bioscope publishable 070419 - europa

BIOSCOPE Final Publishable Report

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BIOs cope

Project no.

NMP4-CT-2003-505211 Project acronym BIOSCOPE

Project full name Self-reporting biological nanosystems to study and control bio-

molecular mechanisms on the single molecule level Instrument type SPECIFIC TARGETED RESEARCH OR INNOVATION PROJECT Priority name Priority 3: Nanotechnology and nanoscience, knowledge-based multifunctional materials, new production processes and devices (NMP)

Final Publishable Report Period covered: from February 1, 2006 to January 31, 2007 Date of preparation: April 19, 2007 Start date of project: Duration: February 1, 2004 36 month Project coordinator name Dr. Tommy Nylander Project coordinator organisation name Revision 2.0 Lund University (Div. Physical Chemistry 1)


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BIOs copewww.BIOSCOPE.FKEM1.LU.SE

Project execution The aim with BIOSCOPE was to pioneer the development of new leading edge nanoscale research tools and methodologies that will allow unprecedented insight into bio-molecular mechanisms at biological interfaces on the molecular scale. BIOSCOPE consider the biomolecular system itself as a part of the nanoscopic instrument, which in various ways reports to the out-side world about its current local state. Thereby it will be possible to study the local effects on the molecular level when e.g. a protein interacts with a biomembrane surface or when a lipase interacts with a lipid surface. The objectives of BIOSCOPE were: • To develop instrumentation and methods for manipulation of enzymes and

enzyme activity at the nanoscale providing insight into the biomolecular mechanisms on a single molecule level.

• To develop novel forms of integration, at the nanoscale level, of enzymes and non-biological systems such as nanoparticles, artificial membranes, electrical field or force field traps.

• To confine several enzymes to surfaces of nanoparticles or membranes on a less than 10 nm scale in order to achieve a self-organized assembly with a concerted bioaction superior to the simple sum of the same individual enzymes. The function of the nanosystems will be optimized for enzymatic action on biosubstrates.

Contractors involved BIOSCOPE is a pan-European consortium where the collective resources both in

terms of knowledge in physics, biology, surface and colloid chemistry, synthesis and

biochemistry, as well as a combination of a range of experimental techniques

reporting on nanoscale organization, theory and modelling will provide the necessary

knowledge-base for the new nanoscopic research tools to study bio-molecular

mechanisms at interfaces on the single molecular level.

Acronym Partner Responsible

scientist

Main role

P1

ULUND

Physical Chemistry 1, Lund

University, Lund, SWEDEN

Dr. Tommy

Nylander

How to build structures from

lipids and showing the

structure of nanoscopic

objects

P2

UCPH

Nano-Science Center (NSC),

University of Copenhagen,

Copenhagen, DENMARK

Prof. Thomas

Björnholm

Showing the effect of

enzymes on the substrate on

the nanoscopic scale

P3

Novozymes

Protein Design, Novozymes A/S,

Bagsvaerd, Denmark

Dr. Allan

Svendsen

Building new enzyme

P4

KUL

Laboratory for Photochemistry

and Spectroscopy, Katholieke

University of Leuven, Leuven,

BELGIUM

Prof. Johan

Hofkens

Watching very fast changes

in enzymes and changes in

the enzymes environment.

Measuring the light signal

from single enzyme

molecule.


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Acronym Partner Responsible

scientist

Main role

P5.

UNILIV

Centre for Nanoscale Science,

Department of Chemistry, The

University of Liverpool,

Liverpool, UK

Dr. Mathias

Brust

Making nanosized particles

that control enzymes on the

nanoscopic scale

P6

PHYSLMU

Biophysics & Molecular

Materials, Ludwig-Maximilians-

Universität, München,Germany

Prof. Hermann

Gaub

Direct measurements of the

force a single enzyme

molecule feels

P7

IBLT

Institute of Biochemistry, Vilnius,

LITHUANIA

Prof.

Valdemaras

Razumas

Making molecules for

connecting enzymes, enzyme

modification and measuring

the electrical signal from

enzymes

P8

SDU

MEMPHYS - Center for

Biomembrane Physics, University

of Southern Denmark Odense,

DENMARK

Prof. Ole. G.

Mouritsen

Telling how the properties of

the soft interface close to the

enzyme affect the function of

the enzyme. Predicting and

calculating enzyme and

substrate interactions

P9

KUN

Dep. of Organic Chemistry,

Radboud University of Nijmegen,

THE NETHERLANDS

Prof. Roeland J.

