Skip to main content

Advertisement

Springer Nature Link
Log in

Detection of human neutrophil elastase (HNE) on wound dressings as marker of inflammation

  • Biotechnologically relevant enzymes and proteins
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

Chronic wound fluids have elevated concentration of human neutrophil elastase (HNE) which can be used as inflammation/infection marker. Our goal is to develop functional materials for fast diagnosis of wound inflammation/infection by using HNE as a specific marker. For that, fluorogenic peptides with a HNE-specific cleavage sequence were incorporated into traditional textile dressings, to allow real-time detection of the wound status. Two different fluorogenic approaches were studied in terms of intensity of the signal generated upon HNE addition: a fluorophore 7-amino-4-trifluormethylcoumarin (AFC) conjugated to a HNE-specific peptide and two fluorophore/quencher pairs (FAM/Dabcyl and EDANS/Dabcyl) coupled to a similar peptide as a Förster resonance energy transfer (FRET) strategy. Also, two immobilization methods were tested: sonochemistry immobilization onto a cotton bandage and glutaraldehyde (GTA)-assisted chemical crosslinking onto a polyamide dressing. The immobilized fluorogenic AFC peptide showed an intense fluorescence emission in the presence of HNE. HNE also induced an enhanced fluorescent signal with the EDANS/Dabcyl FRET peptide which showed to be a more sensitive and effective strategy than the AFC peptide. However, its chemical immobilization onto the polyamide dressing greatly decreased its detection, mainly due to the more difficult access of the enzyme to the cleavage sequence of the immobilized peptide. After optimization of the in situ immobilization, it will be possible to use these fluorescence-functionalized dressings for an effective and specific monitoring of chronic wounds by simply using a portable ultraviolet (UV) light source. We envision that the development of this point-of-care medical device for wound control will have a great impact on patient’s life quality and reduction of costs on health care system.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

Explore related subjects

Discover the latest articles and news from researchers in related subjects, suggested using machine learning.

References

  • Adhikari S, Ghosh A, Mandal S, Sengupta A, Chattopadhyay A, Sanmartín Matalobos J, Lohar S, Das D (2014) Visible light excitable ON fluorescence and naked eye detection of Cu2+ via hydrolysis of rhodamine-thiophene conjugate: human breast cancer cell (MCF7) imaging studies. Dalton Trans 43:7747–7751. doi:10.1039/c4dt00002a

    Article  CAS  PubMed  Google Scholar 

  • Alvarez-Iglesias M, Wayne G, O’Dea KP, Amour A, Takata M (2005) Continuous real-time measurement of tumor necrosis factor-α converting enzyme activity on live cells. Lab Investig 85:1440–1448. doi:10.1038/labinvest.3700340

    Article  CAS  PubMed  Google Scholar 

  • Barros SC, Louro RO, Micaêlo NM, Martins JA, Marcos JC, Cavaco-Paulo A (2013) NMR and molecular modelling studies on elastase inhibitor-peptides for wound management. React Funct Polym 73:1357–1365. doi:10.1016/j.reactfunctpolym.2013.02.011

    Article  CAS  Google Scholar 

  • Begum S, Wu J, Takawira CM, Wang J (2016) Surface modification of polyamide 6, 6 fabrics with an alkaline protease–Subtilisin. J Eng Fiber Fabr 11:64–74

    Google Scholar 

  • Bowers JS (2000) Toluidines. In: Ullmann’s encyclopedia of industrial chemistry. Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, pp. 119–126

    Google Scholar 

  • Brena B, González-Pombo P, Batista-Viera F (2013) Immobilization of enzymes: a literature survey. In: Immobilization of enzymes and cells: third edition. pp 15–31

  • Bru R, Walde P (1991) Product inhibition of alpha-chymotrypsin in reverse micelles. Eur J Biochem 199:95–103

    Article  CAS  PubMed  Google Scholar 

  • Carmona AK, Juliano MA, Juliano L (2009) The use of fluorescence resonance energy transfer (FRET) peptides for measurement of clinically important proteolytic enzymes. An da Acad Bras Ciências 81:381–392

