Synthesis and characterization of TEMPO-oxidized peptide-cellulose conjugate biosensors for detecting human neutrophil elastase

Authors: Mackin, Robert; Fontenot, Krystal; Edwards, Judson - Vince; Prevost, Nicolette; Grimm, Casey; Condon, Brian; French, Alfred - Al; Easson, Michael; Jordan, Jacobs

Submitted to: Cellulose
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/1/2021
Publication Date: 1/9/2022
Citation: Mackin, R.T., Fontenot, K.R., Edwards, J.V., Prevost, N.T., Grimm, C.C., Condon, B.D., French, A.D., Easson, M.W., Jordan, J.H. 2022. Synthesis and characterization of TEMPO-oxidized peptide-cellulose conjugate biosensors for detecting human neutrophil elastase. Cellulose. (2022)29:1293-1305. https://doi.org/10.1007/s10570-021-04362-z.
DOI: https://doi.org/10.1007/s10570-021-04362-z

Interpretive Summary: Being able to effectively detect elevated levels of pathogens in chronic wounds is crucial to early diagnosis and treatment. Human Neutrophil Elastase (HNE) is one such pathogen. When present in normal levels in the body, it can aid in wound repair and regeneration. However, at elevated levels, HNE prevents wound healing and works to degrade healthy tissue, furthering the severity of the wound. In our work, we synthesize and characterize a new bio-sensing material based on a modified cellulose product, TEMPO-oxidized nanofibrillar cellulose, for detecting elevated levels of Human Neutrophil Elastase (HNE) in chronic wound fluids. Our work outlines the structure of the sensing device, calculates the number of sensors attached to the material surface, and compares these results to previous work on other cellulose-based sensor materials as a way to gauge the effectiveness of the diagnostic system. The results suggest that based on characterization and comparison, our TEMPO-oxidized material provides smaller cellulose crystallites and a larger number of sensors which implies it can be effectively used as a sensitive HNE detection device to be integrated into point-of-care diagnostic devices.

Technical Abstract: Here we describe the synthesis and characterization of a peptide-cellulose conjugate biosensor based on TEMPO-oxidized nanofibrillated cellulose for detecting elevated levels of human neutrophil elastase (HNE) in chronic wounds. The fluorescent peptide HNE substrate constructed from n-succinyl-Ala-Pro-Ala-7-amino-4-methyl-coumarin was attached to the TO cellulose surface via polyethylene glycol (PEG) linker. ATR-IR measurements confirm the attachment of the biosensor via spectral shift from a peak representing a carboxyl group to peaks suggesting the presence of amides. The characterization of the biosensor conjugate shows a high degree of peptide incorporation onto the surface with the degree of substitution (DS) sitting at 0.057 which is larger than the DS for any peptide-cellulose conjugate measured previously. The relatively small crystallite size of 26.0 Å compared to other cellulose- and nanocelluose-based materials leads to a large specific surface area (SSA) which promotes access of HNE to the enzyme substrates due to decreased steric interactions. Likewise, the porosity for tNFC was found to be higher than all other samples, including the nanocellulosic aerogel, lending to the hydrogel-like nature of TO cellulose. The volume of each crystallite was calculated and the volume ratio to the largest sample was determined. tNFC was found to occupy the smallest space implying a larger number of tNFC peptide-cellulose conjugates could fit into the same region occupied by a crystallite of another material. Further examination of the integration of PEGylated biosensors onto the tNFC surface showed high amounts of sensors per crystallite unit volume. With a small crystallite volume and large number of sensors, the tNFC peptide-cellulose conjugate biosensor could be a more sensitive system and a good candidate for POC diagnostic devices for detecting elevated protease levels in humans.