Periodic Reporting for period 1 - ZINCLAPS (Light-addressable potentiometric sensors (LAPS) for zinc imaging with high spatiotemporalresolution for elucidating the role of zinc in age related macular degeneration.)
Période du rapport: 2017-06-21 au 2019-06-20
Light-addressable potentiometric sensors (LAPS) have great potential as a tool for functional electrochemical imaging of the attachment area of cells, providing information such as ion concentration, extracellular potentials and ion channel activity. The technique is particularly attractive for analysing cell responses of cells with planar polarisation as the cell-surface attachment area is not accessible to conventional electrophysiological measurements. The technique has the advantage that different spots on a flat, featureless electrolyte/insulator/silicon field effect structure can be addressed by scanning a focused light beam across the sample thereby exciting local photocurrents and generate an electrochemical image. However, current systems suffer either from poor resolution or slow scanning speed to monitor biological processes. In this project, we aimed to develop an electrochemical scanning setup with high spatiotemporal resolution that allows imaging of physiological processes with subcellular resolution and in real time.
The setup can be used with different ion sensitive surfaces to monitor the extracellular ionic flux and its role in cellular processes. The new instrument will lend itself to the investigation of disease mechanisms, the effects of toxicity and efficacy of drugs. In the long term, this could aid the development of organ on-a-chip devices, which can reduce the need for animal models. More specifically, we are planning to develop a zinc sensitive surfaces and use them in conjunction with the high-resolution and high-speed imaging setup to investigate the role of zinc in age related macular degeneration (AMD), the mechanism of which is still not well understood. This may lead to new treatments to prevent, forestall, or reverse the effects of the disease and elucidate zinc's role in other diseases including type 2 diabetes, pancreatic cancer, and Alzheimer disease and is therefore expected to impact the pharmaceutical industry and increase the quality of life for an ageing population.
The main objective of this project was to develop a high-speed and high-resolution LAPS setup with the capability of visualizing dynamic physiological events in a microenvironment. In particular, we were interested in real-time imaging of physiological parameters of living cells and establish protocols for studying their interactions with drugs and toxins.
The setup can be used with different ion sensitive surfaces to monitor the extracellular ionic flux and its role in cellular processes. The new instrument will lend itself to the investigation of disease mechanisms, the effects of toxicity and efficacy of drugs. In the long term, this could aid the development of organ on-a-chip devices, which can reduce the need for animal models. More specifically, we are planning to develop a zinc sensitive surfaces and use them in conjunction with the high-resolution and high-speed imaging setup to investigate the role of zinc in age related macular degeneration (AMD), the mechanism of which is still not well understood. This may lead to new treatments to prevent, forestall, or reverse the effects of the disease and elucidate zinc's role in other diseases including type 2 diabetes, pancreatic cancer, and Alzheimer disease and is therefore expected to impact the pharmaceutical industry and increase the quality of life for an ageing population.
The main objective of this project was to develop a high-speed and high-resolution LAPS setup with the capability of visualizing dynamic physiological events in a microenvironment. In particular, we were interested in real-time imaging of physiological parameters of living cells and establish protocols for studying their interactions with drugs and toxins.
We successfully developed a high-speed high-spatial-resolution LAPS setup using an analogue micromirror for scanning a laser beam across the sensor substrate.
Monolayer modified silicon-on-sapphire (SOS) substrates were modified with zinc chelators using an established “click” chemistry protocol for zinc sensing. Detailed surface characterization (atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), ellipsometry etc.) and the standard LAPS measurement were performed to optimise the surface modification procedure. A zinc sensitivity of 12 mV/p[Zn2+] was achieved. Work to improve the sensor response by increasing the surface loading of chelator on the sensor chip is currently under way.
To validate the new imaging setup, we focused on the fast imaging of single cells and monitoring their responses under the influence of different chemical stimulants using different semiconducting substrates. We also studied dynamic changes of the extracellular pH of living cells to monitor their metabolism. We successfully cultured B50 rat neural cells, MG63 human osteosarcoma cells and mesenchymal stem cells on a number of different semiconductor substrates with and without insulator. AC photocurrent imaging at the traditionally used SOS with a self-assembled organic monolayer as the insulator did not allow imaging of living cells in culture. Single cells were successfully visualized with photoelectrochemical imaging using a semiconductor/electrolyte structures without an insulator, thereby bypassing the difficulties encountered with insulated semiconductor substrates.
