Immobilisation of biomolecules onto organised molecular assemblies: Dissertation

Willem A. Albers

Research output: ThesisDissertation

Abstract

This thesis describes immobilisation techniques for biomolecules on solid surfaces via an intermediate self-assembled or Langmuir-Blodgett (LB) film. Such films are advantageous, because the biological activity can be optimised by tailoring the layer composition. Factors like charge density, density of the linking group and composition of the monolayer matrix have particularly large effects on the activity. In the first part, oxidase enzymes were immobilised on self-assembled, conductive layers of bispyridiniumthiophene oligomers (thienoviologens) on gold substrates. The enzyme was bound by electrostatic interaction of the negatively charged enzyme with the positively charged pyridinium groups. The enzymatic activity of glucose oxidase was dependent on the surface density of the conductor and on the ionic strength during adsorption. In the second part of the thesis, Fab'-fragments were bound to LB-films of various linker lipids and the immobilisation efficiency (relative amount of binding sites) was optimised by variation of the constitution of the lipid LB-film. The films were deposited onto various substrates by vertical contact transfer of a preformed mixed Langmuir film of the linker lipid and a matrix lipid. The immobilisation efficiency was investigated with radioassay, quartz crystal microbalance (QCM) and surface plasmon resonance (SPR) measurements, while also atomic force microscopy (AFM) was used to image the structure of the films at different stages of binding. The immobilisation efficiency of the LB-films appeared to be much higher as that of more conventional methods for antibody immobilisation. Between 20 and 70% of the binding sites could be preserved, depending on the type of linker and film matrix composition, while that of the conventional methods was less than 10%. Films with dipalmitoyl-phosphatidylcholine as the matrix lipid and a maleimide derivative of dipalmitoylphosphatidyl-ethanolamine showed the highest sensitivity (and lowest detection limit) in QCM measurements.
Original languageEnglish
QualificationDoctor Degree
Awarding Institution
  • Cranfield University
Award date21 Sep 1999
Place of PublicationEspoo
Publisher
Print ISBNs951-38-5389-6
Electronic ISBNs951-38-5390-X
Publication statusPublished - 1999
MoE publication typeG5 Doctoral dissertation (article)

Fingerprint

Langmuir Blodgett films
Biomolecules
Lipids
Quartz crystal microbalances
Enzymes
Chemical analysis
Binding Sites
1,2-Dipalmitoylphosphatidylcholine
Glucose Oxidase
Immunoglobulin Fab Fragments
Ethanolamine
Surface plasmon resonance
Substrates
Coulomb interactions
Bioactivity
Ionic strength
Charge density
Oligomers
Gold
Monolayers

Keywords

  • immobilization
  • molecules
  • Langmuir-Blodgett films
  • enzymes
  • oxidases
  • biosensors
  • modelling
  • bilayers
  • lipid membranes

Cite this

Albers, W. A. (1999). Immobilisation of biomolecules onto organised molecular assemblies: Dissertation. Espoo: VTT Technical Research Centre of Finland.
Albers, Willem A.. / Immobilisation of biomolecules onto organised molecular assemblies : Dissertation. Espoo : VTT Technical Research Centre of Finland, 1999. 124 p.
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abstract = "This thesis describes immobilisation techniques for biomolecules on solid surfaces via an intermediate self-assembled or Langmuir-Blodgett (LB) film. Such films are advantageous, because the biological activity can be optimised by tailoring the layer composition. Factors like charge density, density of the linking group and composition of the monolayer matrix have particularly large effects on the activity. In the first part, oxidase enzymes were immobilised on self-assembled, conductive layers of bispyridiniumthiophene oligomers (thienoviologens) on gold substrates. The enzyme was bound by electrostatic interaction of the negatively charged enzyme with the positively charged pyridinium groups. The enzymatic activity of glucose oxidase was dependent on the surface density of the conductor and on the ionic strength during adsorption. In the second part of the thesis, Fab'-fragments were bound to LB-films of various linker lipids and the immobilisation efficiency (relative amount of binding sites) was optimised by variation of the constitution of the lipid LB-film. The films were deposited onto various substrates by vertical contact transfer of a preformed mixed Langmuir film of the linker lipid and a matrix lipid. The immobilisation efficiency was investigated with radioassay, quartz crystal microbalance (QCM) and surface plasmon resonance (SPR) measurements, while also atomic force microscopy (AFM) was used to image the structure of the films at different stages of binding. The immobilisation efficiency of the LB-films appeared to be much higher as that of more conventional methods for antibody immobilisation. Between 20 and 70{\%} of the binding sites could be preserved, depending on the type of linker and film matrix composition, while that of the conventional methods was less than 10{\%}. Films with dipalmitoyl-phosphatidylcholine as the matrix lipid and a maleimide derivative of dipalmitoylphosphatidyl-ethanolamine showed the highest sensitivity (and lowest detection limit) in QCM measurements.",
keywords = "immobilization, molecules, Langmuir-Blodgett films, enzymes, oxidases, biosensors, modelling, bilayers, lipid membranes",
author = "Albers, {Willem A.}",
note = "Project code: K9SU00086",
year = "1999",
language = "English",
isbn = "951-38-5389-6",
series = "VTT Publications",
publisher = "VTT Technical Research Centre of Finland",
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Albers, WA 1999, 'Immobilisation of biomolecules onto organised molecular assemblies: Dissertation', Doctor Degree, Cranfield University, Espoo.

