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Null Ellipsometry and Protein Adsorption to Model Biomaterials
Linköping University, Department of Physics, Measurement Technology, Biology and Chemistry. Linköping University, The Institute of Technology.
2001 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

When implants are inserted into the human body cascades of events become started that will determine the outcome of wound healing and ultimately the success of the implantation. The events start with the adsorption of small molecules, and proteins that may be activated (enzymes) or are able to activate cells of the immune defense and the healing process. In the biomaterials research that is conducted in our group we often ask two questions: "How much?" and "What?" proteins adsorb to a specific surface after incubation in serum or plasma. In the first two papers in this thesis I studied how well we are able to determine the answer to the first question. In the latter two works I tried to answer both questions for two model biomaterial surfaces: oligo(ethylene glycol) terminated self assembled monolayers on gold and chitosan coated silicon.

In many null ellipsometric studies the protein film refractive, Nfilm, is assumed to be close to 1.5. In the first paper we analyzed if the assumption of Nfilm = 1.465 is satisfactory for the determination of the surface mass density of a submonolayer thin protein film. Human serum albumin (HSA) was labeled with 125I and mixed with non-labeled HSA, and hydrophobic and hydrophilic silicon pieces were incubated in the solutions. The surface mass densities on all pieces determined by both ellipsometry and gamma counter measurements, and was pair-wise compared. The above assumption regarding the value of Nfilm for the agreement between the methods was satisfactory, although precautions have to be made not to overestimate the surface mass density when studying radiolabelled proteins, especially at rough surfaces.

Are the assumptions made in Paper I also true for up to 100 nm thick protein films or do we have to use a different protein film refractive index and do the ellipsometric model still hold? Human serum albumin (HSA) and polyclonal anti-HSA were labeled with 125I and mixed with unlabelled proteins. Hydrophobic silicon pieces were alternatingely incubated in the two protein solutions. Again the surface mass density was quantified with null ellipsometry and a gamma counter and the methods compared. The thickest protein layers were also gently scratched and the thickness measured by AFM. It appeared that a protein film refractive index Nfilm = 1.5 was a good choice for the determination of the protein film thickness. However, in order to obtain a good methods agreement for the adsorbed mass density a linear correction term was needed in the Cuypers surface mass density formula for ellipsometry. The physical interpretation of the correction term is presently unclear.

Self assembled monolayers (SAMs) containing oligo (ethyleneglycol) end groups (OEG) have been successfully used to minimize protein adsorption from single protein solutions. We investigated the protein resistance in a fibrinogen solution, serum and plasma of OEG-SAMs with an increasing number of OEG units and with different end groups. It turned out that the adsorbed amounts after 10 minutes of plasma incubation and 15 minutes of fibrinogen incubation decreased with an increasing number of EG units. In serum, the total deposition and subsequent deposition of antibodies towards complement proteins (C3c, C3d and properdin) did not depend on the number of EG units. In summary, the investigated OEG-SAMs were not protein resistant in complex solutions, although the adsorbed amounts varied with the number of EG units and the terminal chemical group. Complement deposition was observed at OEG surfaces.

For the last 50 odd years different polysaccharides, such as heparin and cellulose have been used for clinical applications and in recent years also chitin and its deacetylated form chitosan have gained increasing attention as potential biomaterials. Previous studies on complement activation by chitosan derivatives have focused on the soluble complement factors and not the surface bound ones that may be important for the binding of cells to surfaces. In our study about 10 nm thick chitosan films were incubated in plasma or serum and subsequently in polyclonal antibodysolutions. The films did not activate complement and the intrinsic pathway of coagulational though fibrinogen was detectable after plasma incubations. When the chitosan film was acetylated it became a strong alternative complement pathway activator in serum and fibrinogen was then no longer antibody detectable after plasma incubations.

Place, publisher, year, edition, pages
Linköping: Linköping University , 2001. , p. 43
Series
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 709
National Category
Other Basic Medicine
Identifiers
URN: urn:nbn:se:liu:diva-180146Libris ID: 8374252ISBN: 9173730866 (print)OAI: oai:DiVA.org:liu-180146DiVA, id: diva2:1601593
Public defence
2001-09-21, Planck, Fysikhuset, Linköpings universitet, Linköping, 10:15
Opponent
Available from: 2021-10-08 Created: 2021-10-08 Last updated: 2023-03-09Bibliographically approved
List of papers
1. Quantification of adsorbed human serum albumin at solid interfaces: A comparison between radioimmunoassay (RIA) and simple null ellipsometry
Open this publication in new window or tab >>Quantification of adsorbed human serum albumin at solid interfaces: A comparison between radioimmunoassay (RIA) and simple null ellipsometry
2000 (English)In: Colloids and Surfaces B: Biointerfaces, ISSN 0927-7765, E-ISSN 1873-4367, Vol. 18, no 2, p. 71-81Article in journal (Refereed) Published
Abstract [en]

Radioimmunoassay (RIA) and null ellipsometry are two common methods to quantify adsorbed proteins. However, the accuracy of null ellipsometry with a constant protein refractive index (n=1.465, k=0) at ?=632.8 nm has this far not been explored. The present study compared the methods, and the degree of agreement between the simplified single wavelength null ellipsometry and RIA to quantify adsorbed proteins was explored on different surfaces. The quantification methods agreed well when Angstrom smooth hydrophilic or hydrophobic silicon surfaces, and freshly radio-labelled proteins were used. Some discrepancies were noted when either rough surface or stored and aged labelled proteins were used. The differences decreased when the aged protein solution was equilibrated with freshly dissolved proteins at room temperature (RT) for a few hours prior to the surface incubations. Significant differences were also noted between the methods when albumin was adsorbed at it's iso-electric point (pH 4.8). Copyright (C) 2000 Elsevier Science B.V.

