Quantum Sensing Of Mitochondrial Function

Lead Research Organisation: University of Nottingham
Department Name: Faculty of Engineering

Abstract

Mitochondria are small bacteria like organelles contained inside everybody's cells. Often called the battery pack of a cell, they are responsible for taking the oxygen we breathe and using it to generate a molecule known as ATP, the unit of currency for energy production inside most living organisms. Mitochondria generate ATP using chemical reactions that push protons to one side of a small membrane inside the mitochondria. This generates an imbalance of electrical charge across the membrane called mitochondrial membrane potential (MMP), equivalent in strength to the electrical field required for a bolt of lightning to strike during a thunderstorm. This charge imbalance inside the mitochondria pushes protons back through a small protein motor on the membrane to generate ATP, giving cells the energy required to function in their day-to-day tasks. The importance of this little organelle should not be understated and it is widely held to have underpinned the evolution of all complex life on earth. Dysfunctional mitochondria can cause many problems for health and have been linked to a range of diseases such as Parkinson's, heart disease, cancer and obesity.
A by-product of this energy creating process in mitochondria are molecules called free radicals. The presence of free radicals inside the body are commonly thought to be a bad thing. It is true that in some circumstances they cause damage to the body however, these free radicals are also involved in many different processes in the body that are vital for the maintenance of health. As such free radicals have to be carefully regulated such that they are not being produced at harmful levels, but in sufficient amounts to allow cells to function normally. Collectively, this balance of free radical production and MMP is referred to as the mitochondrial redox state. This redox state can be a very good indicator of whether a cell is healthy or is undergoing stress or dysfunction. For example, a hallmark of many cancers is 'the Warburg effect' in which cancer cells have a very different mechanism for generating energy which implies a change in the function of mitochondria in growing tumours. Researchers have long been interested in how to better understand mitochondria. However, the technologies we use today have certain limitations; one example is the toxic side effects of different chemicals and invasive probes used to measure MMP.
In this work we will develop a new technology that can non-destructively study mitochondria more accurately than existing methods to increase our understanding of these organelles and help develop treatments for diseases more effectively. Our approach is based on a peculiar property of pink diamond that will allow us to use a light microscope to study MMP and free radical production in living cells. Pink diamonds obtain their pinkness due to the presence of Nitrogen impurities lodged in the diamond's usually pure carbon structure. These impurities absorb green light and re-emit red/pink light. Physicists have discovered in the last 10 years that the intensity of this light can be used to measure electromagnetic fields very accurately (~250,000 times smaller than the electric field present in mitochondria) and at very short length scales (about 1 million times smaller than the width of a human hair). Our proposed work involves patterning very thin slabs of diamond with a uniform surface layer of these impurities. Then using a series of controlled pulses of green light, we can take pictures of the red/pink fluorescence using a camera and reconstruct a spatial heat map of electric fields and free radicals produced by mitochondria in cells growing on the diamond surface. We predict this new technology could overcome many of the disadvantages of currently used techniques, and will be able to provide new information about how mitochondria work. This could then lead to new and effective treatments for different diseases for the benefit of all.

Technical Summary

Mitochondria (Mt) are small double walled membrane organelles found in all eukaryotic organisms and are vital for cell survival. Dysfunction of Mt underlies numerous diseases, predominantly those involving cells that have the highest energy demands (e.g. brain, heart, muscle). Our ability however for measurement of Mt function in living biological models at sufficient resolution and sensitivity is lacking. Here a visionary approach to Mt characterisation with single organelle sensitivity, nanoscale spatial resolution and millisecond measurement speed is proposed. This approach will exploit a major advance in fundamental physical science, namely atomic scale quantum sensors based on Nitrogen Vacancy (NV) colour defects in diamond. Central to this work is the hypothesis that the quantum spin dependant optical properties of NV defects can be harnessed to spatially and temporally study electromagnetic fields generated across the Mt membrane and reactive oxygen species (ROS) produced by Mt. The experimental program will combine state of the art advances in nanoscale surface structuring, quantum sensing protocols and optical engineering to develop a technology for non-destructive characterisation of Mt with unprecedented sensitivity. The proposed instrument will be tested and validated to assess its capability for quantum sensing in a controlled biological environment. A further advance will be the implementation of quantum bio-imaging at length scales below the optical diffraction limit using structured illumination microscopy. Following instrument testing and validation, studies of exemplar biological systems will be carried out. Mt extracted from cells and within cells will be characterised and findings compared with current state of the art Mt functional assays. This technology is projected to underpin a transformative step change in measurement capabilities in the life science.

