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A comparison between Dual Polarization Interferometry (DPI) and Surface Plasmon Resonance (SPR) for protein adsorption studies
KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
Novozymes A/S, Bagsvaerd.
KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.ORCID iD: 0000-0003-0578-4003
YKI, Institute for Surface Chemistry, Stockholm.
2007 (English)In: Colloids and Surfaces B: Biointerfaces, ISSN 0927-7765, E-ISSN 1873-4367, Vol. 54, no 2, 236-240 p.Article in journal (Refereed) Published
Abstract [en]

This work was performed with the aim of comparing protein adsorption results obtained from the recently developed dual polarization interferometry (DPI) with the well-established surface plasmon resonance (SPR) technique. Both techniques use an evanescent field as the sensing element but completely different methods to calculate the adsorbed mass. As a test system we used adsorption of the lipase from Thermomyces lanuginosus (TLL) on C18 surfaces. The adsorbed amount calculated with both techniques is in good agreement, with both adsorption isotherms saturating at 1.30-1.35 mg/m(2) at TLL concentrations of 1000 nM and above. Therefore, this supports the use of both SPR and DPI as tools for studying protein adsorption, which is very important when comparing adsorption data obtained from the use different techniques. Due to the spot sensing in SPR, this technique is recommended for initial kinetic studies, whereas DPI is more accurate when the refractive index and thickness of the adsorbed layer is of more interest.

Place, publisher, year, edition, pages
2007. Vol. 54, no 2, 236-240 p.
Keyword [en]
C18-surface; Dual polarization interferometry; Lipase; Protein adsorption; Surface plasmon resonance; Adsorption; Adsorption isotherms; Enzymes; Interferometry; Reaction kinetics; Refractive index; Surface plasmon resonance; triacylglycerol lipase; adsorption; adsorption chromatography; article; calculation; chemical reaction kinetics; interferometry; intermethod comparison; nonhuman; priority journal; surface plasmon resonance; Adsorption; Ascomycota; Interferometry; Lipase; Mitosporic Fungi; Proteins; Surface Plasmon Resonance
National Category
Physical Sciences
Identifiers
URN: urn:nbn:se:kth:diva-6927DOI: 10.1016/j.colsurfb.2006.10.028ISI: 000244473900016Scopus ID: 2-s2.0-33846372015OAI: oai:DiVA.org:kth-6927DiVA: diva2:11778
Note
QC 20100818Available from: 2007-03-27 Created: 2007-03-27 Last updated: 2010-08-18Bibliographically approved
In thesis
1. Dynamics of Enzymes at Interfaces: Lipase adsorption and mobility on solid surfaces
Open this publication in new window or tab >>Dynamics of Enzymes at Interfaces: Lipase adsorption and mobility on solid surfaces
2007 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

This thesis aimed to give more insight in the dynamics of enzymes at interfaces. The adsorption and mobility of adsorbed proteins can e.g. give a better understanding of structure-function properties of interfacially active enzymes. Studied enzyme was the lipase from Thermomyces lanuginosus (TLL).

Adsorption of TLL to surfaces of different hydrophobicity was studied by Dual Polarization Interferometry (DPI), Surface Plasmon Resonance (SPR) and ellipsometry. It was found that TLL had highest affinity and adsorbed to largest adsorbed amount on a hydrophobic, C18 terminated surface. Moreover, activity studies of adsorbed TLL suggested that a larger fraction of the lipases were orientated with the active site facing the surface on hydrophobic surfaces.

Mobility of adsorbed enzymes was studied by means of Fluorescence Recovery After Photobleaching (FRAP) with Confocal Laser Scanning Microscopy (CLSM). CLSM was also used as a tool to image the role of TLL in the detergency of lipids from single cotton fibers. The TLL surface mobility was measured on model surfaces of different hydrophobicity. The rate of TLL surface diffusion was strongly dependent on the surface density of lipase, which was explained by sterical hindrance and intermolecular repulsion. The diffusion was both lowest and decreased as a function of time after adsorption on the most hydrophobic surface. This was thought to be due to a larger fraction of adsorbed TLL oriented with the active site towards the hydrophobic surface and that this fraction increased as a function of time.

The presence of surfactants affected the TLL mobility on hydrophobic surfaces. The diffusion increased more than tenfold when TLL was coadsorbed with C12E6/LAS above the critical micellar concentration (cmc) of the surfactant. This was thought to be due to a surfactant induced desorption-rebinding mechanism of TLL. Total Internal Reflection Fluorescence Correlation Spectroscopy (TIR-FCS) supported this theory and was implemented as a technique to quantify kinetic processes of protein-surfactant interactions at surfaces.

The surface mobility of TLL was higher on a trimyristin substrate surface compared to the model hydrophobic surface. Single particle tracing of lipases could be performed by conjugation of TLL to Quantum Dots (QDs). The microscopic behavior of QD-lipases on trimyristin suggested that the enzyme operated in two different modes on the surface, which gave the trajectories of single lipase molecules a “bead on a string” appearance.

Place, publisher, year, edition, pages
Stockholm: KTH, 2007. 57 p.
Series
Trita-FYS, ISSN 0280-316X ; 2007:20
Keyword
biophysics, surface chemistry, diffusion, enzymes, lipases, adsorption, mobility
National Category
Physical Sciences
Identifiers
urn:nbn:se:kth:diva-4319 (URN)978-91-7178-604-3 (ISBN)
Public defence
2007-04-23, FB53, AlbaNova, Roslagstullsbacken 21, Stockholm, 10:00
Opponent
Supervisors
Note
QC 20100818Available from: 2007-03-27 Created: 2007-03-27 Last updated: 2010-08-18Bibliographically approved

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