    Article  CAS  Google Scholar 

  • Dini V, Salvo P, Janowska A, Di Francesco F, Barbini A, Romanelli M (2015) Correlation between wound temperature obtained with an infrared camera and clinical wound bed score in venous leg ulcers. Wounds a Compend Clin Res Pract 27:274–278

    Google Scholar 

  • Fan H, Jiang X, Zhang T, Jin Q (2012) Peptide-induced fluorescence quenching of conjugated polyelectrolyte for label-free, ultrasensitive and selective assay of protease activity. Biosens Bioelectron 34:221–226. doi:10.1016/j.bios.2012.02.006

    Article  CAS  PubMed  Google Scholar 

  • Gashti MP, Willoughby J, Agrawal P (2011) Surface and bulk modification of synthetic textiles to improve dyeability. Text Dye 18:927–929. doi:10.5772/800

    Google Scholar 

  • Gedanken A (2004) Using sonochemistry for the fabrication of nanomaterials. Ultrason Sonochem 11:47–55. doi:10.1016/j.ultsonch.2004.01.037

    Article  CAS  PubMed  Google Scholar 

  • Gottrup F, Apelqvist J, Price P (eds) (2010) Outcomes in controlled and comparative studies on non-healing wounds: recommendations to improve the quality of evidence in wound management. J Wound Care 19:237–268. doi:10.12968/jowc.2010.19.6.48471

  • Gray CJ, Weissenborn MJ, Eyers CE, Flitsch SL (2013) Enzymatic reactions on immobilised substrates. Chem Soc Rev 42:6378. doi:10.1039/c3cs60018a

    Article  CAS  PubMed  Google Scholar 

  • Harding K, Armstrong DG, Barrett S, Kaufman H, Lázaro-Martínez JL, Mayer D, Moore Z, Romanelli M, Queen D, Schultz G, Serena T, Sibbald G, Snyder R, Strohal R, Vowden K, Vowden P, Paolo Zamboni (2011) International consensus. The role of proteases in wound diagnostics. An expert working group review

  • Hasmann A, Gewessler U, Hulla E, Schneider KP, Binder B, Francesko A, Tzanov T, Schintler M, Van der Palen J, Guebitz GM, Wehrschuetz-Sigl E (2011) Sensor materials for the detection of human neutrophil elastase and cathepsin G activity in wound fluid. Exp Dermatol 20:508–513. doi:10.1111/j.1600-0625.2011.01256.x

    Article  CAS  PubMed  Google Scholar 

  • Herrick SE, Sloan P, McGurk M, Freak L, McCollum CN, Ferguson MW (1992a) Sequential changes in histologic pattern and extracellular matrix deposition during the healing of chronic venous ulcers. Am J Pathol 141:1085–1095

    CAS  PubMed  PubMed Central  Google Scholar 

  • Herrick SE, Sloan P, McGurk M, Freak L, McCollum CN, Ferguson MW (1992b) Sequential changes in histologic pattern and extracellular matrix deposition during the healing of chronic venous ulcers. Am J Pathol 141:1085–1095

    CAS  PubMed  PubMed Central  Google Scholar 

  • Kiel S, Grinberg O, Perkas N, Charmet J, Kepner H, Gedanken A (2012) Forming nanoparticles of water-soluble ionic molecules and embedding them into polymer and glass substrates. Beilstein J Nanotechnol 3:267–276. doi:10.3762/bjnano.3.30

    Article  PubMed  PubMed Central  Google Scholar 

  • Kolaczkowska E, Kubes P (2013) Neutrophil recruitment and function in health and inflammation. Nat Rev Immunol 13:159–175. doi:10.1038/nri3399

    Article  CAS  PubMed  Google Scholar 

  • Korkmaz B, Attucci S, Juliano MA, Kalupov T, Jourdan M-L, Juliano L, Gauthier F (2008a) Measuring elastase, proteinase 3 and cathepsin G activities at the surface of human neutrophils with fluorescence resonance energy transfer substrates. Nat Protoc 3:991–1000. doi:10.1038/nprot.2008.63

