Das Suchergebnis hat sich seit Ihrer Suchanfrage verändert. Eventuell werden Dokumente in anderer Reihenfolge angezeigt.
  • Treffer 9 von 31
Zurück zur Trefferliste
Zitieren Sie bitte immer diesen URN: urn:nbn:de:kobv:b43-545365

Methods for the quantification of particle-bound protein – Application to reagents for lateral-flow immunoassays (LFIA)

  • Protein immobilization for the functionalization of particles is used in various applications, including biosensors, lateral-flow immunoassays (LFIA), bead-based assays, and others. Common methods for the quantification of bound protein are measuring protein in the supernatant before and after coating and calculating the difference. This popular approach has the potential for a significant overestimation of the amount of immobilized protein since layers not directly bound to the surface (soft protein corona) are usually lost during washing and handling. Only the layer directly bound to the surface (hard corona) can be used in subsequent assays. A simplified amino acid analysis method based on acidic hydrolysis and RP-HPLC-FLD of tyrosine and phenylalanine (aromatic amino acid analysis, AAAA) is proposed to directly quantify protein bound to the surface of gold nano- and latex microparticles. The results are compared with indirect methods such as colorimetric protein assays, such asProtein immobilization for the functionalization of particles is used in various applications, including biosensors, lateral-flow immunoassays (LFIA), bead-based assays, and others. Common methods for the quantification of bound protein are measuring protein in the supernatant before and after coating and calculating the difference. This popular approach has the potential for a significant overestimation of the amount of immobilized protein since layers not directly bound to the surface (soft protein corona) are usually lost during washing and handling. Only the layer directly bound to the surface (hard corona) can be used in subsequent assays. A simplified amino acid analysis method based on acidic hydrolysis and RP-HPLC-FLD of tyrosine and phenylalanine (aromatic amino acid analysis, AAAA) is proposed to directly quantify protein bound to the surface of gold nano- and latex microparticles. The results are compared with indirect methods such as colorimetric protein assays, such as Bradford, bicinchoninic acid (BCA), as well as AAAA of the supernatant. For both particle types, these indirect quantification techniques show a protein overestimation of up to 1700% compared to the direct AAAA measurements. In addition, protein coating on latex particles was performed both passively through adsorption and covalently through EDC/sulfo-NHS chemistry. Our results showed no difference between the immobilization methodologies. This finding suggests that usual protein determination methods are no unambiguous proof of a covalent conjugation on particles or beads.zeige mehrzeige weniger

Metadaten exportieren

Weitere Dienste

Teilen auf Twitter Suche bei Google Scholar Anzahl der Zugriffe auf dieses Dokument
Metadaten
Autor*innen:Teodor TchipilovORCiD, Anna RaysyanORCiD, Michael G. WellerORCiD
Dokumenttyp:Sonstiges
Veröffentlichungsform:Graue Literatur
Sprache:Englisch
Titel des übergeordneten Werkes (Englisch):Preprints
Jahr der Erstveröffentlichung:2022
Organisationseinheit der BAM:1 Analytische Chemie; Referenzmaterialien
1 Analytische Chemie; Referenzmaterialien / 1.5 Proteinanalytik
1 Analytische Chemie; Referenzmaterialien / 1.8 Umweltanalytik
Veröffentlichende Institution:Bundesanstalt für Materialforschung und -prüfung (BAM)
Verlag:MDPI
Verlagsort:Basel
Aufsatznummer:2022030332
Erste Seite:1
Letzte Seite:8
DDC-Klassifikation:Naturwissenschaften und Mathematik / Chemie / Analytische Chemie
Freie Schlagwörter:Antibody; Bioconjugation; Covalent conjugation; Gold particles; Hard protein corona; Lateral flow immunoassays; Latex particles; Mikroparticles; Nanoparticles; Protein quantification; Sodium chloride method; Soft protein corona; Supernatant
Themenfelder/Aktivitätsfelder der BAM:Chemie und Prozesstechnik
DOI:10.20944/preprints202203.0332.v1
URN:urn:nbn:de:kobv:b43-545365
Verfügbarkeit des Dokuments:Datei für die Öffentlichkeit verfügbar ("Open Access")
Lizenz (Deutsch):License LogoCreative Commons - CC BY - Namensnennung 4.0 International
Datum der Freischaltung:28.03.2022
Referierte Publikation:Nein
Schriftenreihen ohne Nummerierung:Arbeitspapiere der BAM
Einverstanden
Diese Webseite verwendet technisch erforderliche Session-Cookies. Durch die weitere Nutzung der Webseite stimmen Sie diesem zu. Unsere Datenschutzerklärung finden Sie hier.