Introduction: Wound healing is the very complex and dynamic process of replacing damaged structures with new cells and tissues. While most of the wounds undergo normal healing without delay, chronic wounds require comprehensive therapy and monitoring of the healing status. In standard monitoring procedures the wound dressing is removed for optical investigation and subjective classification. This procedure increases chances of infection, causes skin irritation and can provide only qualitative information. To minimize such risks and promote healing, new smart wound dressings have to be developed. Here we present an innovative wound dressing able to report on the conditions of the wound by converting the changes in essential enzymes of the wound exudate into a fluorescent signal[1].
Figure 1 The design and function of the smart wound dressing. The biosensors in the dressing can detect key parameters for wound healing, like neutrophil elastase concentration, by changes in fluorescent signal.
Materials and Methods: Proteases such as neutrophil elastase were detected using a lab-designed FRET-based protein biosensor. The biosensor is composed of two fluorescent proteins connected with a short peptide linker, which is specifically recognized by the enzyme. The biosensor was recombinantly produced in Escherichia coli and purified. The protease enzyme activity was monitored by fluorescence in a 450-600 nm range upon excitation at 428 nm in the presence of the biosensor.
Results and Discussion: The purified biosensor for neutrophil elastase showed FRET ratio of ~1. Upon incubation with the neutrophil elastase in PBS buffer the FRET ratio decreased from 1 to 0.7 indicating complete dissociation of the two fluorescent proteins. Neutrophil elastase exhibited high affinity to the substrate with KM of 0.7 µM and the kinetic parameters were not affected when enzyme was assayed in a complex medium like DMEM.
Figure 2 Left: decrease of FRET ratio in time after neutrophil elastase addition. Right: changes in fluorescence emission spectra of the protease sensor upon neutrophil elastase addition.
Conclusion: Designed substrate can detect changes of one of the basic parameters which indicate the direction of healing process. It has a potential application as a sensing element in the wound dressing. After immobilization, the biosensor could be used as a screening platform of biological fluids. The detection range might be expanded by engineering of the FRET biosensor.
This work was financed by the Swiss Confederation and funded by Nano-Tera.ch within the Nano-Tera project “Fabrication of fluorescence biosensors in a Textile Dressing for Non-invasive Lifetime Imaging-based Wound Monitoring”, FLUSITEX (RTD 2013) that was scientifically evaluated by the SNSF.
References:
[1] C. Schulenburg, G. Faccio*, D. Jankowska, K. Maniura-Weber and M. Richter*, "A fluorescent biosensor for highly specific neutrophil elastase detection", currently under review.