The aims of the research on chemical probes are to develop chemical probes with longer wavelengths and broader fluorescent life-time capabilities as novel imaging reagents and probes, and to develop new chemistries to expand their possibilities and application for diagnosing and treating disease.
Bacterial and fungal probes
Optimising the design of BAC probes leads to higher specificity and selectivity of pathogen detection. Caspofungin probes allow detection of fungal infections. The new P11 probe family has made significant improvements on the original BAC ONE probe.
See the following publications:
- Megia-Fernandez, A., Klausen, M., Mills, B., Brown, G. E., McEwan, H., Finlayson, N., Dhaliwal, K., & Bradley, M. (2021). Red-Shifted Environmental Fluorophores and Their Use for the Detection of Gram-Negative Bacteria. Chemosensors, 9(6), 117. https://www.mdpi.com/2227-9040/9/6/117
- Baibek, A., Üçüncü, M., Short, B., Ramage, G., Lilienkampf, A., & Bradley, M. (2021). Dyeing fungi: amphotericin B based fluorescent probes for multiplexed imaging [10.1039/D0CC08177A]. Chemical Communications, 57(15), 1899-1902. https://doi.org/10.1039/D0CC08177A
- Baibek, A., Üçüncü, M., Blackburn, E. A., Bradley, M., & Lilienkampf, A. (2021). Wash-free, peptide-based fluorogenic probes for microbial imaging. Peptide Science, 113(1), e24167. https://doi.org/https://doi.org/10.1002/pep2.24167
Longer lifetime dyes: New dyes with long lifetimes have been conjugated to polymyxin to improve imaging of bacteria via FLIM as well as allows the analysis of fluorescent life-time pre- and post-binding. Life-time imaging of protease activity is on-going.
Probes in the deep red (NIR, NIR-I, NIR-II): New NIR-I and NIR-II dyes will allow the ability to ‘see’ deeper inside the body. Research in this area is focusing on synthesising peptide-based probes for Granzyme B, synthesising NIR FRET pairs and evaluating the fluorescence lifetime imaging of these dyes.
The Bradley group developed the concept of localising photodynamic therapy via attachment of a photosensitiser to a bacterial or fungal targeting ligand. A photosensitiser is a dye that locally transforms molecular oxygen into a highly toxic species when it is activated by a specific wavelength of light. In photodynamic therapy (PDT), the reactive oxygen species (ROS) generated by the dye can be exploited for the localised killing of cells and microorganisms exclusively at the location of the illumination.
Antimicrobial PDT (aPDT) has become a promising alternative to classic antibiotic treatment thanks to the absence of possible resistance mechanisms in microorganisms. The challenge is to design aPDT probes with high levels of selectivity to ensure complete eradication of microbes while minimising damage to healthy cells.
Our PDT probes are typically synthesised by covalent attachment of photosensitizers with pathogen-targeting agents. A novel polymyxin-methylene blue conjugate has demonstrated high potential for aPDT against Gram-negative bacteria, and other theranostic probes are currently investigated to detect and kill a wide range of bacteria and fungi.
See the following publications:
- Klausen, M., Ucuncu, M., & Bradley, M. (2020). Design of Photosensitizing Agents for Targeted Antimicrobial Photodynamic Therapy. Molecules, 25(22), 5239. https://www.mdpi.com/1420-3049/25/22/5239
- Ucuncu, M., Mills, B., Duncan, S., Staderini, M., Dhaliwal, K., & Bradley, M. (2020). Polymyxin-based photosensitizer for the potent and selective killing of Gram-negative bacteria. Chemical Communications, 56(26), 3757-3760. https://doi.org/10.1039/D0CC00155D
Proteases are excellent biomarkers for a variety of diseases and offer multiple opportunities for diagnostic applications. Proteases are enzymes with wide-ranging and crucial activities that mediate the hydrolysis of amide bonds and play crucial roles in the cell function. Dysfunction of proteases can be highly detrimental and has been linked to a variety of diseases. We developed a series of sensors that target human neutrophil elastase (hNE), a serine protease implicated in the pathogenesis of acute and chronic inflammatory diseases. The probes provide an OFF/ON fluorescent signal in activated neutrophils and allow the detection of Neutrophil Extracellular Traps (NETs). The first probe developed emits in the green, and current efforts are focused on synthesising a second generation that emits in the NIR region.
