1
Bilaga 2. Survey of scientific, teaching and administrative activities
Survey of Bo Liedberg's scientific, teaching and
administrative experience
Scientific career
My academic career started in 1981 when I made my diploma work for the Ms Sc. degree in Physics, at the Laboratory of Applied Physics, Linköping University, Sweden. Professor I. Lundström, the head of the Laboratory, was at that time heavily involved in setting up methods and technologies for the study of interfacial phenomena of interest in chemistry, biology and medicine. In particular he focused the activities toward the development of novel transducer principles for bio- and chemical sensing. I had during my undergraduate courses been studying a phenomenon called surface plasmon resonance (SPR), an extremely sensitive optical method for the characterization of thin films and molecular adsorbates on metal surfaces. Prof. Lundström and his co-workers offered me and another student a diploma work aiming at investigating the possibilities of using SPR for bio- and chemical sensing. This diploma work turned out to be very interesting, and we showed for the first time that the SPR method could be used to detect small concentrations of the anaesthetic gas halothane. Most importantly, however, SPR could be used to study antigen-antibody interactions at surfaces, an observation which later formed the basis for the development of several commercial systems based on this and related technologies.
I was after the diploma work offered a PhD position at the Laboratory Applied Physics. The first year of my PhD period (1982) was devoted to the SPR physics and to the optimization of the experimental set-up, and we published in 1983 two papers on the use of SPR for chemical and biosensing. Our experimental set-up was at that time very simple, but it enabled us to study the binding between IgG and a-IgG in real time. This paper attracted much interest in the biosensing community. Although it is a very short paper, I do think that it is one of my most important publications, not for its scientific content, but rather for its impact on the development of biosensors based on SPR detection. Another important boundary condition (coincidence) was that a new company was founded within Pharmacia at this time, 1984-85, Phamacia Biosensor AB (now Biacore AB). They were looking for new technologies for the detection of biospecific interactions in real time, and became very interested in SPR, and we started to collaborate with them. After a few years of evaluation the company made a strategic decision to concentrate the efforts on the development of SPR for biospecific interaction analysis (BIA). Our research efforts and know-how were for practical reasons transferred to Biosensor. SPR turned out to be a very efficient detection principle and it is today the key component in all BIA instruments developed by the company including, BIAcore 1000, BIALite, BIAcore 2000, BIAcore X and BIAprobe.
I continued to collaborate with the company and have since then been involved in the development of their systems, as a consultant on transducer physics, spectroscopical characterization and interface analysis. Pharmacia Biosensor AB also offered me a post doc position for three years 1986-1989 to establish a strong activity in the field of transducer physics, artificial biosensing interfaces, molecular films and surface analysis at the laboratory of Applied Physics, Linköping University. Today two of my students are working on joint projects with Biacore AB.
The topics of my PhD studies changed in 1984 from being purely biosensing oriented to include also surface biology, molecular films and surface characterization. In my thesis entitled" Studies of Adsorbed Proteins and Amino Acids: Surface Plasmon Resonance, Infrared and Electron [BL1]Spectroscopy". I used a broad range of modern characterization tools to investigate the structure of adsorbed biomolecules. Many of my papers deal with physical characterization of thin organic films and coatings on metallic substrates. The reason for focusing on metals is that they display many interesting properties for biomaterial applications, cf. the importance titanium and titanium dioxide have had for the development of dental implants. Thus, the physical characterization of metals in complex organic and biological environments is crucial for gaining a deeper understanding of biocompatibility, fouling and related issues. Metals are also extremely important materials for the development of novel transducers based on electrical, electrochemical, gravimetric, and optical detection principles. A large part of the activities within my group is today devoted to development of accurate synthetic protocols for interfacing biomolecules with metallic transducer substrates. I’m convinced that a broad competence within the field of surface modification, molecular films, surface spectroscopy is crucial for establishing a competitive activity in a multidisciplinary field as bio- and chemical sensing. I also have been working with physical characterization of the bulk and interface properties of polymers, in particular so-called conducting polymers. My publications can be broadly divided into four main categories:
- Molecular films and surface spectroscopy (40 papers)
- Artificial biosensing interfaces and bio- and chemical sensors (25 papers)
- Interface biology and biomaterials (20 papers)
- Polymers and polymer interfaces (10 papers)
A part of the molecular film project is also focused on microscopic wetting and the role of interfacial water in molecular recognition and affinity sensing. This is the most fundamental ongoing research project in my group just now. The aim of the project is to establish a fundamental understanding about the organization of strongly bound water at or near biointerfaces and active epitopes in biomolecules. Thus it is closely related to our biosensing projects. Below follows a short presentation of ongoing projects within my group.
