, Volume 23, Issue 2, pp 1297–1309 | Cite as

Human neutrophil elastase detection with fluorescent peptide sensors conjugated to cellulosic and nanocellulosic materials: part II, structure/function analysis

  • Krystal R. Fontenot
  • J. Vincent Edwards
  • David Haldane
  • Elena Graves
  • Michael Santiago Citron
  • Nicolette T. Prevost
  • Alfred D. French
  • Brian D. Condon
Original Paper


Human neutrophil elastase (HNE) is one of a number of proteases that is receiving increased attention as a marker for inflammatory diseases and sensor-based point of care diagnostics. Integral to sensor-based detection is the transducer surface, which is the platform of the sensor’s signal transmittance. Here we describe the bioactivity and related transducer surface properties of cellulose and nanocellulose matrices as peptide–cellulose fluorescent sensors. Detection sensitivity of the sensor signals for HNE levels typically found in chronic wounds is characterized. The fluorescent elastase peptide substrate, Succinamidyl-Ala-Ala-Pro-Val-amidylcoumadin (Pep) was employed in both cellulose and nanocellulose transducer surfaces evaluated for biosensor sensitivity to HNE. The cellulose transducers selected are filter paper (FP) and print cloth (PC) fabric and are comprised of processed cotton fibers. The nanocellulose transducers are the wood cellulose nanocrystals (wCNC) and the wood nanocellulose composites (wNCC). The wNCCs consist of blended quantities of nanocrystalline and microfibrillated cellulose at 66/33 and 50/50, and are characterized as thin films. The biosensor activity was in the order of wCNC-Pep > FP-Pep = NCC-Pep (50/50) > NCC-Pep (66/33) > PC-Pep. Sensor sensitivity correlated with specific surface area. A depiction of peptide substitution on nanocellulosic and cellulosic surfaces is rendered through peptide–cellulose crystallite models derived from X-ray diffraction analysis of the material, and the models discussed in light of biosensor structure activity relationships. In addition, the overall morphology, pore size and porosity of the materials are discussed for their suitability as protease sensors.


Peptides Wood cellulose nanocrystals Wood nanocellulose composites Human neutrophil elastase Biosensors 



We thank Jade Smith for assisting with collecting AFM images.