M. Nolte

Providing self-assembled

supra-molecular structures

for determining single

enzyme molecule action

mechanisms

Co-ordinator contact details Dr. Tommy Nylander

Physical Chemistry 1, Lund University, Center for Chemistry and Chemical

Engineering, P.O.Box 124, S-221 00 Lund, SWEDEN

Email: [email protected]

Phone: +46 46 2228158; Fax: +46 46 2224413 Summary of results BIOSCOPE has provided insight of the biomolecular mechanisms on a single molecule level. This has been made using two approaches: Confocal Microscopy: This is powerful tool to study the activity of single, but it requires that the ezyme is immobilized and does not work for natural substrates. The single enzyme experiment of CalB adsorbed to a hydrophobic surface (Velonia, 2005; Flomenbom, 2005) and TLL linked to the BSA foot labelled with fluorescent probe was studied (Hazitakis, 2006). The waiting times Dt between peaks (bursts of activity) contains the kinetic information. In fact the distribution of waiting times shows non-exponential behavior (Flamenbom, 2006). The appearance of a stretched exponential function implies that the number of conformations is large. This shows that the reaction rate constant, k, is not constant with time, that is TLL shows an oscillating behavior adopting a variety of interconnecting conformations (Figure 1). In fact one might talk of an enzyme molecule that sleeps and works (Engelkamp, 2006). Wide field microscopy: This technique can be used on natural substrates and accounts for the fact that the activity of the enzyme can be affected by its diffusion and vice versa. This method requires imaging of a large area, which means loss of


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resolution. The used approach in BIOSCOPE involves two lasers, one for the labelled enzyme and the labelled substrate layer, with minimal overlap in wavelength of laser wavelength and that of the emitted light (Verheijen, 2006; Hofkens, unpublished data, 2007). The movement of the single enzyme molecule could be followed by analysing the mean square displacement versus time. It was possible to follow the hydrolysis of phospholipid layer by means of fluorescence microscopy and simultaneously track single molecules of enzyme (single molecule resolution) acting on phospholipid layers and to monitor the changes in the phospholipids layers (not single molecule resolution). The results indicate that the enzyme docks on the layer for just short periods of time.

Figure 1. Schematic figure of the different interconnecting conformations of TLL, which in turn have different rate constant for the hydrolysis of the substrate. BIOSCOPE has developed new concepts for integration (linkers) and reporting at the nanoscale-level of enzymes and non-biological systems. A range of variants of Thermomyces lanuginosus lipase (TLL), but also of Candida antarctica lipase B (CalB), mostly for binding signalling, either binding or action, or attachment to surfaces has been prepared. This has been essential for all the bioconjugated system produced within BIOSCOPE. These two lipases has also been the main enzymes used in the work, but some work also includes Phospholipase A2 (PLA or PLA2) In the past there has been a lack of reporter molecules that report on the activity of lipase and at the same time are amphiphilic and self-assembling as the natural substrate for lipases. This challenge has been met by BIOSCOPE by producing lipid like probes (Valincius, 2005; Ignatjev, 2005; Bulovas, 2006), supported bilayers (Simonsen, 2004) as well as well defined lipid based liquid crystalline nanoparticles (Barauskas, 2006; Popescu 2007). In many cases the synthesised substrates can be detected by electrochemical methods (redox activity) as well as optical methods such


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as UV/VIS and fluorescence spectroscopy. Some of the key components of these amphiphilic and self-assembling substrates are presented in Figure 2a.

Figure 2a. Examples of reporter molecules, e.g. substrates for lipase, which can report on the enzymatic activity at different type of interfaces. Apart from profluorescent substrate study of single molecule enzyme activity requires some sort of immobilization of the enzyme. Several approaches have been developed for this purpose involving Direct covalent immobilization as well as Non-specific deposition. Some of the approaches used in BIOSCOPE are summarized in Figure 2b (Dirks, 2005; Hazitakis, 2006; Engelkamp, 2006).