Ongoing research
The activities are currently focused on three main themes: I)Chemical- and Biosensors and artificial biosensing interfaces; II) Molecular Films, Self-Assembled Monolayers and Biomaterials; III) Inorganic Surface Chemistry. The work within inorganic surface chemistry and self-assembled monolayers (SAMs) form the basis for the activities in the sense that the results from this research open for the development of novel model organic surfaces to be used in our applied research projects.
The biological applications of SAMs are of particular interest to us, and we are currently using the above SAMs as model surfaces in our bio- and chemical sensor and biomaterial research. The membrane like structure of these SAMs makes them also very attractive as models for ligand-receptor studies on cell surfaces. For example, the steric requirements for the very specific receptor-ligand reactions can be addressed in detail by designing SAMs with varying tail group separation and mobility.
I. Bio- and Chemical Sensors
I have started to work more actively with biosensors during the last few years. A significant part of the biosensor project is focused on methods to improve the sensitivity of biosensor systems based on surface plasmon resonance (SPR) detection. Patterned surfaces and colloidal particles are used to enhance the sensitivity of SPR equipment. This research also includes theoretical work on the development of efficient algorithms for resonance dip hunt, and evaluation. Considerable research efforts are also directed toward the development of artificial sensing interfaces. Accurate protocols involving SAMs are developed for attachment of receptors (haptens, bioactive carbohydrates, peptides, protein, oligonucleotides, DNA) to the sensing substrate in a proper orientation and with a well-presented active site (epitope). We are also trying to develop a proper surface chemistry which display low levels of non-specific binding. This approach is extremely important for applications in complex biofluids, and we are investigating if oligosaccharide- and/or polyethylene glycol-derivatized SAMs can resolve this problem.
Transducer physics: This research is aiming at improving the sensitivity and widen the applicability of optical transducers, primarily those based on SPR detection. One part of the research is devoted to the design of a multiwavelength imaging SPR apparatus. The multiwavelength approach enables us to obtain an image of the sensing layer and at the same time a depth profile of the layer. This apparatus is developed to meet the increasing interest in multispot and parallel detection in modern bioanalysis. Another project deals with the development of patterned surfaces for multiparameter detection using soft lithograpy “-contact printing”. The idea is to develop an integrated multitransducer sensing chips which enables us to obtain complementary information from the molecular recognition event. We develop for this purpose sensing chips capable of combining SPR with electrochemical, gravimetric, fluorescent, Raman detection principles.
We concentrate also on the development of accurate algorithms for the evaluation of resonance SPR-dip movement. Algorithms that reject noise and linearity artifacts, e.g. artifacts emanating from the discrete separation of photo elements in array detectors have been developed and described. The transducer oriented research is supported by Biacore AB.
Biomimetic membranes, signal transduction and transmembrane sensors: This is one of my recent projects aiming at developing a strategy to generate a supported bilayer structure containing an integral transmembrane protein on a solid surface. Integral membrane bound proteins and receptors play an important role in the complex cascade of interactions occurring at and across cell membranes. Despite this importance, the molecular understanding of the trans membrane processes is still far from complete. On the other hand, operating as molecular machines, they seem to be an attractive approach for molecular device applications, including smart biosensors. One of the main challenges in today's biosensor design is to develop artificial structures that enables us to mimic and monitor these molecular recognition and signal transduction events. The idea of this research is to combine skills and experience from different disciplines (materials and transducer physics, organic and surface chemistry, and molecular biology) in the assembly process of a functional phospholipid bilayer structure, with and incorporated transmembrane protein, on a suitable transducer substrate, Fig. 1. We are today working on various routes capable of attaching the lipid bilayer to the tranducer substrate. A crucial part of that research includes the attachment of a hydrogel layer to the trunducer substrate. This hydrogel should provide sufficient space for the intracellular loops of the transmembrane protein, the active component in the biosensor. The spreading of the lipid bilayer on top of the hydrogel is performed by vesicle fusion techniques.