Immobilisation of biomolecules onto organised molecular assemblies : Dissertation. / Albers, Willem A.

Espoo : VTT Technical Research Centre of Finland, 1999. 124 p.

Research output: ThesisDissertation

TY - THES

T1 - Immobilisation of biomolecules onto organised molecular assemblies

T2 - Dissertation

AU - Albers, Willem A.

N1 - Project code: K9SU00086

PY - 1999

Y1 - 1999

N2 - This thesis describes immobilisation techniques for biomolecules on solid surfaces via an intermediate self-assembled or Langmuir-Blodgett (LB) film. Such films are advantageous, because the biological activity can be optimised by tailoring the layer composition. Factors like charge density, density of the linking group and composition of the monolayer matrix have particularly large effects on the activity. In the first part, oxidase enzymes were immobilised on self-assembled, conductive layers of bispyridiniumthiophene oligomers (thienoviologens) on gold substrates. The enzyme was bound by electrostatic interaction of the negatively charged enzyme with the positively charged pyridinium groups. The enzymatic activity of glucose oxidase was dependent on the surface density of the conductor and on the ionic strength during adsorption. In the second part of the thesis, Fab'-fragments were bound to LB-films of various linker lipids and the immobilisation efficiency (relative amount of binding sites) was optimised by variation of the constitution of the lipid LB-film. The films were deposited onto various substrates by vertical contact transfer of a preformed mixed Langmuir film of the linker lipid and a matrix lipid. The immobilisation efficiency was investigated with radioassay, quartz crystal microbalance (QCM) and surface plasmon resonance (SPR) measurements, while also atomic force microscopy (AFM) was used to image the structure of the films at different stages of binding. The immobilisation efficiency of the LB-films appeared to be much higher as that of more conventional methods for antibody immobilisation. Between 20 and 70% of the binding sites could be preserved, depending on the type of linker and film matrix composition, while that of the conventional methods was less than 10%. Films with dipalmitoyl-phosphatidylcholine as the matrix lipid and a maleimide derivative of dipalmitoylphosphatidyl-ethanolamine showed the highest sensitivity (and lowest detection limit) in QCM measurements.

AB - This thesis describes immobilisation techniques for biomolecules on solid surfaces via an intermediate self-assembled or Langmuir-Blodgett (LB) film. Such films are advantageous, because the biological activity can be optimised by tailoring the layer composition. Factors like charge density, density of the linking group and composition of the monolayer matrix have particularly large effects on the activity. In the first part, oxidase enzymes were immobilised on self-assembled, conductive layers of bispyridiniumthiophene oligomers (thienoviologens) on gold substrates. The enzyme was bound by electrostatic interaction of the negatively charged enzyme with the positively charged pyridinium groups. The enzymatic activity of glucose oxidase was dependent on the surface density of the conductor and on the ionic strength during adsorption. In the second part of the thesis, Fab'-fragments were bound to LB-films of various linker lipids and the immobilisation efficiency (relative amount of binding sites) was optimised by variation of the constitution of the lipid LB-film. The films were deposited onto various substrates by vertical contact transfer of a preformed mixed Langmuir film of the linker lipid and a matrix lipid. The immobilisation efficiency was investigated with radioassay, quartz crystal microbalance (QCM) and surface plasmon resonance (SPR) measurements, while also atomic force microscopy (AFM) was used to image the structure of the films at different stages of binding. The immobilisation efficiency of the LB-films appeared to be much higher as that of more conventional methods for antibody immobilisation. Between 20 and 70% of the binding sites could be preserved, depending on the type of linker and film matrix composition, while that of the conventional methods was less than 10%. Films with dipalmitoyl-phosphatidylcholine as the matrix lipid and a maleimide derivative of dipalmitoylphosphatidyl-ethanolamine showed the highest sensitivity (and lowest detection limit) in QCM measurements.

KW - immobilization

KW - molecules

KW - Langmuir-Blodgett films

KW - enzymes

KW - oxidases

KW - biosensors

KW - modelling

KW - bilayers

KW - lipid membranes

M3 - Dissertation

SN - 951-38-5389-6

T3 - VTT Publications

PB - VTT Technical Research Centre of Finland

CY - Espoo

ER -

Albers WA. Immobilisation of biomolecules onto organised molecular assemblies: Dissertation. Espoo: VTT Technical Research Centre of Finland, 1999. 124 p.