HON3

O

75O

N3

O

75

N

O

O

N340

OOO

40

OO

N3

ON3

O

75

N3

O O

HSi

OH

O O

HSi

O O

HSi

OH

O O

HSi

OH

O O

HSi

O O

HSi

OH

O O

HSi

OH

N3 N3

N3

O

O

O

O

O

N3

OH

O

N

S OO

NH

Clickable Maleimides Clickable Maleimides

Clickable dyes Clickable dyes

Clickable Enzymes-Proteins Clickable Enzymes-Proteins

Clickable Surface Clickable Surface Clickable polymers and Clickable polymers and block-copolymers block-copolymers

HNTLL

O

SBSA

N3

O

O

SBSA

O

O

HNTLL SH

O

SCALB

N3

O

O

SCALB

O

O

Figure 2b. Different linker approaches that have been developed during BIOSCOPE, by KUN. They can be used both to couple to surfaces as well as to form Enzyme dimers. The two new electrochemical (amperometric) techniques to determine the lipase activity have been developed within BIOSCOPE. The main features of these methods are:

• A new interfacial electrochemical technique, based on solid-supported substrates/electrodes (Valincius, 2005). This can in principle be engineered in the form of the nanosensors.


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• A quasi-homogeneous electrochemical technique, where lipid-like synthetic compounds have been employed as the micellar lipase substrates, possessing higher oxidation overvoltage compared to the reporting products of enzymatic reaction (Ignatjev, 2005).

For the first time infrared-visible sum frequency generation (SFG) vibrational spectroscopy applied to probe activity of TLL at the air/water interface using a new, self-assembling substrate, O-palmitoyl-2,3-dicyanohydroquinone (PDCHQ) (Niaura, 2007). The SFG data revealed ordered structure of the PDCHQ monolayer, which was reduced when TLL was present. The PLA2 activity has been determined, using automated image analysis to extract turnover numbers and binding constants on binary and ternary lipid membranes consisting of DOPC and DPPC in solid/fluid mixtures as well as DOPC, DPPC and cholesterol in two fluid membrane phases: liquid disordered (ld) (DOPC rich) and liquid ordered (DPPC + Cholesterol rich) (Simonsen, 2006; Jensen, 2005; Mouritsen, 2006). The results can be related to the phase diagrams of the systems and the main conclusions from the results are that preferential hydrolysis occurs in the fluid phase and that Cholesterol impedes hydrolysis of DPPC in the liquid ordered state. Liquid crystalline lipid based nanoparticles (Barauskas, 2006; Popescu 2007) were used as a well-defined and natural substrates for study of the lipase activity of TLL. The focus is to relate the nanoscopic structure of the substrate to the lipase activity. The studies were carried out using cryo-TEM for imaging and synchrotron X-ray diffraction (Beamline I711 at MAX-lab, Lund University, Sweden) to obtain structure and structure parameters and pH-stat titration with base to follow the lipolytic reaction (Barauskas, unpublished data 2007). The sequence of structural lipid nanoparticle changes due to lipase action is largely dependent on the pH. Lipase induces a transformation of cubic nanoparticles into lamellar/sponge-like aggregates at pH 8.4, whereas at pH 6.5 nanoparticles undergo a phase transition sequence bicontinuous cubic reversed hexagonal reversed micellar cubic emulsion. At the low pH the structural changes of the substrate were in some cases found to affect the pKa of the generated fatty acid, leading to an increase in pH. BIOSCOPE has developed new concepts for enzyme confinement in self-organized assemblies with a concerted bioaction superior to the simple sum of the same individual enzymes – the BIOSCOPE nanomachine, where also the activity can be affecting by external force fields. The new concepts of integration involve preparation of enzyme hetero-dimers by coupling the lipase (Thermomyces Lanuginosus lipase, TLL) to a large hydrophobic protein Bovine Serum Albumin (BSA) (Hatzakis, 2006). BSA is the “protein foot” anchor an enzyme on a surface for single molecule study. The activity of this hetero-dimer can be actually higher than the single TLL. The origin of this increase in activity is not yet clear, but preliminary data shows that it has to do with changes in the local environment of the enzyme. As this open up new possibilities for enzyme regulation on the single molecular scale, ongoing studies that include molecular modelling will further exploit these possibilities.


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Nanoparticles without attached lipase

=>Lipid aggregates were transformed=>Lipid aggregates were transformed

from their initial structure.from their initial structure.