Fig. 1. Artificial transmembrane structure immobilized on surface of an amperometric microelectrode transducer. The bilayer contains a transmembrane protein that, e.g., opens an ion channel Ca2+ because of a recognition (key-lock) event. Vesicle fusion techniques will be used to prepare the bilayer on top of the hydrogel.
I’m planning to use this approach to study G-protein coupled receptors in a joint project with Dr. S. Svensson, Inst. of Pharmacolgy, Linköping University. Preliminary interaction studies between certain fragments of the of the intracellular loop of the receptor and the entire G-protein, as well as its subunits , have already been undertaken using SPR.
Nanoporous assemblies: A new class of SAMs has been developed within the project. Cholesterol, an important molecule in cell membranes, have been modified with a SH group in the 3-position to allow efficient immobilization on gold. This molecule thiocholesterol (TC) forms SAMs with a large fraction of molecular-sized defects on Au(111) terraces, about 5 Å in diameter. These defects can be addressed (filled) by other molecules, including, for example, alkanethiols. Thus, the TC assembly can act as a template for the generation of new SAMs. This project is conducted in collaboration with Professor J.-M. Kauffmann, University Libre, Bruxelles, Belgium. Prof. Kauffmann is interested in the electrochemical properties of these defect-rich structures, and we are trying to develop them for pharmacological sensing applications.
Oligothiophenes and Molecular Wires: A new set of SAMs based on alkanethiols and oligothiophenes have been initiated in collaboration with Prof. P. Bäuerle, University of Ulm, and Prof. W. Göpel, University of Tübingen. Professsor Bäuerle is an organic synthetic chemist working with organic conductors and he is interested in the development of new devices based on highly organized oligothiophenes. The molecular electronic materials display many interesting properties, and we are primarily interested in using them as molecular wires for charge transfer and signal transduction between the biological recognition system and the transducer substrate. Thus, we hope to be able to used them in combination with our research efforts on electrochemical sensing (see oligonucleatide and DNA sensing). The project is a spin-off of a visiting fellowship for Bo Liedberg at the institute of Physical and Theoretical Chemistry, University of Tübingen.
Affinity sensing and structure-function analysis of cytokines: A new project on receptor-ligand (antigen-antibody) interactions also has been initiated together with Professor R. Revoltella, CNR, Pisa, Italy. The project deals with a structure-function analysis of recombinant human Granulozyte Machrophage Colony Stimulating Factor (rhGM-CSF), an important growth factor for proliferation, differentiation, maturation of cells. Biosensor technology developed at out laboratory has been used to identify important epitopes for the normal function of rhGM-CSF by using monoclonal antibodies directed against the entire molecule as well as against active sequences (10-15 amino acid long) of the molecule. We have also synthesized mutant peptides where the native sequence has been changed by selective substitution of alanine. This type of experiments have enabled us to identify the critical amino acid sequence of GM-CSF.
Another project with Professor Revoltella deals with pesticide detection and monitoring. A combined RIA , ELISA and SPR approach for the study of cross reactivity and epitope mapping was undertaken using [(2,4-dichlorophenoxy)-acetic acid]-BSA conjugates and produced monoclonal antibodies.
