Peptide-cellulose conjugates designed for use as optical protease sensors have recently gained interest for point-of-care (POC) detection. Elevated serine protease levels are often found in patients with chronic illnesses, leading to increased enthusiasm for optimizing biosensor design for POC assessment. Biocompatible nanocellulose provides a platform for protease sensors as a transducer surface, and the employment of nanocellulose which has a high specific surface area confers sensitive detection of highly dilute biomarkers. However, a basic understanding of the spatiotemporal relationships of the transducer surface and sensor disposition is needed to improve protease sensor design and development. Therefore, balancing sensor detection sensitivity with selectivity poses fundamental questions about the molecular orientation of the sensor to the cellulose surface. Here we examine a model elastase biosensor attached to the glucan C6 hydroxyl of nanofibrillated, TEMPO-oxidized cellulose. The sensor is elongated with respect to the transducer surface by a PEGylated linker attached to an elastase substrate composed of a tripeptide and capped by a coumarin fluorophore. The conjugate was synthesized, characterized, and found to be active in the presence of Human Neutrophil Elastase. Computational models examined the relationship of the biosensor to the transducer surface by systematically varying the dihedral of the TEMPO-oxidized unit. The results of this modeling demonstrated preferred conformations for each surface based on energy minimization calculations. These calculations suggested notable differences in two crystallite transducer surfaces ((110) vs. (1-10) crystal planes) and revealed preferred orientations of the peptide with respect to the cellulose crystallite based on Boltzmann distribution determinations. On the (110) surface conformations, the rigid structure of the PEG oligomers holds the biosensor much closer to the transducer surface resulting in an average distance from the surface of only 6.9 Å. However, the distance of the protease sensor to the (1-10) surface was 24.4 Å, providing ample room for the protease to interact with the fluorophore. These results offer insight into potential design approaches for biosensors using specific peptide/cellulose crystallite orientations to provide the highest level of sensitivity for POC diagnostics.

Point-of-care protease sensors: Modeling and application of cellulose-based diagnostic devices

ACS Spring Meeting 2021

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