Peptide conjugated cellulose nanocrystals with sensitive human neutrophil elastase sensor activity

In chronic wounds, elevated human neutrophil 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 peptide conjugated cotton cellulose nanocrystals (CCN) is reported here. For this purpose a HNE tripeptide substrate, n-Succinyl-Alanine-Alanine-Valine-para-nitroanilide (Suc-Ala-Ala-Val-pNA), was covalently attached to glycine esterified CCN and compared with a similar tetrapeptide analog for colorimetric HNE sensor activity. 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 Glycine-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 that react with pNA were compared for their ability to enhance the visual sensor response to HNE activity. The colorimetric detection of HNE with CCN tripeptide conjugates was sensitive at HNE levels previously reported in chronic wound fluid (0.05 U/mL HNE). The HNE sensor and the chromogen amplifying dyes were interfaced with 50 and 10 kD dialysis cellulose membranes (DCM) to model filtration of HNE and chromogen (pNA) from a model wound dressing surface before and after sensor reactivity. The detection sensitivity to HNE activity was assessed with the CCN-tripeptide conjugate interfaced at the DCM surface distal and proximal to a dressing surface. The HNE sensor interfaced proximal to the dressing surface was most efficient with 10 kD membrane filtration of pNA and subsequent reaction with amplifying dyes. When interfaced with the 10 kD cellulose membrane, elastase sensor activity remained sensitive to 0.05 U/mL HNE. The nanocellulose surface properties, performance and design issues of the biosensor approach are discussed.