Here we describe the synthesis and characterization of peptide conjugated cellulose and nanocellulose materials as sensors for fluorescent detection of human neutrophil elastase (HNE). The cellulose sensor surfaces selected are filter paper (FP) and print cloth (PC) fabric, which are composed of processed cotton fibers. The nanocellulose based sensors have transducer surfaces comprised of cellulose nanocrystals (wCNC) and microfibrillated cellulose (MFC) derived from wood that are fabricated as wood nanocellulose composites (wNCCs) consisting of blended quantities of nanocrystalline and MFC at 66/33 and 50/50 ratios. These ratios were selected to examine the effect of significantly different CNC loadings had on surface area and peptide uptake. The fluorescent peptide HNE substrate, n-succinyl-Ala-Pro-Ala-4-amido-7-methylcoumarin (Pep) was attached to both cellulosic and nanocellulosic matrices and conjugated peptide analogs were confirmed by mass spectrometry (MS) and infrared (IR). The nanocellulose biosensors wCNCPep (3c) and wNCC-Pep (4c, 66/33 and 5c, 50/50) have higher levels of peptide incorporation than the cellulosic biosensors FP-Pep (1c) and PC-Pep (2c). The range of incorporation for the cellulosic sensors is 7-25 lg/mg and for the nanocellulose sensors 30-80 lg/mg. The degree of substitution of peptide was found to be in the order of approximate number of peptides per 200 anhydroglucose residues, 1 in PC-Pep (2c), 2 in FP-Pep (1c), 4 in wNCC-Pep (5c, 50/50), 6 in wNCC-Pep (4c, 66/33), and 12 in wCNC-Pep (3c). The specific surface areas of the sensors ranged from 0.016 to 261 m 2 g -1 and correlated with degree of substitution of peptide on the cellulosic and nanocellulosic surfaces. Of the cellulose and nanocellulose biosensors, the wCNC-Pep (3c) has the highest level of peptide incorporation and the highest specific surface area, which makes it the preferred sensor matrix for human neutrophil elastase.
Nanocrystalline cellulose is an amphiphilic, high surface area material that can be easily functionalized and is biocompatible and eco-friendly. It has been used singularly and in combination with other nanomaterials to optimize biosensor design. The attachment of peptides and proteins to nanocrystalline cellulose and their proven retention of activity provide a route to bioactive conjugates useful in designs for point of care biosensors. Elastase is a biomarker for a number of inflammatory diseases including chronic wounds, and its rapid sensitive detection with a facile approach to sensing is of interest. An increased interest in the use of elastase sensors for point of care diagnosis is resulting in a variety of approaches to elsastase sensors utilizing different detection technologies. Here elastase substrate peptide-celluose conjugates synthesized as colorimetric and fluorescent sensors on cotton cellulose nanocrystals are compared. The structure of the sensor peptide-nanocellulose crystals when modeled with computational crystal structure parameters demonstrates the spatio-stoichiometric features of the nanocrystalline surface that allows ligand to active site protease interacttion. An understanding of the structure/function relations of enzyme and conjugate substrate of the peptides covalently attached to nancellulose has implications for enhancing the biomolecular transducer. The potential applications of both fluorescent and colorimetric detection to markers like elastase using peptide cotton cellulose nanocrystals as a transducer surface to model point of care biosensors for protease detection are discussed.
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