Planned Impact

The expected beneficiaries of the proposed quantum sensing technology are detailed below.

Healthcare sector: Dysfunction of Mt underlies numerous diseases, predominantly those involving cells that have the highest energy demands (e.g. brain, heart, muscle). The spectrum of disease types is broad including inherited disorders (e.g. mitochondrial syndrome), metastatic cancers, acute conditions (e.g. cardiovascular disease) and chronic conditions (e.g. neurodegenerative). There is currently no cure for many of these diseases and whilst the past decade has seen major advances in the understanding disease genetic basis and pathology, these findings are yet to translate to new therapies. Deployment of the proposed enabling technology to study Mt function and dysfunction with unprecedented sensitivity and resolution will deliver new and complementary information to accelerate the pace towards effective treatments and support the identification of translatable biomarkers of disease progression. The successful application of the proposed quantum sensor in the life sciences will deliver benefit to patients through the provision of new therapies and economic benefit by reducing the burden of disease on an overstretched healthcare sector.

Pharmaceutical industry: Drug induced perturbation of Mt function is recognised as a contributing factor to the late stage attrition of several pharmaceuticals. Driven by the enormous costs associated with late stage failure, pharmaceutical companies are developing in vitro cellular models for detection of drug induced Mt dysfunction early in the development pipeline. Correspondingly, there is a demand for assays capable of characterising Mt function in intact cells to enable drug testing in an environment that aims at recapitulating the complex state of internal cellular signalling interactions found in vivo. The pharmaceutical industry also stands to benefit from the availability of Mt functional assays in intact cells capable of facilitating the identification and validation of new molecular drug targets. Such assays will also support the development of drugs that prevent the downstream damage associated with diseases, particularly in the case of mitochondrial syndrome where drugs could be designed to block for example cardiovascular damage. Overall it is anticipated that deployment of the proposed technology to assay Mt function in a manner accessible to the pharmaceutical industry will have significant impact on the development and manufacture of medicines that will not only create better outcomes for patients but will drive economic growth in the pharmaceutical industry.

Quantum technology: Entirely new industries are emerging due to the advent of quantum technologies. Quantum sensing achieve unprecedented sensitivity, accuracy and resolution in measurement by coherently manipulating quantum objects. In the context of this work, the quantum object is the unpaired electron associated with the NV centre in diamond which can be exploited as an extraordinarily sensitive room temperature magnetometer, deployed for nanoscale temperature measurements and in the near future play a major role in the semiconductor industry. Indeed, quantum sensors are expected to have a significant economic impact in the coming years in a number of sectors, including health care, defence and encrypted quantum communication networks. Government projections in 2015 cite the use of quantum diamond sensors for medical diagnostics as an integral part of the developing quantum imaging sector, projected to be worth £33B globally by 2020. This economic incentive, coupled with the opportunity to understand biological systems at an entirely new and fundamental level will no doubt have a significant contribution to the future development of the rapidly advancing field of quantum technologies and help identify routes for commercial deployment of a new set of products from medical devices to sensors and safer communication.

Publications

10 25 50
 
Description Early work has produced diamond sensors with patterned surfaces on a micron and nanoscale.
We have also demonstrated detection of free radicals through the use of a spin probe and NV sensing schemes. This has been verified with studies in known chemical reactions and then carried out in parallel with respirometry studies tracking changes in mitochondrial function in the presence of substrates and reagents that uncouple the electron transport chain.
Exploitation Route The very early findings could be taken forward by others to increase the sensitivity of current diamond sensing platforms. This has application not only in biology but also the findings are pertinent to biochemistry and nanomaterials. The free radical sensing protocol is not only applicable to the study of mitochondria but as free radicals are produced in many chemical reactions and physiological processes there is applicablity to other areas.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description Chair in Emerging Technologies
Amount £2,500,000 (GBP)
Funding ID CiET-2223-102 
Organisation Royal Academy of Engineering 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2023 
End 02/2033
 
Description Correlative Optical, Magnetic & Electron Miscopy Imaging and Analysis.
Amount £60,000 (GBP)
Funding ID 2443607 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 09/2020 
End 09/2024
 
Description ERC Consolidator Grant
Amount € 2,400,000 (EUR)
Organisation European Research Council (ERC) 
Sector Public
Country Belgium
Start 06/2016 
End 06/2021
 
