Incorporation of model proteins into lipid layers

Aspects of biosensors: Dissertation

Inger Vikholm

Research output: ThesisDissertationCollection of Articles

Abstract

In this work different approaches of the Langmuir-Blodgett (LB) technique have been studied in order to obtain an oriented and sensitive protein layer onto a solid surface. LB films of Cd arachidate or self-assembled films of octadecylmercaptan have been used as supports for the artificial protein layers. It is essential that the support is defect-free in order to minimise non-specific binding. The quality of LB layers of Cd arachidate was noticed to be strongly influenced by how the solid slide was positioned in relation to the compressing barrier during vertical deposition. Atomic force microscopy, AFM, revealed that the layers were more homogeneous, if the slide was placed parallel to the compressing barrier. The amount of layer transferred could be determined by using either a surface acoustic wave device or a quartz crystal as a microbalance. Moreover, surface plasmon resonance, SPR, could be used to evaluate monolayer transfer. Unilamellar vesicles fused to form monolayers when spread onto the air-water interface. The layers could be transferred to hydrophobic supports as visualised by AFM. This could be a means to incorporate membrane proteins into lipid layers, as membrane proteins easily can be embedded into vesicles. Aged vesicles were transferred as large domains. Antibodies of the C-reactive protein, CRP, formed a monolayer, when spread directly onto the air-water interface. The antibodies seemed to take up a slanted orientation or have a random distribution in the monolayer. Antibodies were also adsorbed from the subphase onto various monolayers. Binding of anti-CRP was dependent on the packing of the film, on the antibody concentration in the aqueous subphase, but also on the monolayer matrix. The binding was highest to a monolayer of octadecylamine. The monolayer was, furthermore, stabilised by the antibodies. Evaluation of the specific interaction between the antigen-antibody complex with a quartz crystal microbalance indicated that the antigen could be determined in the concentration range of 0.1 - 5 µg/ml. In order to obtain site-directed immobilization, various amounts of a biosynthetically produced lipid-tagged single-chain antibody were incorporated into phospholipid monolayers preformed at the air-water interface. Incorporation of the single-chain antibodies, transfer of the layer onto solid slides, amount of non-specific adsorption and thus the amount of specific binding depended on the composition of the lipid matrix. Studies by AFM revealed that the film consisted of antibody-enriched lipid domains and that the films were not stable when stored long times in aqueous solution. The binding of hapten, which was used as an antigen, could be detected with SPR in the concentration range of 0.1 - 100 µg/ml.
Original languageEnglish
QualificationDoctor Degree
Awarding Institution
  • Åbo Akademi University
Supervisors/Advisors
  • Teleman, Olle, Advisor, External person
Award date27 May 1996
Place of PublicationEspoo
Publisher
Print ISBNs951-38-4931-7
Publication statusPublished - 1996
MoE publication typeG5 Doctoral dissertation (article)

Fingerprint

Biosensors
Monolayers
Lipids
Proteins
Antibodies
Single-Chain Antibodies
Water
Membrane Proteins
Air
Antigens
Acoustic surface wave devices
Unilamellar Liposomes
Quartz
Haptens
Quartz crystal microbalances
Langmuir Blodgett films
Surface plasmon resonance
Antigen-Antibody Complex
C-Reactive Protein
Atomic force microscopy

Keywords

  • Langmuir-Blodgett films
  • layers
  • immobilization
  • proteins
  • antibodies
  • biosensors
  • detectors
  • surface acoustic wave device
  • quartz crystal microbalance
  • surface plasmon resonance
  • atomic force microscopy
  • lipids

