chemosensors Article Red-Shifted Environmental Fluorophores and Their Use for the Detection of Gram-Negative Bacteria Alicia Megia-Fernandez 1, *, Maxime Klausen 1 , Bethany Mills 2 , Gillian E. Brown 3 , Heather McEwan 1 , Neil Finlayson 3 , Kevin Dhaliwal 2 and Mark Bradley 1   Citation: Megia-Fernandez, A.; Klausen, M.; Mills, B.; Brown, G.E.; McEwan, H.; Finlayson, N.; Dhaliwal, K.; Bradley, M. Red-Shifted Environmental Fluorophores and Their Use for the Detection of Gram-Negative Bacteria. Chemosensors 2021, 9, 117. https:// doi.org/10.3390/chemosensors9060117 Academic Editor: Mark Lowry Received: 27 April 2021 Accepted: 18 May 2021 Published: 21 May 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1 EaStCHEM School of Chemistry, University of Edinburgh, David Brewster Road, Edinburgh EH9 3FJ, UK; mklausen@ed.ac.uk (M.K.); H.McEwan-1@sms.ed.ac.uk (H.M.); Mark.Bradley@ed.ac.uk (M.B.) 2 EPSRC Proteus IRC Hub, Centre for Inflammation Research, Queen’s Medical Research Institute, University of Edinburgh, 47 Little France Crescent, Edinburgh EH16 4TJ, UK; Beth.Mills@ed.ac.uk (B.M.); Kev.Dhaliwal@ed.ac.uk (K.D.) 3 Institute for Integrated Micro and Nano Systems, School of Engineering, University of Edinburgh, Edinburgh EH9 3FF, UK; g.e.brown@ed.ac.uk (G.E.B.); N.Finlayson@ed.ac.uk (N.F.) * Correspondence: A.Megia@ed.ac.uk Abstract: Two novel, water-soluble, merocyanine fluorophores were readily prepared by microwave- assisted synthesis. Full optical characterization was performed in a series of protic and aprotic solvents, and the dyes displayed fluorescence in the red region with up to a 20-fold decrease in brightness in water, demonstrating a strong environmental sensitivity hereby termed as solvato- fluorogenicity (to distinguish from solvatochromism). Shorter fluorescent lifetimes were also mea- sured in water, which confirmed this character. These dyes were conjugated to a modified polymyxin scaffold that allowed fluorescence “switch-on” upon binding to Gram-negative bacterial membranes, and selective fluorescence detection of bacteria in a wash-free protocol. Keywords: merocyanines; solvato-fluorogenicity; environmental fluorophores; optical imaging; bacterial detection; fluorescent labelling; fluorescence lifetime 1. Introduction Bacterial infections are one of the world’s leading cause of human disease and death [1,2] and current diagnostic methods for identification of microbial infections often involve time-consuming culture and staining of micro-organisms, which do not provide immediate results. However, a “belt-and-braces” approach to treatment is often initiated empirically before confirmed diagnosis, which leads to over/misuse of antibiotics. Many research efforts are focusing on the development of rapid and reliable point of care diagnos- tics techniques as a means to optimize therapy, and reduce the emergence of antimicrobial resistance. Although optical methods such as classic Gram staining have been used for decades, real-time “molecular imaging” of bacteria is still in its infancy compared to the achievements in cancer imaging [3]. Considerable potential lies in the translation of bacterial imaging probes into clinical practice, but their contribution is still scarce in practice [4]. Several review papers have covered the topic of fluorescent antibiotic-based probes [5–7] and their potential use in the area of antimicrobial resistance. In this regard, such pathogen-binding probes have the potential to detect bacterial infections, elucidate the mode of action of the antimicro- bial agents and resistance mechanisms, and assess drug susceptibilities etc. Among the pathogen-targeting agents available, polymyxins (PMX) are an example of a naturally occurring class of cyclic antibiotic lipopeptides that bind to lipid A on the Gram-negative bacterial outer membrane through electrostatic interactions while also anchoring into the lipid bilayer. Fluorescent probes based on polymyxin have been reported incorporating fluorophores such as dansyl [8,9] with the purpose of gaining a better understanding Chemosensors 2021, 9, 117. https://doi.org/10.3390/chemosensors9060117 https://www.mdpi.com/journal/chemosensors