Nanoparticles with attached lipase

Figure 3 LEFT: Nanoparticles prepared with the functionalized Lipase (TLL) provided, using Biotin/Streptavidin binding to gold nanoparticles. RIGHT: Cryo-TEM images shows that gold nanoparticles without the lipase do not interact with the liquid crystalline cubic nanoparticles, but the lipase-gold nanoparticles do interact with the liquid crystalline cubic nanoparticles and transform the aggregate from a cubic phase to vesicle-like structures due to the hydrolyses of the GMO in the substrate particles (Baruaskas, 2007). A substantial effort was put in preparing bioconjugated systems where several enzymes can be put together on well-defined particle, e.g. gold particle. Several concepts were investigated and in this process, we found that it is important to consider the non-specific binding of linkers, e.g. DNA, and the aggregation of the particles (Cardenas, 2006, 2007). The working lipase (TLL)-gold nanoparticle hybrids (Brennan, 2006), one of BIOSCOPE nano-machine prototypes, were tested by using liquid crystalline lipid based nanoparticles (Barauskas, 2006; Popescu 2007) as a substrate. The cryo-TEM images (Figure 3) shows that gold nanoparticles without the lipase do not interact with the liquid crystalline cubic nanoparticles, but the lipase-gold nanoparticles do interact with the liquid crystalline cubic nanoparticles and transform the aggregate from a cubic phase to vesicular structures due to the hydrolyses of the GMO in the substrate particles (Barauskas, 2007). This proof of concept has inspired ongoing studies where we are linking several different enzymes to one gold nanoparticle. A system of Fluorescence Microscope with Fluorescence Correlation Spectroscopy (FCS) and a Langmuir trough for lateral pressure control has been set up. The FCS is modified to be capable of doing wide field single molecule microscopy (with an EMCCD Camera). With this setup we have been able to visualize enzymatic processes, such phospholipase A (PLA) activity on lipid monoalyers (DPPC) at the air/water interface with Single molecule resolution and in Real time at the same time as we can regulated lateral pressure on the substrate and thereby the domain


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formation. Thorough analyses of the images versus time reveal that the enzyme seems to attack on one side only of the DPPC domains (Gudmundsen et al., unpublished data 2007). The aim of the combined AFM/TIRFM work is to correlate the turnover of CalB with an external force, which is applied to the enzyme. This setup can be used to vary the distance between surface and AFM tip and therefore can be used to stretch or compress the enzyme. The cleavage of a profluorescent substrate that diffuses free in solution results in one single fluorescent molecule per turnover. One of the major challenges to achieve this objective was to come up with a strategy on how to attach the enzyme to a surface. This was successfully done by a combination of enzyme engineering and advanced linker technology (Blank, Morfill, Gumpp and Gaub, 2006; Blank, Morfill, and Gaub, 2006). An externally applied force most likely shifts the equilibrium between different conformations of enzyme, which was revealed by the single molecule enzyme kinetics measurements. The activity can either increase or decrease upon stretching of the enzyme. Preliminary results show that the enzymatic activity of CalB can be manipulated by an agarose bead approaching the immobilized enzyme. It seems that CalB is switched on, when the bead is retracted from the surface (Blank et al., unpublished data 2007). We can therefore conclude that the BIOSCOPE team indeed has managed to build a nanomachine!!! Concluding remarks As a unifying end-goal BIOSCOPE has been to design and test a preorganized nanosystem consisting of several components with properties superior to the simple sum of the individual components. For this purpose single enzyme activity measurements have been performed. BIOSCOPE has significantly increase our fundamental knowledge in this area and showed that the reaction rate constant, k, is not constant in time, that is TLL shows an oscillating behavior adopting a variety of interconnecting conformations. The “nano-machines” have superior enzyme activity compared to traditional means of immobilisation. We have shown that the length of the linker and the point of attachment are crucial and one can in fact achieve a higher activity also for enzyme homo-dimers. We have also demonstrated that we can modulate the enzyme activity on single molecule scale by means of atomic force microscope. This has been achieved by sophisticated modification protocol including state of the art enzyme engineering. In the summary of the results above, the contribution of the different groups has not explicitly stated, since all the BISCOPE activities, and in particularly the project meetings, have been characterized by a true team spirit. All has contributed in there own way and style and the exchanged has been deep and sincere. Only the atom and the sky have marked the limits for our discussions. The impact goes beyond the publications and dissemination. BIOSCOPE references T. Nylander “Self-reporting biological nano-systems to study and control biomolecular acion on the single molecule level (BIOSCOPE)” Materials Technology 20 (2005), 32-35