    Article  CAS  PubMed  Google Scholar 

  • Korkmaz B, Moreau T, Gauthier F (2008b) Neutrophil elastase, proteinase 3 and cathepsin G: physicochemical properties, activity and physiopathological functions. Biochimie 90:227–242. doi:10.1016/j.biochi.2007.10.009

    Article  CAS  PubMed  Google Scholar 

  • Lakowicz JR (2006) Principles of fluorescence spectroscopy. Springer US, Boston

    Book  Google Scholar 

  • Laurent N, Haddoub R, Flitsch SL (2008) Enzyme catalysis on solid surfaces. Trends Biotechnol 26:328–337. doi:10.1016/j.tibtech.2008.03.003

    Article  CAS  PubMed  Google Scholar 

  • Macgregor L, Calne S, Day K, Jones J, Pugh A (2008) Wound infection in clinical practice. Int Wound J 5:iii–i11. doi:10.1111/j.1742-481X.2008.00488.x

    Google Scholar 

  • Masters BR (2014) Paths to Förster’s resonance energy transfer (FRET) theory. Eur Phys J H 39:87–139. doi:10.1140/epjh/e2013-40007-9

    Article  Google Scholar 

  • Medintz I, Hildebrandt N (2013) FRET-Förster Resonance Energy Transfer. Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

    Book  Google Scholar 

  • Mersmann A (2001) Crystallization technology handbook, a. CRC Press, Mersman

    Book  Google Scholar 

  • Migneault I, Dartiguenave C, Bertrand MJ, Waldron KC (2004) Glutaraldehyde: behavior in aqueous solution, reaction with proteins, and application to enzyme crosslinking. BioTechniques 37(790–6):798–802. doi:10.2144/3705A0790

    Google Scholar 

  • Monro TM, Moore RL, Nguyen MC, Ebendorff-Heidepriem H, Skouroumounis GK, Elsey GM, Taylor DK (2012) Sensing free sulfur dioxide in wine. Sensors (Switzerland) 12:10759–10773. doi:10.3390/s120810759

    Article  CAS  Google Scholar 

  • Moore K, McCallion R, Searle RJ, Stacey MC, Harding KG (2006) Prediction and monitoring the therapeutic response of chronic dermal wounds. Int Wound J 3:89–98. doi:10.1111/j.1742-4801.2006.00212.x

    Article  PubMed  Google Scholar 

  • Mustoe TA, O’Shaughnessy K, Kloeters O (2006) Chronic wound pathogenesis and current treatment strategies: a unifying hypothesis. Plast Reconstr Surg 117:35S–41S. doi:10.1097/01.prs.0000225431.63010.1b

    Article  CAS  PubMed  Google Scholar 

  • Niles AL, Moravec RA, Eric Hesselberth P, Scurria MA, Daily WJ, Riss TL (2007) A homogeneous assay to measure live and dead cells in the same sample by detecting different protease markers. Anal Biochem 366:197–206. doi:10.1016/j.ab.2007.04.007

    Article  CAS  PubMed  Google Scholar 

  • Niles AL, Moravec RA, Riss TL (2009) In vitro viability and cytotoxicity testing and same-well multi-parametric combinations for high throughput screening. Curr Chem Genomics 3:33–41. doi:10.2174/1875397300903010033

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Owen CA, Campbell EJ (1999) The cell biology of leukocyte-mediated proteolysis. J Leukoc Biol 65:137–150

    CAS  PubMed  Google Scholar 

  • Perelshtein I, Applerot G, Perkas N, Wehrschetz-Sigl E, Hasmann A, Guebitz GM, Gedanken A (2009) Antibacterial properties of an in situ generated and simultaneously deposited nanocrystalline ZnO on fabrics. ACS Appl Mater Interfaces 1:361–366. doi:10.1021/am8000743

