Submitted to: Carbohydrate Polymers
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/20/2014
Publication Date: 2/13/2015
Citation: Edwards, J.V., Prevost, N.T., French, A.D., Concha, M.C., Condon, B.D. 2015. Kinetic and structural analysis of fluorescent peptides on cotton cellulose nanocrystals as elastase sensors. Carbohydrate Polymers. 116:278-285.
Interpretive Summary: Cotton nanocrystalline cellulose possesses a very high surface area that has many potential uses in sensor technology. Biosensors were prepared to illustrate the highly sensitive levels of detection that are achievable. Fluorescent sensors were prepared and shown to possess sensitivity to proteases that are of diagnostic value in chronic diseases. The biosensor developed has potential as a point of care biomarker for numerous diseases. Thus, the manuscript discusses an approach to designing, 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. The fluorescent sensor is sensitive enough to be useful as a point of care diagnostic for elastase which is a biomarker for a number of chronic diseases as well.
Technical Abstract: Both human neutrophil elastase (HNE) and porcine pancreatic elastase (PPE) are serine proteases that have been associated with destructive proteolytic activity when their levels are elevated in chronic diseases. Thus there is considerable interest in the development of elastase sensors. Nanocyrstalline cellulose provides a biocompatible high surface area matrix for sensor-based detection of proteases. This paper reports the synthesis, characterization, and kinetic profiles of minimal peptide substrates of HNE and PE as conjugate fluorescent analogs of cotton cellulose nanocrystalline (CCN) for elastase detection. Elastase tri- and tetrapeptide substrates, N-Succinyl-Alaninyl-Prolinyl-Alaninyl-4-methyl-7-coumarylamide, and N-Succinyl-Alaninyl-Alaninyl-Prolinyl-Valinyl-4-methyl-7-coumarylamide were covalently attached to Glycine-esterified cotton cellulose nanocrystals (CCN). The degree of substitution (DS levels) of covalently linked peptide incorporated was found to be 3-4 peptides per 100 anhydroglucose units. X-ray diffraction analysis of the glycine and peptide-cellulose-nanocrystals revealed crystallinity indices of 79 and 76 percent respectively, and a crystallite size of 58.5 Å. Based on crystallite size and substitution levels of the peptides a model of the peptide-cellulose conjugate within a cellulose crystallite was constructed. The tripeptide conjugate analog attached to CCN demonstrated five-fold greater efficiency in detecting HNE than found for the same analog in solution as judged by its kcat/Km of 33,515. The sensor limits of detection at 2mg of the tri- and tetrapeptide CCN conjugates over a 10 minute reaction time course were found to be 0.03 U/mL PE and 0.05 U/mL HNE respectively.