DNA and oligonucleotide sensing: This research was recently initiated as a collaborative project between my group and Sangtec Medical AB, Bromma. The idea is to use electrochemical detection principles (capacitive or impedance) for the study hybridization phenomena at electrode surfaces. Again, we intend to utilize our knowledge in molecular engineering and self-assembly to design and develop novel sensing surfaces. The research is also devoted to the development of microelectrode arrays for fast and efficient detection of hybridization phenomena. The capacitance method in particular has proven to be very sensitive in antigen/antibody experiments. Recent studies indicate that the detection level fall in the femtomolar range, which is quite impressing for a non-labeling technique.
Chemical sensors: We are also using SAMs to develop novel interfaces for gas sensing applications. We are using a surface acoustic wave (SAW) transducer to develop sensing interfaces based on hydrogen-bond interaction with the molecule to be detected. In this particular case we are using dimethylmethylphosphonate (DMMP), which is used as a model molecule for the highly toxic sarin molecule. SAMs terminated with functionalities of different hydrogen bonding/accepting properties are investigated to optimize the sensor action and response. We are, in particular, investigating the role of water on sensor response and function because water is always present in real situations. The SAW results have been correlated with spectroscopical data to obtain a structural understanding of the interaction events at the sensor substrate. Infrared and mass spectroscopy techniques have been employed.
II. Molecular Films, Self-Assembled Monolayers and Biomaterials
One of the important break-through in surface chemistry during the eighties was the development of the preparation strategies for Self Assembled Monolayers (SAMs) based on long chain n-alkyl thiols HS(CH2)n-X on gold (silver and copper). These molecules, 10-22 methylene units long, were shown to form highly organized, densely packed and very stable monolayers on gold with the SH group acting as an anchor to the surface and the tail group (X=COOH, CN, OH, CH3 etc.) pointing towards the surrounding medium. The monolayers exhibit a low defect density and the alkyl chains are organized in an almost perfect all trans (zig-zag) conformation. The structural properties of the thiol monolayers are, in fact, fully comparable with those obtained for the more well known Langmuir-Blodgett(LB) films of, e.g., long-chain carboxylic acids (-OOC-(CH2)n-CH3) on metal oxides. They display, at the same time, the chemical and mechanical robustness of alkyltrichlorosilanes on Si/SiO2. Thus, SAMs based on n-alkyl thiols on gold (RSH/Au) are believed to have a great potential for applications in very aggressive environments where a predefined chemical identity is of vital importance.
Fundamental studies on the structure of SAMs: The specificity and high binding strength of the SH group to gold, as compared to otherfunctional groups like COOH, CN and OH, implies that the adsorption characteristics is almost independent of the chemical properties of the tail group X. Thus, substituted n-alkyl thiols also form highly organized assemblies on Au. This unique property opens the possibility of forming mixed monolayers with predefined composition and distribution from binary mixtures of HS-(CH2)n-X and HS(CH2)m-Y. Another very important experimental parameter is the mobility of the tail group. By preparing SAMs from binary mixtures of thiols with varying chain length it is possible to alter the properties of the outermost surface from a crystalline-like (n=m) to a more liquid-like (nm) character. Two fundamentally very important questions can be raised about mixed SAMs.First, what is the relation between the solution composition of the two components and the ultimate surface composition ? Secondly, do the components aggregate into single component domains (macroscopic islands) or are they distributed randomly onthe surface ? We have found these questions very interesting and much of our fundamental research is focused on compositional and structural characterization of mixed SAMs.
Molecular interaction studies on SAMs: We have used mixed SAMs of HS(CH2)16-OH and HS-(CH2)15-CH3 to prepare model organic surfaces with varying wettabilty (0°<H2O<114°). These SAMs are used substrates to study wettability induced phase transitions condensed overlayers of D2O. Both infrared and temperature programmed desorption spectroscopy have shown that thermal annealing (82-140 K) of the initially condensed D2O layer (82 K) undergoes a phase transition from amorphous solid D2O ice to polycrystalline Ih ice in the above temperature range. The exact transition temperature depends on the wettability of the surface. It is also found that the microscopic wetting phenomena are closely related to those observed in the macroscopic world, i.e., 3-D (droplet-like) nucleation occurs on low energy surfaces and 2-D (flat) nucleation at high energy surfaces.