Label-free electrochemical detection of enzymatic activity using peptide microarrays
Lead Research Organisation:
University of Leeds
Department Name: Sch of Chemistry
Abstract
The sequencing of the human genome has provided a wealth of valuable information regarding the genetic basis of disease. However, the existence of a mere 30.000 human genes that encode an equal number of proteins cannot explain the complexity of human life and disease. Protein function is tightly regulated by a vast number of modifications and by interactions with other molecules. A comprehensive understanding of cellular function, in both healthy and diseased cells, requires knowledge of the functional state of proteins. Because enzymes are proteins with a crucial role in many physiological processes and diseases, the measurement of multiple enzymatic activities in biological samples and their correlation with cellular function can improve the understanding disease. Therefore, methods for the straightforward measurement of enzyme activity profiles are in high demand.
In this project we will develop a new method for the measurement of multiple enzymatic activities simultaneously using microarrays. The new technology will rely on a simple and cheap detection method, label-free electrochemical spectroscopy, which will enable its application in the development of point-of-care diagnostics.
In this project we will focus on proteases, enzymes that cleave the amide bonds of proteins (and shorter peptides). This process often leads to the activation or inactivation of the substrate proteins. Misregulation of protease activity is linked to many diseases such as cancer and cardiovascular disease. Furthermore, viruses such as HIV and Hepatitis C encode proteases that they use to successfully infect their hosts. Therefore, the measurement of specific protease activity profiles in biological samples such as blood may assist the early diagnosis of such diseases, and help monitor progress during treatment.
The project will involve the synthesis of protease substrates and their immobilisation on gold electrodes. We will measure the cleavage of the immobilised substrates by electrochemical spectroscopy. Once the conditions for the electrochemical detection of protease activity have been established, we will immobilise a number of protease substrates on a microarray and measure multiple protease activities simultaneously. In future projects, this methodology will be used as the basis for microarray-based diagnostics that profile protease activities in biological samples (e.g., blood or cell lysates).
In this project we will develop a new method for the measurement of multiple enzymatic activities simultaneously using microarrays. The new technology will rely on a simple and cheap detection method, label-free electrochemical spectroscopy, which will enable its application in the development of point-of-care diagnostics.
In this project we will focus on proteases, enzymes that cleave the amide bonds of proteins (and shorter peptides). This process often leads to the activation or inactivation of the substrate proteins. Misregulation of protease activity is linked to many diseases such as cancer and cardiovascular disease. Furthermore, viruses such as HIV and Hepatitis C encode proteases that they use to successfully infect their hosts. Therefore, the measurement of specific protease activity profiles in biological samples such as blood may assist the early diagnosis of such diseases, and help monitor progress during treatment.
The project will involve the synthesis of protease substrates and their immobilisation on gold electrodes. We will measure the cleavage of the immobilised substrates by electrochemical spectroscopy. Once the conditions for the electrochemical detection of protease activity have been established, we will immobilise a number of protease substrates on a microarray and measure multiple protease activities simultaneously. In future projects, this methodology will be used as the basis for microarray-based diagnostics that profile protease activities in biological samples (e.g., blood or cell lysates).
Planned Impact
Who?
In this project we will develop a new method for the measurement of multiple enzymatic activities simultaneously using microarrays. In addition to the impact the proposed project will have on academic research (see 'Academic beneficiaries'), the main beneficiaries will be:
1. Companies developing and selling diagnostic devices
2. The public
How?
Funding of this new investigator grant will pump-prime research into the development of electrochemical biosensors as diagnostics in the research group of the PI. The proposed project will deliver the first demonstration of a label-free capacitive biosensor of enzymatic activity. The new technology will be generally applicable for the detection of a number of enzymatic activities that lead to a change in charge/size of the substrates, such as (de)phosphorylation and acylation. The generally applicable technology will rely on a simple and cheap detection method - label-free electrochemical spectroscopy - which will enable its application in the future development of new point-of-care diagnostics. Such diagnostics could be used for the early detection of disease, the monitoring of progress during treatment and to influence the choice of medication.
In month 14 of this 15-month project, the PI will organise a workshop at Leeds with potential end-users of the developed methodology. In addition to future academic and clinical collaborators, these potential end-users will include industrial partners with enterprises in the diagnostic sector, the University spin-out eTect, and the University's Technology Transfer Agency (TechTran). During this workshop, we will explore opportunities for application and future commercialisation of the technology.
In order to ensure the development of the technology in a clinically relevant direction, the investigators engage (and will continue to engage) with clinicians in the field of cardiovascular disease (through the Multidisciplinary Cardiovascular Research Centre) and cancer (through the Biomedical and Health Research Centre) through regular meetings.
In this project we will develop a new method for the measurement of multiple enzymatic activities simultaneously using microarrays. In addition to the impact the proposed project will have on academic research (see 'Academic beneficiaries'), the main beneficiaries will be:
1. Companies developing and selling diagnostic devices
2. The public
How?
