Submitted to: Cellulose
Publication Type: Peer reviewed journal
Publication Acceptance Date: 3/5/2013
Publication Date: 3/16/2013
Citation: Edwards, J.V., Prevost, N.T., Sethumadhavan, K., Ullah, A.H., Condon, B.D. 2013. Colorimetric elastase sensor with peptide-conjugated cellulose nanocrystals interfaced to dialysis membranes. Cellulose. 20:1223-1235. Interpretive Summary: The creation of new markets for value-added, cotton materials is currently an agriculture issue. Work in Southern Regional Research Center’s Cotton Chemistry Utilization Unit is addressing the application of bio-active molecules to cotton materials as a model to explore the creation of new value-added cotton products with highly selective properties and environmentally acceptable finishes. Previously we developed FDA approved cotton dressings that remove the destructive protease human neutrophil elastase from chronic wound and thus have an important use in chronic wound patients. In the course of our work to explore the potential uses of cotton cellulose nanocrystals (CCN) in wound care we found that they were very effective when combined with small peptides to ‘color visualize’ the presence of the destructive enzyme human neutrophil elastase which the cotton dressings remove from the wound. In addition it may be possible to use the cotton cellulose nanocrystals in combination with the cotton dressing to determine when the dressing has reached its capacity to remove elastase from the chronic wound. Thus, the manuscript discusses an approach to design, preparing and assessing the activity of CCN peptides to detect human neutrophil elastase at levels found in chronic wounds, and an approach to using the technology as a biosensor.
Technical Abstract: In chronic wounds, elevated human neutorphil elastase (HNE) is a destructive protease that has been proposed as a biomarker. Numerous wound dressing designs have been introduced in an effort to lower HNE levels. The clinical detection of HNE as a point of care biomarker or an in situ colorimetric adjuvant to chronic wound dressings presents potential advantages in the management of chronic wounds. A colorimetric approach to the detection of HNE using cotton cellulose nanocrystals (CCN) is reported here. For this purpose a HNE tripeptide substrate (Succinyl-Ala-Ala-Val-pNA) was covalently attached to glycine esterified CCN and compared with a tetrapeptide analog as a colorimetric HNE sensor. Visible HNE activity was significantly higher on CCN tripeptide conjugates when compared with similar analogs synthesized on paper. Upon enzymatic release of para-nitroaniline (pNA) from the CCN conjugate of succinyl-Ala-Ala-Val-pNA, amplification of the colorimetric response from pNA with reactive dyes enhanced visible absorption of the chromogen. Two color amplifying dyes which react with pNA were compared for their ability to enhance the visual sensor response to HNE activity. Thus, colorimetric detection of HNE with CCN peptide conjugates was sensitive at HNE levels typically found in chronic wound fluid (0.05 U/mL HNE). The HNE sensor and the chromogen amplifying dyes were interfaced with both a 50 and 10 kD dialysis cellulose membrane (DCM) to model filtration of HNE and chromogen (pNA) from a putative wound dressing surface before and after biosensor reactivity, respectively. The detection sensitivity to HNE activity was assessed with the CCN-tripeptide conjugate interfaced at the DCM surface distal and proximal to the dressing surface. The HNE sensor interfaced proximal to the dressing surface was most efficient with the 10kD membrane filtration of pNA followed by reaction with the amplifying dyes. When interfaced with the 10kD membrane elastase biosensor sensitivity of detection at 0.05 U/mL HNE was observed. The performance issues of each biosensor/DCM design approach are discussed.