Cite this

Vikholm, I. (1996). Incorporation of model proteins into lipid layers: Aspects of biosensors: Dissertation. Espoo: VTT Technical Research Centre of Finland.
Vikholm, Inger. / Incorporation of model proteins into lipid layers : Aspects of biosensors: Dissertation. Espoo : VTT Technical Research Centre of Finland, 1996. 53 p.
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abstract = "In this work different approaches of the Langmuir-Blodgett (LB) technique have been studied in order to obtain an oriented and sensitive protein layer onto a solid surface. LB films of Cd arachidate or self-assembled films of octadecylmercaptan have been used as supports for the artificial protein layers. It is essential that the support is defect-free in order to minimise non-specific binding. The quality of LB layers of Cd arachidate was noticed to be strongly influenced by how the solid slide was positioned in relation to the compressing barrier during vertical deposition. Atomic force microscopy, AFM, revealed that the layers were more homogeneous, if the slide was placed parallel to the compressing barrier. The amount of layer transferred could be determined by using either a surface acoustic wave device or a quartz crystal as a microbalance. Moreover, surface plasmon resonance, SPR, could be used to evaluate monolayer transfer. Unilamellar vesicles fused to form monolayers when spread onto the air-water interface. The layers could be transferred to hydrophobic supports as visualised by AFM. This could be a means to incorporate membrane proteins into lipid layers, as membrane proteins easily can be embedded into vesicles. Aged vesicles were transferred as large domains. Antibodies of the C-reactive protein, CRP, formed a monolayer, when spread directly onto the air-water interface. The antibodies seemed to take up a slanted orientation or have a random distribution in the monolayer. Antibodies were also adsorbed from the subphase onto various monolayers. Binding of anti-CRP was dependent on the packing of the film, on the antibody concentration in the aqueous subphase, but also on the monolayer matrix. The binding was highest to a monolayer of octadecylamine. The monolayer was, furthermore, stabilised by the antibodies. Evaluation of the specific interaction between the antigen-antibody complex with a quartz crystal microbalance indicated that the antigen could be determined in the concentration range of 0.1 - 5 µg/ml. In order to obtain site-directed immobilization, various amounts of a biosynthetically produced lipid-tagged single-chain antibody were incorporated into phospholipid monolayers preformed at the air-water interface. Incorporation of the single-chain antibodies, transfer of the layer onto solid slides, amount of non-specific adsorption and thus the amount of specific binding depended on the composition of the lipid matrix. Studies by AFM revealed that the film consisted of antibody-enriched lipid domains and that the films were not stable when stored long times in aqueous solution. The binding of hapten, which was used as an antigen, could be detected with SPR in the concentration range of 0.1 - 100 µg/ml.",
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Vikholm, I 1996, 'Incorporation of model proteins into lipid layers: Aspects of biosensors: Dissertation', Doctor Degree, Åbo Akademi University, Espoo.

Incorporation of model proteins into lipid layers : Aspects of biosensors: Dissertation. / Vikholm, Inger.

Espoo : VTT Technical Research Centre of Finland, 1996. 53 p.

Research output: ThesisDissertationCollection of Articles

TY - THES

T1 - Incorporation of model proteins into lipid layers

T2 - Aspects of biosensors: Dissertation

AU - Vikholm, Inger

PY - 1996

Y1 - 1996

N2 - In this work different approaches of the Langmuir-Blodgett (LB) technique have been studied in order to obtain an oriented and sensitive protein layer onto a solid surface. LB films of Cd arachidate or self-assembled films of octadecylmercaptan have been used as supports for the artificial protein layers. It is essential that the support is defect-free in order to minimise non-specific binding. The quality of LB layers of Cd arachidate was noticed to be strongly influenced by how the solid slide was positioned in relation to the compressing barrier during vertical deposition. Atomic force microscopy, AFM, revealed that the layers were more homogeneous, if the slide was placed parallel to the compressing barrier. The amount of layer transferred could be determined by using either a surface acoustic wave device or a quartz crystal as a microbalance. Moreover, surface plasmon resonance, SPR, could be used to evaluate monolayer transfer. Unilamellar vesicles fused to form monolayers when spread onto the air-water interface. The layers could be transferred to hydrophobic supports as visualised by AFM. This could be a means to incorporate membrane proteins into lipid layers, as membrane proteins easily can be embedded into vesicles. Aged vesicles were transferred as large domains. Antibodies of the C-reactive protein, CRP, formed a monolayer, when spread directly onto the air-water interface. The antibodies seemed to take up a slanted orientation or have a random distribution in the monolayer. Antibodies were also adsorbed from the subphase onto various monolayers. Binding of anti-CRP was dependent on the packing of the film, on the antibody concentration in the aqueous subphase, but also on the monolayer matrix. The binding was highest to a monolayer of octadecylamine. The monolayer was, furthermore, stabilised by the antibodies. Evaluation of the specific interaction between the antigen-antibody complex with a quartz crystal microbalance indicated that the antigen could be determined in the concentration range of 0.1 - 5 µg/ml. In order to obtain site-directed immobilization, various amounts of a biosynthetically produced lipid-tagged single-chain antibody were incorporated into phospholipid monolayers preformed at the air-water interface. Incorporation of the single-chain antibodies, transfer of the layer onto solid slides, amount of non-specific adsorption and thus the amount of specific binding depended on the composition of the lipid matrix. Studies by AFM revealed that the film consisted of antibody-enriched lipid domains and that the films were not stable when stored long times in aqueous solution. The binding of hapten, which was used as an antigen, could be detected with SPR in the concentration range of 0.1 - 100 µg/ml.

