Periodic Reporting for period 1 - Illuminate-AMR (Single-molecule visualization of individual multi-drug efflux pump activity in living bacteria with nanophotonic biochips)
Okres sprawozdawczy: 2022-01-01 do 2023-12-31
The problem being addressed in this project is multidrug resistance (MDR) in bacteria, which poses a significant threat to global health. MDR arises when bacteria develop the ability to resist multiple antimicrobial drugs, reducing the effectiveness of treatments and complicating the management of infections. A key mechanism behind this resistance is drug efflux, where bacteria actively pump out antibiotic molecules, decreasing their intracellular concentration and rendering treatments ineffective. Nevertheless, current imaging techniques are insufficient to visualize and understand the molecular processes driving this resistance, particularly at the bacterial membrane level. This technological gap similarly limits research in other membrane-controlled bioprocesses such as viral infections, cell signaling, and nutrient acquisition. Thus, a deeper understanding of the mechanisms behind drug resistance is essential for maintaining the efficacy of existing drugs and developing novel therapeutic strategies to combat resistant infections.
To meet this challenge, the project objectives are set to development and testing of a photonic biochip-based imaging platform for visualization and tracking of membrane-bound proteins i.e. multidrug efflux pumps in individual Escherichia coli cells. The biochip is designed for use with conventional light microscopes, making the single-molecule imaging accessible to many labs without the need to invest in costly and difficult-to-handle equipment. The project further aims to establish a direct assay for imaging and analyzing the activity of multidrug efflux pumps in E. coli cell membrane at the single-cell level. In addition, integration of the nanophotonic chip platform with a microfluidic control system is envisioned. Lastly, performance of the proposed imaging platform in detailing the resistance mechanisms in genetically engineered E. coli cells is to be compared with current single-molecule imaging methods on an industrial scale. To this end, the applications of this platform can be expanded in life sciences and material research, with potential integration into medical diagnostics and other industrial sectors.
To meet this challenge, the project objectives are set to development and testing of a photonic biochip-based imaging platform for visualization and tracking of membrane-bound proteins i.e. multidrug efflux pumps in individual Escherichia coli cells. The biochip is designed for use with conventional light microscopes, making the single-molecule imaging accessible to many labs without the need to invest in costly and difficult-to-handle equipment. The project further aims to establish a direct assay for imaging and analyzing the activity of multidrug efflux pumps in E. coli cell membrane at the single-cell level. In addition, integration of the nanophotonic chip platform with a microfluidic control system is envisioned. Lastly, performance of the proposed imaging platform in detailing the resistance mechanisms in genetically engineered E. coli cells is to be compared with current single-molecule imaging methods on an industrial scale. To this end, the applications of this platform can be expanded in life sciences and material research, with potential integration into medical diagnostics and other industrial sectors.
The main framework of this project is divided into three work packages: i) development and testing of a photonic chip with which to visualize and track membrane bound proteins and their activity in single living bacterial cells; ii) development of a direct assay for imaging the presence and activity of multi-drug efflux pumps in bacterial membranes at the single-cell level and single efflux pump resolution; and iii) implementation and comparison of the chip platform with modern single-molecule approaches. Despite the effort to adhere to the original schedule, the project encountered several unforeseen challenges mainly due to technical arrangements in implementing nanofabrication protocols and restrictions in building open-system microscopy and spectroscopy setups for ideal measurements. As a result, the arrangement for prototyping the chip has been delayed. Nevertheless, the inclusive scope of the project in introducing a micromachined optical setup for imaging live cells was not affected by the setbacks and the project delivered additional results in mapping the picosecond-scale viscoelastic anisotropy in diverse cell types.