  1. Brunauer S, Emmett PH, Teller E (1938) Adsorption of gases in multimolecular layers. J Am Chem Soc 60:309–319. doi: 10.1021/ja01269a023 CrossRefGoogle Scholar
  2. Canetta E, Montiel K, Ady AK (2009) Morphological changes in textile fibres exposed to environmental stresses: atomic force microscopic examination. Forensic Sci Int 191:6–14CrossRefGoogle Scholar
  3. Dargaville TR, Farrugia BL, Broadbent JA, Pace S, Upton Z, Voelcker NH (2013) Sensors and imaging for wound healing: a review. Biosens Bioelectron 41:30–42. doi: 10.1016/j.bios.2012.09.029 CrossRefGoogle Scholar
  4. Das G, Talukdar P, Matile S (2002) Fluorometric detection of enzyme activity with synthetic supramolecular pores. Science 298:1600–1602. doi: 10.1126/science.1077353 CrossRefGoogle Scholar
  5. De Oliveria RRL, Albuquerque DAC, Cruz TGS, Yamaji FM, Leite FL (2012) Measurement of the nanoscale roughness by atomic force microscopy: basic principles and applications. In: Bellitto V (ed) Atomic force microscopy—imaging, measuring and manipulating surfaces at the atomic scale. InTech. doi: 10.5772/2673
  6. Deka G, Wu W-W, Kao F-J (2013) In vivo wound healing diagnosis with second harmonic and fluorescence lifetime imaging. J Biomed Opt 18:061222-061221–061222-061228Google Scholar
  7. Diegelmann RF, Evans MC (2004) Wound healing: an overview of acute, fibrotic and delayed healing. Front Biosci 9:283–289CrossRefGoogle Scholar
  8. Dodson B (2012) Wood pulp extract stronger than carbon fiber or Kevlar. Accessed 01/05/2015
  9. Edwards JV, Goheen SC (2006) Performance of bioactive molecules on cotton and other textiles. Res J Text Appar 10:19–32Google Scholar
  10. Edwards JV, Prevost N, Sethumadhavan K, Ullah A, Condon B (2013) Peptide conjugated cellulose nanocrystals with sensitive human neutrophil elastase sensor activity. Cellulose 20:1223–1235. doi: 10.1007/s10570-013-9901-y CrossRefGoogle Scholar
  11. Edwards JV, Prevost NT, French AD, Concha M, Condon BD (2015) Kinetic and structural analysis of fluorescent peptides on cotton cellulose nanocrystals as elastase sensors. Carbohydr Polym 116:278–285. doi: 10.1016/j.carbpol.2014.04.067 CrossRefGoogle Scholar
  12. Edwards JV, Fontenot KR, Haldane D, Prevost NT, Condon BD (2016) Human neutrophil elastase peptide sensors conjugated to cellulosic and nanocellulosic materials: part I, synthesis and characterization of fluorescent analogs. Cellulose. doi: 10.1007/s10570-016-0869-2 Google Scholar
  13. Eichhorn SJ (2011) Cellulose nanowhiskers: promising materials for advanced applications. Soft Matter 7:303–315. doi: 10.1039/C0SM00142B CrossRefGoogle Scholar
  14. French A, Santiago Cintrón M (2013) Cellulose polymorphy, crystallite size, and the Segal crystallinity index. Cellulose 20:583–588. doi: 10.1007/s10570-012-9833-y CrossRefGoogle Scholar
  15. International consensus (2011) The role of proteases in wound diagnostics. An expert working group review. Wounds International, LondonGoogle Scholar
  16. Henares TG, Takaishi M, Yoshida N, Terabe S, Mizutani F, Sekizawa R, Hisamoto H (2006) Integration of multianalyte sensing functions on a capillary-assembled microchip: simultaneous determination of ion concentrations and enzymatic activities by a “drop-and-sip” technique. Anal Chem 79:908–915. doi: 10.1021/ac061245i CrossRefGoogle Scholar
  17. Hyungki K, Petryayeva E, Algar WR (2014) Enhancement of quantum dot Forster resonance energy transfer within paper matrices and application to proteolytic assays. IEEE J Sel Top Quantum Electron 20:141–151. doi: 10.1109/JSTQE.2013.2280498 Google Scholar
  18. Korkmaz B, Horwitz MS, Jenne DE, Gauthier F (2010) Neutrophil elastase, proteinase 3, and cathepsin G as therapeutic targets in human diseases. Pharmacol Rev 62:726–759. doi: 10.1124/pr.110.002733 CrossRefGoogle Scholar
  19. Lagerwall JPF, Schutz C, Salajkova M, Noh J, Hyun Park J, Scalia G, Bergstrom L (2014) Cellulose nanocrystal-based materials: from liquid crystal self-assembly and glass formation to multifunctional thin films. NPG Asia Mater 6:e80. doi: 10.1038/am.2013.69 CrossRefGoogle Scholar
  20. Lahiji RR, Reifenberger R, Moon RJ, Rudie A (2008) Characterization of cellulose nanocrystals by SPM. In: NSTI nanotechnology conference and trade show: life sciences, medicine & bio materials, Boston, MA. Nano Science and Technology Institute, Inc., Boca Raton, FL, USA, pp 704–707. 1–5 June 2008Google Scholar
  21. Liu Q, Wang J, Boyd BJ (2015) Peptide-based biosensors. Talanta 136:114–127. doi: 10.1016/j.talanta.2014.12.020 CrossRefGoogle Scholar