    Article  CAS  PubMed  Google Scholar 

  • Perelshtein I, Ruderman E, Francesko A, Fernandes MM, Tzanov T, Gedanken A (2014) Tannic acid NPs—synthesis and immobilization onto a solid surface in a one-step process and their antibacterial and anti-inflammatory properties. Ultrason Sonochem 21:1916–1920. doi:10.1016/j.ultsonch.2013.11.022

    Article  CAS  PubMed  Google Scholar 

  • Perelshtein I, Lipovsky A, Perkas N, Tzanov T, Аrguirova M, Leseva M, Gedanken A (2015) Making the hospital a safer place by sonochemical coating of all its textiles with antibacterial nanoparticles. Ultrason Sonochem 25:82–88. doi:10.1016/j.ultsonch.2014.12.012

    Article  CAS  PubMed  Google Scholar 

  • Perkas N, Amirian G, Dubinsky S, Gazit S, Gedanken A (2007) Ultrasound-assisted coating of nylon 6,6 with silver nanoparticles and its antibacterial activity. J Appl Polym Sci 104:1423–1430. doi:10.1002/app.24728

    Article  CAS  Google Scholar 

  • Pol VG, Gedanken A, Calderon-Moreno J, Palchik V, Slifkin MA, Weiss AM (2003) Deposition of gold nanoparticles on silica spheres: a sonochemical approach. Chem Mater 15:3402–3402. doi:10.1021/cm033005e

    Article  CAS  Google Scholar 

  • Scharpé S, De Meester I, Vanhoof G, Hendriks D, van Sande M, Van Camp K, Yaron A (1988) Assay of dipeptidyl peptidase IV in serum by fluorometry of 4-methoxy-2-naphthylamine. Clin Chem 34:2299–2301

    PubMed  Google Scholar 

  • Schneider LA, Korber A, Grabbe S, Dissemond J (2007) Influence of pH on wound-healing: a new perspective for wound-therapy? Arch Dermatol Res 298:413–420. doi:10.1007/s00403-006-0713-x

    Article  PubMed  Google Scholar 

  • Sen CK, Gordillo GM, Roy S, Kirsner R, Lambert L, Hunt TK, Gottrup F, Gurtner GC, Longaker MT (2010) Human skin wounds: a major and snowballing threat to public health and the economy. Wound Repair Regen 17:763–771. doi:10.1111/j.1524-475X.2009.00543.x

    Article  Google Scholar 

  • Shang L, Zhang S, Yang X, Sun J, Li L, Cui Z, He Q, Guo Y, Sun Y, Yin Z (2015) Biochemical characterization of recombinant enterovirus 71 3C protease with fluorogenic model peptide substrates and development of a biochemical assay. Antimicrob Agents Chemother 59:1827–1836. doi:10.1128/AAC.04698-14

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Silva C, Silva CJ, Zille A, Guebitz GM, Cavaco-Paulo A (2007) Laccase immobilization on enzymatically functionalized polyamide 6,6 fibres. Enzym Microb Technol 41:867–875. doi:10.1016/j.enzmictec.2007.07.010

    Article  CAS  Google Scholar 

  • Sunartio D, Yasui K, Tuziuti T, Kozuka T, Iida Y, Ashokkumar M, Grieser F (2007) Correlation between Na* emission and “chemically active” acoustic cavitation bubbles. ChemPhysChem 8:2331–2335. doi:10.1002/cphc.200700426

    Article  CAS  PubMed  Google Scholar 

  • Szöllosi J, Damjanovich S, Mátyus L (1998) Application of fluorescence resonance energy transfer in the clinical laboratory: routine and research. Cytometry 34:159–179. doi:10.1002/(SICI)1097-0320(19980815)34:4<159::AID-CYTO1>3.0.CO;2-B

    Article  PubMed  Google Scholar 

  • Tabassum S, Al-Asbahy WM, Afzal M, Arjmand F, Hasan Khan R (2012) Interaction and photo-induced cleavage studies of a copper based chemotherapeutic drug with human serum albumin: spectroscopic and molecular docking study. Mol BioSyst 8:2424. doi:10.1039/c2mb25119a