National Category
Engineering and Technology
Identifiers
urn:nbn:se:liu:diva-47606 (URN)10.1016/S0927-7765(99)00136-8 (DOI)
Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2021-10-08
2. The determination of thickness and surface mass density of mesothick immunoprecipitate layers by null ellipsometry and protein 125Iodine labeling
Open this publication in new window or tab >>The determination of thickness and surface mass density of mesothick immunoprecipitate layers by null ellipsometry and protein 125Iodine labeling
2002 (English)In: Journal of Colloid and Interface Science, ISSN 0021-9797, E-ISSN 1095-7103, Vol. 249, no 1, p. 84-90Article in journal (Refereed) Published
Abstract [en]

The aim of the present study was to ellipsometrically determine the thickness and surface mass density in air for up to 110-nm-thick organic layers made of alternatingly deposited layers of HSA and polyclonal anti-HSA on hydrophobic silicon. The ellipsometrically determined thickness was compared to that obtained by AFM and the deposited surface mass density calibrated with 125I-labeled proteins. The results indicate a good agreement in protein layer thickness between AFM and ellipsometry when the protein film refractive index Nfilm = 1.5 -0i, although then the calculated surface mass density from the ellipsometry data became grossly overestimated by the Cuypers one-component formula. A good agreement in the surface mass density was obtained when the M/A ratio in this formula was lowered from 4.14 to 2.35. This approach indicates a convenient means of determining the refractive indices and surface mass densities of mesothick organic layers proteins on solid supports. © 2002 Elsevier Science (USA).

Keywords
Ellipsometry, Mass, Protein adsorption, Radio labeling, Thickness
National Category
Engineering and Technology
Identifiers
urn:nbn:se:liu:diva-46903 (URN)10.1006/jcis.2002.8247 (DOI)
Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2021-10-08
3. Protein adsorption to oligo(ethylene glycol) self-assembled monolayers: Experiments with fibrinogen, heparinized plasma, and serum
Open this publication in new window or tab >>Protein adsorption to oligo(ethylene glycol) self-assembled monolayers: Experiments with fibrinogen, heparinized plasma, and serum
Show others...
2001 (English)In: Journal of Biomaterials Science. Polymer Edition, ISSN 0920-5063, E-ISSN 1568-5624, Vol. 12, no 6, p. 581-597Article in journal (Refereed) Published
Abstract [en]

Low protein adsorption is believed advantageous for blood-contacting materials and ethylene glycols (EG)-based polymeric compounds are often attached to surfaces for this purpose. In the present study, the adsorption of fibrinogen, serum, and plasma were studied by ellipsometry on a series of well-defined oligo(EG) terminated alkane-thiols self-assembled on gold. The layers were prepared with compounds of the general structure HS-(CH2)15-CONH-EGn, where n = 2, 4, and 6. Methoxy-terminated tri(EG) undecanethiol and hydroxyl-terminated hexadecanethiol self-assembled monolayers (SAMs) were used as references. The results clearly demonstrate that the adsorption depends on the experimental conditions with small amounts of fibrinogen adsorbing from a single protein solution, but larger amounts of proteins from serum and plasma. The adsorption of fibrinogen and blood plasma decreased with an increasing number of EG repeats and was temperature-dependent. Significantly less serum adsorbed to methoxy tri(EG) than to hexa(EG) and more proteins remained on the latter surface after incubation in a sodium dodecyl sulfate (SDS) solution, indicating a looser protein binding to the methoxy-terminated surface. All surfaces adsorbed complement factor 3(C3) from serum and plasma, although no surface-mediated complement activation was observed. The present study points to the importance of a careful choice of the protein model system before general statements regarding the protein repellant properties of potential surfaces can be made.

Place, publisher, year, edition, pages
Taylor & Francis, 2001
Keywords
Complement, Fibrinogen, Heparinized plasma, Oligo(ethylene glycol), Protein adsorption, Self-assembled monolayers, Serum
National Category
Engineering and Technology
Identifiers
urn:nbn:se:liu:diva-47265 (URN)10.1163/156856201316883421 (DOI)
Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2021-10-08Bibliographically approved
4. Blood protein adsorption onto chitosan
Open this publication in new window or tab >>Blood protein adsorption onto chitosan
2002 (English)In: Biomaterials, ISSN 0142-9612, E-ISSN 1878-5905, Vol. 23, no 12, p. 2561-2568Article in journal (Refereed) Published
Abstract [en]

Chitosan was recently indicated to enhance osteogenesis, improve wound healing but to activate the coagulation and the complement systems. In the present study approximately 10nm thick chitosan film were prepared on aminopropyltriethoxysilane (APTES) coated silicon. The surfaces were incubated in serum or plasma and subsequently in antibodies towards key complement and contact activation of coagulation proteins. The deposited amounts were compared with those on hydrophilic and hydrophobic silicon, APTES and IgG coated reference samples. Although large amounts of serum deposited to chitosan only a weak transient activation of the complement system and no activation of the intrinsic pathway was observed. Upon acetylation the chitosan layer became a strong activator of the alternative pathway of the complement. After incubation in human plasma anti-fibrinogen deposited onto chitosan but not onto the acetylated chitosan, a finding that may explain previous observations of procoagulant activity by chitosan. Copyright © 2002 Elsevier Science Ltd.

Keywords
Chitosan, Complement activation, Contact activation, Ellipsometry, Serum, Surface immobilization
National Category
Engineering and Technology
Identifiers
urn:nbn:se:liu:diva-47038 (URN)10.1016/S0142-9612(01)00391-X (DOI)
Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2021-10-08

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