  22. Maver T, Maver U, Mostegel F, Griesser T, Spirk S, Smrke D, Stana-Kleinschek K (2015) Cellulose based thin films as a platform for drug release studies to mimick wound dressing materials. Cellulose 22:749–761. doi: 10.1007/s10570-014-0515-9 CrossRefGoogle Scholar
  23. McCarty SM, Percival SL (2013) Proteases and delayed wound healing. Adv Wound Care 2:438–447. doi: 10.1089/wound.2012.0370 CrossRefGoogle Scholar
  24. Moreau C, Beury N, Delorme N, Cathala B (2012) Tuning the architecture of cellulose nanocrystal–poly(allylamine hydrochloride) multilayered thin films: influence of dipping parameters. Langmuir 28:10425–10436. doi: 10.1021/la301293r CrossRefGoogle Scholar
  25. Mushi NE, Butchosa N, Zhou Q, Berglund LA (2014) Nanopaper membranes from chitin–protein composite nanofibers—structure and mechanical properties. J Appl Polym Sci. doi: 10.1002/app.40121 Google Scholar
  26. Naseri N, Mathew A, Girandon L, Fröhlich M, Oksman K (2015) Porous electrospun nanocomposite mats based on chitosan–cellulose nanocrystals for wound dressing: effect of surface characteristics of nanocrystals. Cellulose 22:521–534. doi: 10.1007/s10570-014-0493-y CrossRefGoogle Scholar
  27. Paul DW, Ghassemi P, Ramella-Roman JC, Prindeze NJ, Moffatt LT, Alkhalil A, Shupp JW (2015) Noninvasive imaging technologies for cutaneous wound assessment: a review. Wound Repair Regen 23:149–162. doi: 10.1111/wrr.12262 CrossRefGoogle Scholar
  28. Scherrer P (1912) Bestimmung der inneren Struktur und der Größe von Kolloidteilchen mittels Röntgenstrahlen. In: Kolloidchemie Ein Lehrbuch. Chemische Technologie in Einzeldarstellungen. Springer, Berlin, pp 387–409. doi: 10.1007/978-3-662-33915-2_7
  29. Schyrr B, Pasche S, Voirin G, Weder C, Simon YC, Foster EJ (2014) Biosensors based on porous cellulose nanocrystal–poly(vinyl alcohol) scaffolds. ACS Appl Mater Interfaces 6:12674–12683. doi: 10.1021/am502670u CrossRefGoogle Scholar
  30. Sehaqui H, Zhou Q, Ikkala O, Berglund LA (2011) Strong and tough cellulose nanopaper with high specific surface area and porosity. Biomacromolecules 12:3638–3644. doi: 10.1021/bm2008907 CrossRefGoogle Scholar
  31. Sehaqui H, Zimmermann T, Tingaut P (2014) Hydrophobic cellulose nanopaper through a mild esterification procedure. Cellulose 21:367–382. doi: 10.1007/s10570-013-0110-5 CrossRefGoogle Scholar
  32. Serena TE (2014) Development of a novel technique to collect proteases from chronic wounds. Adv Wound Care 3:729–732. doi: 10.1089/wound.2013.0463 CrossRefGoogle Scholar
  33. Shen B, Shimmon S, Smith MM, Ghosh P (2003) Biosensor analysis of the molecular interactions of pentosan polysulfate and of sulfated glycosaminoglycans with immobilized elastase, hyaluronidase and lysozyme using surface plasmon resonance (SPR) technology. J Pharm Biomed Anal 31:83–93. doi: 10.1016/S0731-7085(02)00606-4 CrossRefGoogle Scholar
  34. Stanislaw B, Halina K, Alina K, Katarzyna K, Marek K, de Manu G (2012) Wound dressings and cosmetic materials from bacterial nanocellulose. In: Gama M, Gatenholm P, Klemm D (eds) Bacterial nanocellulose. Perspectives in nanotechnology series. CRC Press, Inc., Boca Raton, pp 157–174. doi: 10.1201/b12936-9 Google Scholar
  35. Still JM, Law EJ, Klavuhn KG, Island TC, Holtz JZ (2001) Diagnosis of burn depth using laser-induced indocyanine green fluorescence: a preliminary clinical trial. Burns 27:364–371. doi: 10.1016/S0305-4179(00)00140-6 CrossRefGoogle Scholar
  36. Wang Y, Zagorevski DV, Stenken JA (2008) In situ and multisubstrate detection of elastase enzymatic activity external to microdialysis sampling probes using LC-ESI-MS. Anal Chem 80:2050–2057. doi: 10.1021/ac702047w CrossRefGoogle Scholar
  37. Wang J, Bowie D, Zhang X, Filipe C, Pelton R, Brennan JD (2014) Morphology and entrapped enzyme performance in inkjet-printed sol–gel coatings on paper. Chem Mater 26:1941–1947. doi: 10.1021/cm500206s CrossRefGoogle Scholar
  38. Yager DR, Chen SM, Ward SI, Olutoye OO, Diegelmann RF, Kelman Cohen I (1997) Ability of chronic wound fluids to degrade peptide growth factors is associated with increased levels of elastase activity and diminished levels of proteinase inhibitors. Wound Repair Regen 5:23–32. doi: 10.1046/j.1524-475X.1997.50108.x CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht (outside the USA)  2016

Authors and Affiliations

  • Krystal R. Fontenot
    • 1
  • J. Vincent Edwards
    • 1
  • David Haldane
    • 2
  • Elena Graves
    • 1
  • Michael Santiago Citron
    • 1
  • Nicolette T. Prevost
    • 1
  • Alfred D. French
    • 1
  • Brian D. Condon
    • 1
  1. 1.Southern Regional Research Center, USDANew OrleansUSA
  2. 2.Innovatech-EngineeringTallahasseeUSA

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