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Protein-Functionalized Gold Nanoparticles as Refractometric Nanoplasmonic Sensors for the Detection of Proteolytic Activity of Porphyromonas gingivalis
Örebro University, School of Medical Sciences. Laboratory of Molecular Materials, Division ofBiophysics and Bioengineering, Department of Physics,Chemistry and Biology (IFM), Linköping University, Linköping, Sweden; Cardiovascular Research Centre (CVRC), School of Medical Sciences, Örebro University, Örebro, Sweden.ORCID iD: 0000-0002-6554-3554
Department of Oral Biology, Faculty of Odontology, Malmö University, Malmö, Sweden.
Örebro University, School of Medical Sciences. Cardiovascular Research Centre (CVRC).
Department of Oral Biology, Faculty of Odontology, Malmö University, Malmö, Sweden.
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2020 (English)In: ACS Applied Nano Materials, E-ISSN 2574-0970, Vol. 3, no 10, p. 9822-9830Article in journal (Refereed) Published
Abstract [en]

Periodontitis is an inflammatory oral disease that affects a large part of the adult population, causing significant costs and suffering. The key pathogen, Porphyromonas gingivalis, secretes gingipains, which are highly destructive proteases and the most important virulence factors in the pathogenesis of the disease. Currently, periodontitis is diagnosed mainly by mechanical manual probing and radiography, often when the disease has already progressed significantly. The possibilities of detecting gingipain activity in gingival fluid could enable early-stage diagnosis and facilitate treatment. Here, we describe a sensitive nanoparticle-based nanoplasmonic biosensor for the detection of the proteolytic activity of gingipains. Gold nanoparticles (AuNPs) were self-assembled as a submonolayer in multiwell plates and further modified with casein or IgG. The proteolytic degradation of the protein coating was tracked by monitoring the shift in the localized surface plasmon resonance (LSPR) peak position. The sensor performance was investigated using model systems with trypsin and purified gingipains (subtypes Kgp and RgpB) and further validated using supernatants from cultures of P. gingivalis. Proteolytic degradation by proteases in buffer results in a concentration- and time-dependent blueshift of the LSPR band of about 1-2 nm when using casein as a substrate. In bacterial supernatants, the degradation of the protein coating resulted in unspecific binding of proteins present in the complex sample matrix to the nanoparticles, which instead triggered a redshift of about 2 nm of the LSPR band. A significant LSPR shift was seen only in samples with gingipain activity. The sensor showed a limit of detection < 0.1 mu g/mL (4.3 nM), which is well below gingipain concentrations detected in severe chronic periodontitis cases (similar to 50 mu g/mL). This work shows the possibility of developing cost-effective nanoparticle-based biosensors for rapid detection of protease activity for chair-side periodontal diagnostics.

Place, publisher, year, edition, pages
American Chemical Society , 2020. Vol. 3, no 10, p. 9822-9830
Keywords [en]
gold nanoparticles, localized surface plasmon resonance, gingipains, proteolytic activity, P. gingivalis, periodontitis
National Category
Microbiology in the medical area
Identifiers
URN: urn:nbn:se:oru:diva-87946DOI: 10.1021/acsanm.0c01899ISI: 000583331600088Scopus ID: 2-s2.0-85096515317OAI: oai:DiVA.org:oru-87946DiVA, id: diva2:1507903
Funder
Swedish Research Council, 2016-04874 2017-04475Swedish Foundation for Strategic Research, FFL15-0026Knut and Alice Wallenberg Foundation, KAW 2016.0231Available from: 2020-12-09 Created: 2020-12-09 Last updated: 2024-03-21Bibliographically approved
In thesis
1. Novel methods for diagnosis and treatment of periodontal disease
Open this publication in new window or tab >>Novel methods for diagnosis and treatment of periodontal disease
2024 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Periodontitis is an inflammatory condition affecting tooth-supportive structures, the periodontium. Gingival tissues respond to biofilm formation by initiating an inflammatory process. If left untreated, inflammation progresses to a non-reversible condition in susceptible individuals, causing degradation of the underlying bone structures, where teeth become mobile and tooth-loss eventually occurs. Periodontitis is very common, affecting about half of the adult population. The disease is not only a concern for oral health but is linked to many systemic conditions, such as cardiovascular disease, diabetes, rheumatoid arthritis, and Alzheimer’s.

The studies included in this thesis aim to develop new strategies for the diagnosis and treatment of periodontal disease. Current diagnostic practices rely on probing and x-ray examination. There is a need for more sophisticated diagnostic tools. We have developed different sensing strategies for detection of a key-pathogen in periodontitis, Porphyromonas gingivalis, and its secreted enzymes, gingipains. The proteolytic activity of this pathogen, which drives the destructive effects of the disease, can be detected at low levels by utilizing gold nanoparticles and nanoplasmonic sensing in different ways.

Treatment of periodontitis is basically limited to the removal of soft and hard biofilms. This requires significant resources, from patients as well as clinicians, and maintenance phase is often life-long. We have identified and tested the antibacterial effect of an antimicrobial peptide derived from Lactobacillus plantarum. This bacteriocin, plantaricin NC8αβ, has been shown to effectively inhibit P. gingivalis. The bacteriocin has been further optimized into a short peptide sequence, C5, which can rupture outer membrane vesicles from P. gingivalis. Taken together, these results suggest new sensing strategies for novel diagnostic tools for periodontal disease and define a promising antibacterial agent for periodontal treatment in the future.

Place, publisher, year, edition, pages
Örebro: Örebro University, 2024. p. 58
Series
Örebro Studies in Medicine, ISSN 1652-4063 ; 289
Keywords
Periodontitis, inflammation, Porphyromonas gingivalis, gingipains, biosensing, gold nanoparticles, antimicrobial treatment, NC8
National Category
Other Basic Medicine
Identifiers
urn:nbn:se:oru:diva-111438 (URN)9789175295510 (ISBN)
Public defence
2024-04-12, Örebro universitet, Campus USÖ, hörsal X3, Södra Grev Rosengatan 32, Örebro, 09:00 (Swedish)
Opponent
Supervisors
Available from: 2024-02-07 Created: 2024-02-07 Last updated: 2024-03-25Bibliographically approved

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Svärd, AnnaPalm, EleonorBengtsson, Torbjörn

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