AB - In this work different approaches of the Langmuir-Blodgett (LB) technique have been studied in order to obtain an oriented and sensitive protein layer onto a solid surface. LB films of Cd arachidate or self-assembled films of octadecylmercaptan have been used as supports for the artificial protein layers. It is essential that the support is defect-free in order to minimise non-specific binding. The quality of LB layers of Cd arachidate was noticed to be strongly influenced by how the solid slide was positioned in relation to the compressing barrier during vertical deposition. Atomic force microscopy, AFM, revealed that the layers were more homogeneous, if the slide was placed parallel to the compressing barrier. The amount of layer transferred could be determined by using either a surface acoustic wave device or a quartz crystal as a microbalance. Moreover, surface plasmon resonance, SPR, could be used to evaluate monolayer transfer. Unilamellar vesicles fused to form monolayers when spread onto the air-water interface. The layers could be transferred to hydrophobic supports as visualised by AFM. This could be a means to incorporate membrane proteins into lipid layers, as membrane proteins easily can be embedded into vesicles. Aged vesicles were transferred as large domains. Antibodies of the C-reactive protein, CRP, formed a monolayer, when spread directly onto the air-water interface. The antibodies seemed to take up a slanted orientation or have a random distribution in the monolayer. Antibodies were also adsorbed from the subphase onto various monolayers. Binding of anti-CRP was dependent on the packing of the film, on the antibody concentration in the aqueous subphase, but also on the monolayer matrix. The binding was highest to a monolayer of octadecylamine. The monolayer was, furthermore, stabilised by the antibodies. Evaluation of the specific interaction between the antigen-antibody complex with a quartz crystal microbalance indicated that the antigen could be determined in the concentration range of 0.1 - 5 µg/ml. In order to obtain site-directed immobilization, various amounts of a biosynthetically produced lipid-tagged single-chain antibody were incorporated into phospholipid monolayers preformed at the air-water interface. Incorporation of the single-chain antibodies, transfer of the layer onto solid slides, amount of non-specific adsorption and thus the amount of specific binding depended on the composition of the lipid matrix. Studies by AFM revealed that the film consisted of antibody-enriched lipid domains and that the films were not stable when stored long times in aqueous solution. The binding of hapten, which was used as an antigen, could be detected with SPR in the concentration range of 0.1 - 100 µg/ml.

KW - Langmuir-Blodgett films

KW - layers

KW - immobilization

KW - proteins

KW - antibodies

KW - biosensors

KW - detectors

KW - surface acoustic wave device

KW - quartz crystal microbalance

KW - surface plasmon resonance

KW - atomic force microscopy

KW - lipids

M3 - Dissertation

SN - 951-38-4931-7

T3 - VTT Publications

PB - VTT Technical Research Centre of Finland

CY - Espoo

ER -

Vikholm I. Incorporation of model proteins into lipid layers: Aspects of biosensors: Dissertation. Espoo: VTT Technical Research Centre of Finland, 1996. 53 p.