Description Quantum Sensing Of Mitochondrial Function
Amount £147,439 (GBP)
Funding ID BB/T012226/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 08/2020 
End 02/2022
 
Title Magnetic modulation of NV 
Description A method in which a a large off-axis magnetic field is used to set the NV sensing quantisation axis and in doing so reduced the NV photoluminescence is developed. Under these conditions the eigenstates of the spin Hamiltonian are described by superpositions of the NV spin sublevels leading to spin state mixing. Experimentally this shortens the transverse relaxation time and increases the mean probability for non-radiative intersystem crossing (ISC) transitions for all spin states from the excited state to the metastable level, leading to a reduction in NV photoluminescence. The presence of proximal paramagnetic spin centres further reduces the efficiency of spin polarisation and the excited level lifetime, as observed as reduced contrast between a magnet on and off state. Importantly, this approach can be employed with a basic electromagnet and light emitting diode significantly simplifying the quantum sensing protocol and exapanding the accessibility of this method to non-specialists. 
Type Of Material Technology assay or reagent 
Year Produced 2022 
Provided To Others? No  
Impact This method enables quantum sensing to be performed using non-specialist equipment. In the context of studies performed within this research group the method has enabled oxidative phosphorylation to be monitored in mitochondrial extracts and live cells, the uptake of paramagnetic agents within cells and the magnetic transformation in spin cross over nanomaterials. Publications are in preparation and one is currently under review. 
 
Title Spin probe based free radical detection 
Description A new methodology for in culture detection of free radicals has been developed using Nitrogen Vacancy centres within diamond. The methodology involves the use of an electron paramagnetic resonance spin probe in combination with optical NV sensing schemes. The method has been compared with respirometry to study changes in mitochondrial function caused by the addition of substrates and electron transport chain uncouplers. This respresents as significant, new approach to detection of free radicals in biological systems compatible with physiological settings. Mitochondrial dysfunction is one of the many hallmarks of disease and in particular cancer. The use of the spin probe provides a means to target specific free radicals, in this example hydroxyl radicals. 
Type Of Material Technology assay or reagent 
Year Produced 2022 
Provided To Others? No  
Impact These are recent results however we are now testing this methodology to study mitochondrial dysfunction in well characterised cell lines. We wish to move this forward to studying disease samples, namely cancer and parkinsons disease. We are seeking funding to pursue this work. 
 
Description Collaboration with the University of Melbourne 
Organisation University of Melbourne
Country Australia 
Sector Academic/University 
PI Contribution Melissa Mather visited the University of Melbourne to meet with Dr Liam Hall. Dr Hall is a world leader in NMR implementations of diamond based quantum sensing. He has a current fellowship funded by the Australian government specifically to study fluids and the reactions within these using diamond based NMR. We have plans for Dr Hall to visit Nottingham to work with us and implement his methodology to increase the spectral resolution of diamond based NMR in our experimental system.
Collaborator Contribution Dr Hall provided Melissa Mather with technical advice on the implementation of high spectral resolution diamond sensing based NMR relevant to the Cancer Research UK grant.
Impact Melissa Mather visited the University of Melbourne and carried out initial experiments for NMR diamond based sensing of fluids.
Start Year 2020
 
Description Element Six 
Organisation De Beers Group
Department Element Six
Country Luxembourg 
Sector Private 
PI Contribution With funding from EPSRC we are developing a new technique for correlative microscopy linking electron microscopy with diamond based quantum microscopy. Element Six are arguably the world's largest supplier of quantum grade diamond. The work we are developing would provide a new appliation for their diamond products.
Collaborator Contribution Element Six are providing diamond materials that will be tested. They are also providing expert knowledge through technical support on diamond materials from Dr Matthew Markham.
Impact Invitation to speak at a European Union funded flagship progject on quantum sensing using diamond. Physics, Engineering, Life Sciences, Chemistry
Start Year 2020
 
Description Excel in Science 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Undergraduate students
Results and Impact Excel in Science aims to tackle the challenges faced in supporting more BAME students and those from disadvantaged backgrounds to progress into research careers and bring about this much needed, sustained change, through a series of events, internships, and a Nottingham Advantage Award module. I spoke at the kick off event for the current cohort describing my research journey. The hope is this will inspire or at least educate students on the course about careers in research.
Year(s) Of Engagement Activity 2022