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G. Valincius, I. Ignatjev, G. Niaura, M. Ka em kait , Z. Talaikyt , V. Razumas and A. Svendsen. A new electrochemical method for the detection of lipase activity. Anal. Chem. 77 (2005), 2632-2636. I. Ignatjev, G. Valincius, I. vedait , E. Gaidamauskas, M. Ka emekait , V. Razumas and Allan Svendsen. Direct amperometric determination of lipase activity. Anal. Biochem. 344 (2005), 275-277. J. L. Brennan, N. S. Hatzakis, T. R. Tshikhudo, N. Dirvianskyt , V. Razumas, S. Patkar, J. Vind, A. Swendsen, R. J. M. Nolte, A. E. Rowan, M. Brust. Bioconjugation via click chemistry: The creation of functional hybrids of lipases and gold nanoparticles. Bioconjugate Chem. 17(2006), 1373-1375. A. Bulovas, N. Dirvianskyt , Z. Talaikyt , G. Niaura, S. Valentukonyt , E. Butkus and V. Razumas. Electrochemical and structural properties of self-assembled monolayers of 2-methyl-3-( -mercaptoalkyl)-1,4-naphthoquinones on gold. J. Electroanal. Chem., 591 (2006), 175-188 M. Cardenas, J. Barauskas, K. Schillen, J.L. Brennan, M. Brust, T. Nylander. Thiol-Specific and Non-Specific Interactions between DNA and Gold Nanoparticles, Langmuir 2006, 22, 3294-3299. J. Barauskas, M. Johnsson, T. Nylander, F. Tiberg. Hexagonal Liquid-Crystalline Nanoparticles in Aqueous Mixtures of Glyceryl Monooleyl Ether and Pluronic F127, Chem. Lett. 2006, 35, 830-831. G. Popescu, J. Barauskas, T. Nylander, F. Tiberg. Liquid Crystalline Phases and their Dispersionsin Aqueous Mixtures of Glycerol Monooleate and Glyceryl Monooleyl Ether, Langmuir 2007, 23, 496-503. G. Niaura, Z. Kuprionis, I. Ignatjev, M. Kazemekaite, G. Valincius, Z. Talaikyt , V. Razumas, and Allan Svendsen. Probing of lipase activity at air/water interface by Sum-Frequency Generation Spectroscopy J. Am. Chem. Soc., 2007, submitted. W. Verheijen, H. Uji-i, C. Flors, J. Hotta, J. Hofkens, “Probing dynamics of individual bio molecules by single molecule spectroscopy” In Ultrasensitive and Single-Molecule Detection Technologies (Eds. Jörg Enderlein, Zygmunt K Gryczuznski) Proc. SPIE, Vol 6092, 2006, in press. Wendy Verheijen, Davey Loos, Frans C. De Schryver, Johan Hofkens, Book chapter in Springer Series in Biophysics, Volume: Single Molecules and Nanotechnology, 2007. J. (Ton) Dirks, Sander S. van Berkel, Nikos S. Hatzakis, Joost A. Opsteen, Floris L. van Delft, Jeroen J. L. M. Cornelissen, Alan E. Rowan, Jan C. M. van Hest, Floris P. J. T. Rutjes and Roeland J. M. Nolte, “Preparation of biohybrid amphiphiles via the copper catalysed Huisgen [3+2] dipolar cycloaddition reaction.” Chem. Commun. 2005, 4172-4174.


