and PCA provides critical insight into the spatial distribution of metabolites and their role in organizing microbial communities.

 

For more information see:

 

Baig, N.F., et al. “Multimodal Chemical Imaging of Molecular Messengers in Emerging Pseudomonas aeruginosa Bacterial Communities,” Analyst 2015, 140, 6544-6552.

 

Polisetti, S., et al. “Raman Chemical Imaging of the Rhizosphere Bacterium Pantoea sp. YR343 and its Co-Culture with Arabidopsis thaliana,” Analyst 2016, 141, 2175-2182.

 

Polisetti, S., et al.  “Spatial Mapping of Pyocyanin in Pseudomonas aeruginosa Bacterial Communities by Surface Enhanced Raman Scattering,” Appl. Spectrosc. 2017, 71, 215-223.

 

 

 

CRM imaging of a 48h P. aeruginosa biofilm. (a) Composite Raman image includes integration over 1338-1376 cm-1 (pink) and 1638-1676 cm-1 (blue) showing the co-location of two distinct chemical entities; (b)PC1 and (c) PC2 contain features characteristic of PQS and HQNO standards, respectively; heat maps of PC1 (d) and PC2 (e) .

 

Spatially- and Temporally-Organized Antibiotic Responses

For surface-growing bacteria, QS and other functions are not spatially uniform. Indeed, bacteria growing in biofilms are heterogeneous in func­tion. To address this challenge, we are using multimodal CRM/MSI chemical imaging to study spatial heteroge­neity of P. aeruginosa QS response to antibiotics. We find that motile “pre-biofilm” swarm communities of P. aeruginosa exhibit differing responses to inhibitory levels of select antibiotics. While exposure to the aminoglycoside tobramycin (TOB) and the β-lactam carbenicillin (CARB) produce analogous reductions in plate-assay swarming, distinct responses in the secreted chemical messengers are demonstrated by straightforward application of CRM/MSI. These experiments allow us to conclude that, in contrast to existing dogma, exposure to TOB elicits a complex, spatially-organized hierarchy of chemical messages to community members in response to this single stimulus in P. aeruginosa.

 

For more information see:

 

Morales-Soto, N., et al. “Antibiotic exposure induces spatially-dependent variations in alkyl quinolone signaling during Pseudomonas aeruginosa swarming,” J. Biol. Chem. 2018, 293, 9544-9552.

 

 

 

 

Impact of antibiotic treatment on P. aeruginosa swarm colonies. Exposure to (A) 0 μg antibiotic, (B) 25μg tobramycin (TOB), or (C) 400μg carbenicillin (CAR) by 5 μl liquid added to 10-mm diam. filter disc (at left); scale bar=5mm. CRM images from the edge proximal to the antibiotic treatment exhibit chemically specific responses to TOB (both PQS and AQNO subclasses), and PCA (loading plots and score images on PC1 and PC2) identifies chemically significant variations within samples. Loading plots for PC1 and PC2 include features corresponding to Raman spectra from cellular components (black), PQS/C9-PQS (blue), and AQNOs (HQNO/NQNO; red). Score images show the distribution of principal components.

BRG

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www.bohnresearchgroup.com

Email: bohngrp@nd.edu

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© Bohn Research Group.  Last updated July 1, 2015.