Fluorescent Hydrogel Sensor Particles for Detection of Protease Activity
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Fluorescent Hydrogel Sensor Particles for Detection of Protease Activity Alison Patrick, and Rein V Ulijn School of Materials & Manchester Interdisciplinary Biocentre (MIB), The University of Manchester, Grosvenor Street, Manchester, M1 7HS, United Kingdom ABSTRACT We demonstrate the design of novel sensor particles that display fluorescence in the presence of elastase, an enzyme that is present at elevated levels in chronic (non-healing) wounds. Poly(ethyleneglycol acrylamide) hydrogel particles, approximately 200µm in diameter are used as a polymeric matrix, to which a peptide based sensing element is attached. This sensing element consists of a Förster resonance energy transfer (FRET) pair separated by an enzyme cleavable linker. In addition, negatively charged Glutamic acid (Glu) residues are incorporated into the hydrogel structure to facilitate diffusion of the positively charged enzyme into the hydrogel matrix. Enzymatic hydrolysis of the enzyme cleavable linker results in fluorescence of the donor molecule being switched on. We have shown that these particles can detect elastase to a concentration of 100ng/ml, a concentration found in chronic wound fluids. These particles simultaneously detect and address balances in elastase levels and may therefore find applications as smart wound dressings. INTRODUCTION Enzymes play a vital role in the state of both healthy and diseased tissues. Imbalances in levels of enzymes can encourage the development of serious illness such as cancers[1, 2] or arthritis[3]. Due to the importance of these molecules in disease progression, sensors that can detect their presence and also systems that can address the imbalance by removal of excess enzymes are of significant interest. One example of a condition that is worsened by enzyme imbalance is the state of chronic wounds[4]. In this case elastase, a protease, is over-expressed leading to continual breakdown of tissue in the wound and hindering progression of the healing process[5, 6]. Work outlined here describes the design of sensor particles that can simultaneously detect elastase activity and absorb excess elastase. Elastase is a protease that naturally acts to degrade elastin. The sensor particles described here use a peptide derived from elastin, Ala-Ala-Pro-Val, that is specifically cleaved by elastase, as the enzyme cleavable linker (ECL) sensing element. This peptide is used as a linker to connect a FRET pair, EDANS (donor) and DABCYL
FRET
Elastase Fluorescence
Fig. 1 Sensor Design layout: FRET Donor is denoted by grey circle, FRET Acceptor is denoted by black circle, ECL is shown by black rectangle
(acceptor)[7, 8]. DABCYL is a dark quencher so when the FRET pair is intact no fluorescence is seen. This FRET-ECL component is coupled into PEGA1900 (poly (ethyelene glycol acrylamide) particles, ~200µm diameter, at the donor molecule end of the FRET pair, as shown in Fig.1. Elastase activity leads to hydrolysis of the ECL, removing the acceptor molecule and allowing fluorescence of the donor molecule to be
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