Human neutrophil elastase peptide sensors conjugated to cellulosic and nanocellulosic materials: part I, synthesis and characterization of fluorescent analogs

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 wCNC-Pep (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 μg/mg and for the nanocellulose sensors 30–80 μg/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 m² 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.