The theoretical designs of the diffractive structures were performed using finite-difference time-domain method. The electromagnetic modes in various periodic media were solved analytically using open-source simulation packages in a Python environment. Models based on diffraction grating theory and Helmholtz wave equations were employed to study two different types of holograms, each supporting two modes: leaky and waveguide modes in a periodic structure, and two distinct waveguide modes in a biperiodic structure. By tuning the diffraction orders and subsequently confining the local density of optical states at two distinct resonant wavelengths, an optical sensing platform that can resolve 35.5 to 41.3 nm/RIU of spectral shift for two separate bioanalytes was then realized. The fabrication of the nanostructured chip was carried out at CEITEC Nano. The layer structure consisted of a 200 nm thin-film of patterned Si₃N₄ deposited on a standard microscope glass coverslip. The relief grating nanostructure was milled by focused ion beam lithography. The grating amplitude was subsequently optimized by in-situ correlative analysis of the surface topology using atomic force microscopy. The chip was tested with E. coli cells, in which the membrane protein AcrB is fused with the green fluorescent protein (AcrB-GFP). Images obtained in 3D from a confocal laser scanning microscope (CLSM Leica SP8), along with a wide field-of-view construct under total internal reflection fluorescence microscopy (TIRFM) resolved a selective measurement of the tagged efflux pump. To this end, the optical performance of the developed imaging platform can be optimized by an attenuation of the total reflection, where light is partially confined in the waveguide. The imaging setup for such adjustment is accessible at a project partner’s lab and a protocol for direct measurement of AcrAB-TolC efflux pump activity can be developed in the future.
The results of the project have been published in an article in a renowned, peer-reviewed scientific journal, at 3 international conferences/workshops, and in internal and external institutional seminars.
The theoretical designs of the diffractive structures were performed using finite-difference time-domain method. The electromagnetic modes in various periodic media were solved analytically using open-source simulation packages in a Python environment. Models based on diffraction grating theory and Helmholtz wave equations were employed to study two different types of holograms, each supporting two modes: leaky and waveguide modes in a periodic structure, and two distinct waveguide modes in a biperiodic structure. By tuning the diffraction orders and subsequently confining the local density of optical states at two distinct resonant wavelengths, an optical sensing platform that can resolve 35.5 to 41.3 nm/RIU of spectral shift for two separate bioanalytes was then realized. The fabrication of the nanostructured chip was carried out at CEITEC Nano. The layer structure consisted of a 200 nm thin-film of patterned Si₃N₄ deposited on a standard microscope glass coverslip. The relief grating nanostructure was milled by focused ion beam lithography. The grating amplitude was subsequently optimized by in-situ correlative analysis of the surface topology using atomic force microscopy. The chip was tested with E. coli cells, in which the membrane protein AcrB is fused with the green fluorescent protein (AcrB-GFP). Images obtained in 3D from a confocal laser scanning microscope (CLSM Leica SP8), along with a wide field-of-view construct under total internal reflection fluorescence microscopy (TIRFM) resolved a selective measurement of the tagged efflux pump. To this end, the optical performance of the developed imaging platform can be optimized by an attenuation of the total reflection, where light is partially confined in the waveguide. The imaging setup for such adjustment is accessible at a project partner’s lab and a protocol for direct measurement of AcrAB-TolC efflux pump activity can be developed in the future.
The results of the project have been published in an article in a renowned, peer-reviewed scientific journal, at 3 international conferences/workshops, and in internal and external institutional seminars.
The main potential of the project outcomes is the integration of diffractive supercell nanostructures into a microfluidic chip, a platform with real-life applications in biomedical research. The prototyped chip is compatible with fiber-coupled optical imaging systems and can offer an improved illumination uniformity, a smaller footprint, and a more accurate knowledge of penetration depth of the evanescent field. Such capabilities compliment the current single-molecule approaches in measuring MDR activity in single cells. In conclusion, the scalable chip technology is projected to provide a wide-field imaging platform for measuring drug efflux kinetics in E. coli cells, ready for use in lab settings. These outcomes have immediate implications for the study of MDR in bacteria and for the investigation of other biological membrane processes.
The project has socio-economic impacts. The technology will bring about faster and affordable diagnostic and prognostic devices with commercial interests in the optical microscopy market. This is particularly relevant considering global healthcare challenges, where diagnostic tools are in high demand.
The project has socio-economic impacts. The technology will bring about faster and affordable diagnostic and prognostic devices with commercial interests in the optical microscopy market. This is particularly relevant considering global healthcare challenges, where diagnostic tools are in high demand.