    Article  CAS  PubMed  Google Scholar 

  • Trevan MD (1988) Enzyme immobilization by covalent bonding. In: New Protein Techniques. Humana Press, New Jersey, pp. 495–510

    Chapter  Google Scholar 

  • Vasconcelos A, Pêgo AP, Henriques L, Lamghari M, Cavaco-Paulo A (2010) Protein matrices for improved wound healing: elastase inhibition by a synthetic peptide model. Biomacromolecules 11:2213–2220. doi:10.1021/bm100537b

    Article  CAS  PubMed  Google Scholar 

  • Vasconcelos A, Azoia NG, Carvalho AC, Gomes AC, Güebitz G, Cavaco-Paulo A (2011) Tailoring elastase inhibition with synthetic peptides. Eur J Pharmacol 666:53–60. doi:10.1016/j.ejphar.2011.05.056

    Article  CAS  PubMed  Google Scholar 

  • Weiss SJ, Curnutte JT, Regiani S (1986) Neutrophil-mediated solubilization of the subendothelial matrix: oxidative and nonoxidative mechanisms of proteolysis used by normal and chronic granulomatous disease phagocytes. J Immunol 136:636–641

    CAS  PubMed  Google Scholar 

  • Weitz JI, Landman SL, Crowley KA, Birken S, Morgan FJ (1986) Development of an assay for in vivo human neutrophil elastase activity. Increased elastase activity in patients with alpha 1-proteinase inhibitor deficiency. J Clin Invest 78:155–162. doi:10.1172/JCI112545

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

We would like to acknowledge the European project InFact—Functional materials for fast diagnosis of wound infection (FP7-NMP-2013-SME-7—grant agreement no. 604278) for funding the work and also the grant for Joana Cunha. Ana Ferreira would also like to acknowledge the Portuguese Foundation for Science and Technology (FCT) for the PhD student grant SFRH/BD/113247/2015. The work done at Centre of Biological Engineering (CEB) was supported by FCT under the scope of the strategic funding of UID/BIO/04469/2013 unit, COMPETE 2020 (POCI-01-0145-FEDER-006684) and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by European Regional Development Fund under the scope of Norte 2020—Programa Operacional Regional do Norte.

Author information

Authors and Affiliations

  1. Centre of Biological Engineering (CEB), University of Minho, 4710-057, Braga, Portugal

    Ana V. Ferreira, Joana Cunha & Artur Cavaco-Paulo

  2. Department of Chemistry, Bar-Ilan Institute of Nanotechnology and Advanced Materials, Bar-Ilan University, IL-52900, Ramat-Gan, Israel

    Ilana Perelshtein, Nina Perkas & Aharon Gedanken

Authors
  1. Ana V. Ferreira
    View author publications

    Search author on:PubMed Google Scholar

  2. Ilana Perelshtein
    View author publications

    Search author on:PubMed Google Scholar

  3. Nina Perkas
    View author publications

    Search author on:PubMed Google Scholar

  4. Aharon Gedanken
    View author publications

    Search author on:PubMed Google Scholar

  5. Joana Cunha
    View author publications

    Search author on:PubMed Google Scholar

  6. Artur Cavaco-Paulo
    View author publications

    Search author on:PubMed Google Scholar

Corresponding author

Correspondence to Artur Cavaco-Paulo.

Ethics declarations

Funding

This study was funded by the European project InFact—Functional materials for fast diagnosis of wound infection (FP7-NMP-2013-SME-7—grant agreement no. 604278). The work done at Centre of Biological Engineering (CEB) was also supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit, COMPETE 2020 (POCI-01-0145-FEDER-006684) and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by European Regional Development Fund under the scope of Norte 2020—Programa Operacional Regional do Norte.

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Electronic supplementary material

ESM 1

(PDF 796 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ferreira, A.V., Perelshtein, I., Perkas, N. et al. Detection of human neutrophil elastase (HNE) on wound dressings as marker of inflammation. Appl Microbiol Biotechnol 101, 1443–1454 (2017). https://doi.org/10.1007/s00253-016-7889-6

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-016-7889-6

Keywords