Funding of this new investigator grant will pump-prime research into the development of electrochemical biosensors as diagnostics in the research group of the PI. The proposed project will deliver the first demonstration of a label-free capacitive biosensor of enzymatic activity. The new technology will be generally applicable for the detection of a number of enzymatic activities that lead to a change in charge/size of the substrates, such as (de)phosphorylation and acylation. The generally applicable technology will rely on a simple and cheap detection method - label-free electrochemical spectroscopy - which will enable its application in the future development of new point-of-care diagnostics. Such diagnostics could be used for the early detection of disease, the monitoring of progress during treatment and to influence the choice of medication.
In month 14 of this 15-month project, the PI will organise a workshop at Leeds with potential end-users of the developed methodology. In addition to future academic and clinical collaborators, these potential end-users will include industrial partners with enterprises in the diagnostic sector, the University spin-out eTect, and the University's Technology Transfer Agency (TechTran). During this workshop, we will explore opportunities for application and future commercialisation of the technology.
In order to ensure the development of the technology in a clinically relevant direction, the investigators engage (and will continue to engage) with clinicians in the field of cardiovascular disease (through the Multidisciplinary Cardiovascular Research Centre) and cancer (through the Biomedical and Health Research Centre) through regular meetings.
Publications
Koutsoumpeli E
(2015)
Probing molecular interactions with methylene blue derivatized self-assembled monolayers
in Sensing and Bio-Sensing Research
Koutsoumpeli E
(2017)
Antibody Mimetics for the Detection of Small Organic Compounds Using a Quartz Crystal Microbalance
in Analytical Chemistry
Murray J
(2014)
Solid phase synthesis of functionalised SAM-forming alkanethiol-oligoethyleneglycols.
in Journal of materials chemistry. B
Description | We have developed an efficient synthesis of functionalised long-chain alkanethiol-oligoethyleneglycols (LCAT-OEGs), molecules that form well-defined self-assembled monolayers on gold. Our molecules are compatible with pre- or post-assembly conjugation of (bio)molecules. We have demonstrated the versatility of our synthetic route by synthesising LCAT-OEGs with a range of functional moieties, including peptides, electro-active redox groups, chemical handles for post-assembly conjugation of (bio)molecules. We also demonstrated the application of our LCAT-OEG monolayers in immunosensing, where they show good biocompatibility with minimal biofouling. These results have been published in J. Mater. Chem. B 2014, 2, 3741. We have also used one of our new, redox-active LCAT-OEGs to sense local pH changes, chemical reactions and (bio)molecular interactions on surfaces using a combination of cyclic voltammetry and QCM-D. These findings have been published in Sen. Bio-Sens. Res. 2015, 6, 1. We have used enzymatic ligations and bioorthogonal reactions for the site-specific immobilisation of (bio)molecules on self-assembled monolayers (unpublished). Finally, we have detected activities of clinically relevant proteases on our surfaces (unpublished). |
Exploitation Route | The successful development of synthetic routes to (redox-acitve) LCAT-OEGs has led to a number of collaborative spin-off projects, some of which have already attracted funding (AstraZeneca). |
Sectors | Chemicals Electronics Environment Healthcare Pharmaceuticals and Medical Biotechnology |
Title | methylene blue SAM |
Description | A long chain alkanethiol-oligoethyleneglycol modified with the redox-active group methylene blue |
Type Of Material | Technology assay or reagent |
Year Produced | 2013 |
Provided To Others? | Yes |
Impact | -initial results for a new grant application in electrochemical detection of environmental contaminants -paper in Sensing and Biosensing Research -paper in Analytical Chemistry |
URL | http://pubs.acs.org/doi/abs/10.1021/acs.analchem.6b04790 |
Description | Antibody-mimetic immunosensors for emerging environmental contaminants |
Organisation | University of Leeds |
Department | School of Molecular & Cellular Biology |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | We have provided biotinylated small molecules for binding protein selection |
Collaborator Contribution | The Tomlinson group has selected binding proteins against small molecules which will be used in our new project, for which we are currently seeking funding from NERC. |
Impact | Not yet |
Start Year | 2013 |
Description | Modified quantum-dots to measure NADH/NAD+ redox balance in cells |
Organisation | AstraZeneca |
Country | United Kingdom |
Sector | Private |
PI Contribution | Previous work on surface modification has led to a collaborative PhD project at Leeds: October 2014-September 2017 AstraZeneca top-up funding for a University of Leeds PhD studentship to Andrew Harvie, £24k 'Modified quantum-dots to measure NADH/NAD+ redox balance in cells' K. Critchley (PI), L. Jeuken, R.S. Bon In my group, Andrew has made redox-active ligands for his quantum dots. |
Collaborator Contribution | AZ provides experience in cellular delivery of quantum dots, and the PhD student has learned this in their labs in Cambridge |
Impact | none yet |
Start Year | 2014 |