The main results achieved in this project are listed below:
i) We have developed a high-speed high lateral resolution electrochemical setup that can visualize the surface charge of a single-cell and monitor the dynamics of cell-lysis events.
The setup achieved a temporal resolution of 8 frames per second and a spatial resolution of 1.68 µm2 with SOS as a sensor plate.
ii) We use ac-photocurrent imaging at ITO coated glass substrates for mapping of cell surface charges of the basal, substrate facing side of various viable mammalian cells both in buffered electrolyte solution and cell culture medium. The technique was also used to monitor the lysis of mesenchymal stem cells.
iii) We investigated InGaN/GaN on sapphire as a new substrate for AC photoelectrochemical imaging of single cells. Photocurrent imaging at a low bias (0 V) and at high frequencies was demonstrated and photocurrent imaging of a cell was achieved. The low bias and the high frequency make InGaN a promising material for high-speed imaging with minimal damage to living cells.
iv) We introduced iron oxide on ITO-coated glass as a new substrate for fast photoelectrochemical imaging of single cells using the analogue micro-mirror based scanning technique. The dynamic activity of cells at different concentrations of surfactant TX-100 were studied. We also monitored the extracellular pH activity of B50 rat neuron cell after injecting glucose in a low buffer measurement solution.
Monolayer modified silicon-on-sapphire (SOS) substrates were modified with zinc chelators using an established “click” chemistry protocol for zinc sensing. Detailed surface characterization (atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), ellipsometry etc.) and the standard LAPS measurement were performed to optimise the surface modification procedure. A zinc sensitivity of 12 mV/p[Zn2+] was achieved. Work to improve the sensor response by increasing the surface loading of chelator on the sensor chip is currently under way.
To validate the new imaging setup, we focused on the fast imaging of single cells and monitoring their responses under the influence of different chemical stimulants using different semiconducting substrates. We also studied dynamic changes of the extracellular pH of living cells to monitor their metabolism. We successfully cultured B50 rat neural cells, MG63 human osteosarcoma cells and mesenchymal stem cells on a number of different semiconductor substrates with and without insulator. AC photocurrent imaging at the traditionally used SOS with a self-assembled organic monolayer as the insulator did not allow imaging of living cells in culture. Single cells were successfully visualized with photoelectrochemical imaging using a semiconductor/electrolyte structures without an insulator, thereby bypassing the difficulties encountered with insulated semiconductor substrates.
The main results achieved in this project are listed below:
i) We have developed a high-speed high lateral resolution electrochemical setup that can visualize the surface charge of a single-cell and monitor the dynamics of cell-lysis events.
The setup achieved a temporal resolution of 8 frames per second and a spatial resolution of 1.68 µm2 with SOS as a sensor plate.
ii) We use ac-photocurrent imaging at ITO coated glass substrates for mapping of cell surface charges of the basal, substrate facing side of various viable mammalian cells both in buffered electrolyte solution and cell culture medium. The technique was also used to monitor the lysis of mesenchymal stem cells.
iii) We investigated InGaN/GaN on sapphire as a new substrate for AC photoelectrochemical imaging of single cells. Photocurrent imaging at a low bias (0 V) and at high frequencies was demonstrated and photocurrent imaging of a cell was achieved. The low bias and the high frequency make InGaN a promising material for high-speed imaging with minimal damage to living cells.
iv) We introduced iron oxide on ITO-coated glass as a new substrate for fast photoelectrochemical imaging of single cells using the analogue micro-mirror based scanning technique. The dynamic activity of cells at different concentrations of surfactant TX-100 were studied. We also monitored the extracellular pH activity of B50 rat neuron cell after injecting glucose in a low buffer measurement solution.
The key achievement of this project is the development of a high-speed high lateral resolution electrochemical scanning set-up having great potential to visualize the dynamic changes in physiological functions in a microenvironment. The present setup is highly applicable to gain insight in the details of the dynamics of cell surface charge, extracellular potentials or ion channels at the subcellular level. The results of this project will inform drug-induced cell responses in unprecedented detail which are key to understanding how disease-associated biochemistry can take place even under a single cell and are directly relevant to the theme priority of personalizing health and care in Horizon 2020 - producing knowledge that will be applied in the area of health and medicine