Abstract
Nanocellulose has high specific surface area, hydration properties, and ease of derivatization to prepare protease sensors. A Human Neutrophil Elastase sensor designed with a nanocellulose aerogel transducer surface derived from cotton is compared with cotton filter paper, and nanocrystalline cellulose versions of the sensor. X-ray crystallography was employed along with Michaelis–Menten enzyme kinetics, and circular dichroism to contrast the structure/function relations of the peptide-cellulose conjugate conformation to enzyme/substrate binding and turnover rates. The nanocellulosic aerogel was found to have a cellulose II structure. The spatiotemporal relation of crystallite surface to peptide-cellulose conformation is discussed in light of observed enzyme kinetics. A higher substrate binding affinity (Km) of elastase was observed with the nanocellulose aerogel and nanocrystalline peptide-cellulose conjugates than with the solution-based elastase substrate. An increased Km observed for the nanocellulosic aerogel sensor yields a higher enzyme efficiency (kcat/Km), attributable to binding of the serine protease to the negatively charged cellulose surface. The effect of crystallite size and β-turn peptide conformation are related to the peptide-cellulose kinetics. Models demonstrating the orientation of cellulose to peptide O6-hydroxymethyl rotamers of the conjugates at the surface of the cellulose crystal suggest the relative accessibility of the peptide-cellulose conjugates for enzyme active site binding.
Highlights
Nanocellulose-Based Protease SensorsThe sophistication and sensitivity of point of care diagnostic detectors underlies much of the progress in the rapidly growing field of biosensors as is found with point of care protease sensors [1].The sensor’s transducer serves to ground and facilitate transformation of the sensor molecule into a signature signal, which in this study is a fluorescence response indicating proteolytic activity
The intended application of the nanocellulose aerogel sensor is as an interface with chronic wound dressings to detect protease levels in situ
The functional properties of a protease sensor are determined by the composition of the transducer surface, the bioactivity of the enzyme substrate attached, and how the substrate is immobilized
Summary
The sophistication and sensitivity of point of care diagnostic detectors underlies much of the progress in the rapidly growing field of biosensors as is found with point of care protease sensors [1]. The sensor’s transducer serves to ground and facilitate transformation of the sensor molecule (in this study, a peptide fluorophore) into a signature signal, which in this study is a fluorescence response indicating proteolytic activity. The composition of the transducer surface material and its interaction at the biomolecular interface (protease binding on the transducer surface) is of equal.
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