Label-free electrochemical detection of enzymatic activity using peptide microarrays

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).

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.