We are also working on fluorogenic probes that target Granzyme B, a protease involved in the T-immune response, mediating the immune response in pathologies such as transplant rejection or cancer. We are developing a series of pH insensitive sensors that can detect Cathepsin D activity, another protease that gets activated in macrophages upon bacterial infection.
Enzyme probes include:
- A library of Near-infrared (NIR) probes for human neutrophil elastase
- Probes targetting granzyme B probe to image activation of T cells
- A pH insensitive probe for Cathepsin D
- Probes for Fibroblast Activation Protein (FAP)
- Matrix Metalloproteinase (MMP) probes that target lung fibrosis/cancer
See the following publications:
- Optical Detection of Distal Lung Enzyme Activity in Human Inflammatory Lung Disease. A.Megia-Fernandez, A.Marshall, A.R.Akram, B.Mills, S.V.Chankeshwara, E.Scholefield, A.Miele, B.C.McGorum, C.Michaels, N.Knighton, T.Vercauteren, F.Lacombe, V.Dentan, A.M.Bruce, J.Mair, R.Hitchcock, N.Hirani, C.Haslett, M.Bradley, K.Dhaliwal, BMEFrontiers 2021, 9834163.
- Rios, M. R., Garoffolo, G., Rinaldi, G., Megia-Fernandez, A., Ferrari, S., Robb, C. T., Rossi, A. G., Pesce, M., & Bradley, M. (2021). A fluorogenic peptide-based smartprobe for the detection of neutrophil extracellular traps and inflammation [10.1039/D0CC07028A]. Chemical Communications, 57(1), 97-100. https://doi.org/10.1039/D0CC07028A
- Mills, B., Norberg, D., Dhaliwal, K., Akram, A. R., Bradley, M., & Megia-Fernandez, A. (2020). A matrix metalloproteinase activation probe for painting human tumours [10.1039/D0CC03886E]. Chemical Communications, 56(69), 9962-9965. https://doi.org/10.1039/D0CC03886E
- Bimodal fluorogenic sensing of matrix proteolytic signatures in lung cancer. A.Megia-Fernandez, B.Mills, C.Michels, S.V.Chankeshwara, N.Krstajić, C.Haslett, K.Dhaliwal and M.Bradley Org Biomol Chem, 2018, 16, 8056-8063. Featured on the cover.
Sensor functionalised particles support pH and oxygen sensing in the lung
- High fidelity fibre-based physiological sensing deep in tissue. T.R.Choudhary, M.G.Tanner, A.Megia-Fernandez, K.Harrington, H.A.Wood, A.Marshall, P.Zhu, S.V.Chankeshwara, D.Choudhury, G.Monro, M.Ucuncu, F.Yu, R.R. Duncan, R.R.Thomson, K.Dhaliwal and M.Bradley Sci Rep 2019, 9, 7713
Light-responsive compounds: Visible-light-responsive polymers can be used to support light-powered clinical devices. Under exploration are thiol-modified light-responsive compounds and polymer microarrays that can detect novel photochromic polymers as well an NIR photochromic compound and light-driven nanoparticle (NP) drug release (NIR-I and II).
Diagnostic tools: Diagnostic tools can allow rapid detection and classification of bacteria and fungi and could have clinical relevance in guiding antibiotic selection. The proof-of-concept technology for a non-antibody based lateral flow test is proven, and includes antibiotic based chemistries for selective binding of bacteria to surfaces in flow.