Systematic studies of protein immobilization by surface plasmon field-enhanced fluorescence spectroscopy [Elektronische Ressource] / vorgelegt von Jing Liu

Administrator

Informations

• Publié par: johannes_gutenberg-universitat_mainz
• Publié le: 01 janvier 2005
• Nombre de lectures: 9
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

Systematic Studies of Protein Immobilization
by Surface Plasmon Field-Enhanced
Fluorescence Spectroscopy




Dissertation zur Erlangung des Grades
“Doktor der Naturwissenschafen”





am Fachbereich Biologie
der Johannes Gutenberg-Universität
in Mainz





vorgelegt von
Jing Liu
aus Chengdu, Sichuan, V. R. China



Mainz, Februar, 2005




























Die vorliegende Arbeit wurde unter Betreuung von Herrn Prof. Dr. H. Paulsen und Herrn
Prof. Dr. W. Knoll im Zeitraum zwischen August 2001 bis Februar 2005 am Max-Planck-
Institute für Polymerforschung, Mainz, Deutschland angefertigt.

Abstract
The research interest of this study is to investigate surface immobilization strategies for proteins and other
biomolecules by the surface plasmon field-enhanced fluorescence spectroscopy (SPFS) technique.
The recrystallization features of the S-layer proteins and the possibility of combining the S-layer lattice arrays with
other functional molecules make this protein a prime candidate for supramolecular architectures. The
recrystallization behavior on gold or on the secondary cell wall polymer (SCWP) was recorded by SPR. The optical
thicknesses and surface densities for different protein layers were calculated. In DNA hybridization tests performed
in order to discriminate different mismatches, recombinant S-layer-streptavidin fusion protein matrices showed their
potential for new microarrays. Moreover, SCWPs coated gold chips, covered with a controlled and oriented
assembly of S-layer fusion proteins, represent an even more sensitive fluorescence testing platform. Additionally, S-
layer fusion proteins as the matrix for LHCII immobilization strongly demonstrate superiority over routine
approaches, proving the possibility of utilizing them as a new strategy for biomolecular coupling.
In the study of the SPFS hCG immunoassay, the biophysical and immunological characteristics of this glycoprotein
hormone were presented first. After the investigation of the effect of the biotin thiol dilution on the coupling
efficiently, the interfacial binding model including the appropriate binary SAM structure and the versatile
streptavidin-biotin interaction was chosen as the basic supramolecular architecture for the fabrication of a SPFS-
based immunoassay. Next, the affinity characteristics between different antibodies and hCG were measured via an
equilibrium binding analysis, which is the first example for the titration of such a high affinity interaction by SPFS.
The results agree very well with the constants derived from the literature. Finally, a sandwich assay and a
competitive assay were selected as templates for SPFS-based hCG detection, and an excellent LOD of 0.15 mIU/ml
was attained via the “one step” sandwich method. Such high sensitivity not only fulfills clinical requirements, but is
also better than most other biosensors.
Fully understanding how LHCII complexes transfer the sunlight energy directionally and efficiently to the reaction
center is potentially useful for constructing biomimetic devices as solar cells. After the introduction of the structural
and the spectroscopic features of LHCII, different surface immobilization strategies of LHCII were summarized
next. Among them the strategy based on the His-tag and the immobilized metal (ion) affinity chromatography
(IMAC) technique were of great interest and resulted in different kinds of home-fabricated His-tag chelating chips.
Their substantial protein coupling capacity, maintenance of high biological activity and a remarkably repeatable
binding ability on the same chip after regeneration was demonstrated. Moreover, different parameters related to the
stability of surface coupled reconstituted complexes, including sucrose, detergent, lipid, oligomerization,
temperature and circulation rate, were evaluated in order to standardize the most effective immobilization
conditions. In addition, partial lipid bilayers obtained from LHCII contained proteo-liposomes fusion on the surface
were observed by the QCM technique. Finally, the inter-complex energy transfer between neighboring LHCIIs on a
gold protected silver surface by excitation with a blue laser ( λ = 473nm) was recorded for the first time, and the
factors influencing the energy transfer efficiency were evaluated.
- i -
CONTENTS
CONTENTS...................................................................................................................................................I
1 INTRODUCTION............................................................................................................................... 1
1.1 BIOSENSOR ....................................................................................................................................... 1
1.2 OPTICAL BIOSENSOR......................................................................................................................... 3
1.3 AIM OF THE STUDY ........................................................................................................................... 4
2 THEORETICAL BACKGROUND................................................................................................... 6
2.1 SURFACE PLASMON OPTICS .............................................................................................................. 6
2.1.1 Evanescent Wave and Surface Polaritons....................................................................... 6
2.1.1.1 Total Internal Reflection (TIR) and Evanescent Wave.......................................................................... 6
2.1.1.2 Surface Polaritons at a Noble Metal/Dielectric Interface ...................................................................... 7
2.1.2 Surface Plasmon Spectroscopy with Prism Coupling .................................................. 10
2.1.3 SPR Response to a Thin Film Deposition...................................................................... 13
2.2 FLUORESCENCE............................................................................................................................... 15
2.2.1 Fluorescence Process....................................................................................................... 15
2.2.2 nce Resonance Energy Transfer (FRET) ..................................................... 16
2.2.3 Surface Plasmon Field-Enhanced Fluorescence Spectroscopy (SPFS)................... 18
2.2.3.1 Field Enhancement.............................................................................................................................. 18
2.2.3.2 Fluorescence at the Metal/Dielectric Interface .................................................................................... 19
2.3 LANGMUIR ISOTHERM..................................................................................................................... 22
3 EXPERIMENTAL METHODS 26
3.1 SPFS SETUP AND LIQUID HANDLING SYSTEM................................................................................ 26
3.1.1 Home-Made SPFS Setup ................................................................................................. 26
3.1.2 Flow Cell and Liquid Handling System ........................................................................... 28
3.2 SURFACE MODIFICATION TECHNIQUES........................................................................................... 29
3.2.1 Substrate and Metal Film Preparation ............................................................................ 29
3.2.2 Self-Assembled Monolayers............................................................................................. 31
3.2.3 Spin coating for Deposition of Polystyrene Film............................................................ 32
3.2.4 Sol-gel Procedure for Deposition of Silica Film ............................................................. 33
3.2.5 SCWP Activation and Self Assembly on Gold............................................................... 34
3.3 PREPARATION OF RECONSTITUTED LHCII SOLUTION FOR SURFACE
IMMOBILIZATION ............................................................................................................................... 35
4 S-LAYER MATRIX: ANOTHER STRATEGY FOR BIOMOLECULAR COUPLING.......... 38
4.1 INTRODUCTION ............................................................................................................................... 38
4.2 MATERIALS...... 40
4.3 RECRYSTALLIZATION OF S-LAYER PROTEINS ON GOLD OR SCWPS ............................................... 41
4.3.1 Recrystallization of rSbpA on Gold.................................................................................. 42
4.3.2 Rezation of rSbsB-HT on SCWPs...................................................................... 42
4.3.3 Recrystallization of rSbpA-HT on Gold and on SCWPs 43
4.4 GRAFTING EXPERIMENTS................................................................................................................ 44
4.5 DNA HYBRIDIZATION ON S-LAYER-STREPTAVIDIN FUSION PROTEINS........................................... 46
4.5.1 Instability Problem.............