Detection of Human Neutrophil Elastase by Fluorescent Peptide Sensors Conjugated to TEMPO-Oxidized Nanofibrillated Cellulose

Abstract

Peptide-cellulose conjugates designed for use as optical protease sensors have gained interest for point-of-care (POC) detection. Elevated serine protease levels are often found in patients with chronic illnesses, necessitating optimal biosensor design for POC assessment. Nanocellulose provides a platform for protease sensors as a transducer surface, and the employment of nanocellulose in this capacity combines its biocompatibility and high specific surface area properties to confer sensitive detection of 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. Here, we examine a tripeptide, fluorogenic elastase biosensor attached to TEMPO-oxidized nanofibrillated cellulose via a polyethylene glycol linker. The synthetic conjugate was found to be active in the presence of human neutrophil elastase at levels comparable to other cellulose-based biosensors. Computational models examined the relationship of the sensor molecule to the transducer surface. The results illustrate differences in two crystallite transducer surfaces ((110) vs. (1-10)) and reveal preferred orientations of the sensor. Finally, a determination of the relative (110) vs. (1-10) orientations of crystals extracted from cotton demonstrates a preference for the (1-10) conformer. This model study potentiates the HNE sensor results for enhanced sensor activity design.

Keywords: TEMPO-oxidized nanofibrillated cellulose; biosensors; computational modeling; human neutrophil elastase; peptides.

Figure 1

(a) Optical and (b) FE-SEM images of tNFC. The FE-SEM images were produced at 80,000× magnification with a 500 nm scale, and (c) the major components of the linker–fluorogenic elastase substrate, which is composed of PEG linker, Suc-APA elastase substrate, and AMC fluorophore. The AMC cleavage point is highlighted by the arrow.

Figure 2

Response curves of 2 mg of tNFC biosensors upon detection of 7-amino-4-methylcoumarin released with HNE at 0.5 U/mL substrate hydrolysis at 37 °C.

Figure 3

(a) Fluorescence intensity based on the number of sensors per crystallite volume, and (b) sensors per crystallite volume as a function of the calculated crystallite volume.

Figure 4

(a) Top-down view and (b) side view of the 5 × 5 TEMPO-oxidized transducer surface ((1−10) crystal plane) model without attached biosensor. In this model, the grey atoms correspond to carbon, red is for oxygen, and black for hydrogen.

Figure 5

Side view of the optimized peptide–cellulose conjugates (tNFC-Pep) for the (a) (1−10) crystal plane and the (b) (110) crystal plane.

Figure 6

(a) Minimized energy for each crystal surface with attached biosensor unit (tNFC-Pep) as a function of the dihedral angle, (b) Boltzmann distributions for each surface based on their optimized energies, and (c) the distance from the AMC cleavage point to the transducer surface (red dashed line) based on the optimized geometries for each crystal surface. The symbols with an “X” indicate geometries, which did not converge. Thus, the symbols with an “X” indicate calculations that did not fully converge, often because the initial geometry resulted in atomic overlap and are not considered viable structures.

Figure 7

(a) Experimental diffraction patterns for lyophilized CNC powder, and (b–d) results of Rietveld refinement of the entire diffraction pattern for each nanocrystalline material.