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O. G. Mouritsen, T. L. Andresen, A. Halperin, P. L. Hansen, A. F., Jakobsen, U. B. Jensen, M. Ø. Jensen, K. Jørgensen, T. Kaasgaard, C. Leidy, A. C. Simonsen, G. H. Peters and M. Weiss "Activation of interfacial enzymes at membrane surfaces” (Review) Journal of Physics: Condensed Matter, 18, 2006, S1293–S1304 U. B. Jensen, A. C. Simonsen “Shape relaxations in a fluid supported membrane during hydrolysis by phospholipase A2” BBA - Rapid Report 1715, 2005 1 – 5 A. C. Simonsen, L. A. Bagatolli “Structure of spin-coated lipid films and domain formation in supported membranes formed by hydration”. (2004) Langmuir, 20, 9720-9728. A. C. Simonsen, U. B. Jensen, P. L. Hansen “Hydrolysis of fluid supported membrane islands by phospholipase A(2): Time-lapse imaging and kinetic analysis” J. Colloid Interface Sci. 2006, 107-115. K. Velonia, O. Flomenbom, D. Loos, S. Masuo, M. Cotlet, Y. Engelborghs, J. Hofkens, A. E. Rowan, J. Klafter, R. J. M. Nolte, F. C. Schryver, “Single-enzyme kinetics of CalB-catalyzed hydrolyzes” Angew. Chem. Int. Ed. 2005, 44, 560-564, O. Flomenbom, K. Velonia, D. Loos, S., Masuo¶ M. Cotlet, Y. Engelborghs, J. Hofkens, A. E. Rowan, R. J. M. Nolte, M. Auweraer, F. C. Schryver, J. Klafter, “Stretched exponential decay and correlations in the catalytic activity of fluctuating single lipase molecules” PNAS, 2005, 102, 2368-2372. O. Flomenbom, J. Hofkens, K. Velonia, F. C. Schryver, A. E. Rowan, R. J. M. Nolte, J. Klafter, R. J. Silbey. “Correctly validating results from single molecule data: The case of stretched exponential decay in the catalytical activity of single lipase B molecules” Chem. Phys. Lett. 432 (2006), 371-374. Hans Engelkamp, Nikos S. Hatzakis, Johan Hofkens, Frans C. de Schryver, Roeland J. M. Nolte and Alan E. Rowan, “Do enzymes sleep and work?”Chem. Commun. 2006, 935-940 Nikos S. Hatzakis, Hans Engelkamp, Kelly Velonia, Johan Hofkens, Peter C. M. Christianen, Allan Svendsen, Shamkantr A. Patkar, Jesper Vind, Jan C. Maan, Alan E. Rowan and Roeland J. M. Nolte, “Synthesis and single enzyme activity of a clicked lipase–BSA hetero-dimer.”Chem. Commun. (2006) 2012-2014. Nørgaard, K., Weygand, M.J., Kjaer, K.,Brust, M., Bjørnholm, T. Adaptive chemistry

of bifunctional gold nanoparticles at the air/water interface. A synchrotron X-ray study

of giant amphiphiles. Faraday Discussions 125, 221-233 (2004).

Balashev, K., DiNardo, J., Callisen, T., Svendsen, A., Bjørnholm, T. Atomic Force

Microscope Visualization of Lipid Bilayer Degradation due to Action of

Phospholipase A2 and Humicola Lanuginosa Lipase Biochemica et Biophysics Acta –

Biomenbranes, 1768, 90-99, (Jan 2007)


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K. Blank, J. Morfill, H. Gumpp and H. E. Gaub. Functional Expression of Candida antarctica Lipase B in Eschericha coli. Journal of Biotechnology, 125, 2006, 474-483. K. Blank, J. Morfill, H. E. Gaub. Site-Specific Immobilization of Genitcally Engineered Variants of Candida Antarctica Lipase B. (2006) ChemBioChem 7: 1349-1351. M Cardenás and T. Nylander “Adsorption of DNA and DNA-surfactant complexes at solid surfaces” In Interaction of DNA with Surfactants and Polymers (Eds. B. Lindman and R. Dias), 2007, Blackwell Publishing, In Press. J. Barauskas, A. Kanaras, J. L. Brennan, A. Svendsen, M. Brust, T. Nylander. Direct

visualization of Lipase-Functionalized Gold Nanoparticle Activity on Self-Assembled

Lipid Superstructures, 2007, manuscript. Plan for using and disseminating knowledge It is the obligation of the partners to disseminate the generic know-how obtained

during the project during training of PhD-students and post docs as well as in teaching

to students and through publication in journals or via the Internet. Distribution of the

results to interested partners within the European Union will be ensured by:

By daily papers, media, general meetings etc. to reach the general public. 9th of January 2005, Vilnius – On the first national TV channel, the Lithuanian National Radio and Television (LRT) broadcasted the information about the “BIOSCOPE” project during the most popular news program “Panorama”. In the telecast, prof. Valdemaras Razumas (IBLT) briefly introduced the project. SDU has been very engaged in science play in Danish for the general public entitle “Den Magiske Kugle” (The magic ball) (www.magiskekugle.sdu.dk) which presents some of the BIOSCOPE topics. The actors are scientists including Ole G. Mouritsen and Adam Cohen Simonsen who are in the BIOSCOPE team. Thomas Bjørnholm (UCPH) gave invited talks at Seminar & Partnering Event: Bio-nanotechnology, British Embassy Copenhagen "Bio-nanotechnology research at The Nano-Science Center", 2004 for an audience of government official and business representative. He also gave a popular science lecture at the Day of Research at the Danish University of Pharmaceutical Sciences "Nanoscience", 2005. The BIOSCOPE home page. The homepage contain one part open for the public and

another part for internal use. The open part will as soon as the final reports are

approved by European Commission feature this report. A full and updated publication

list for work published from BIOSCOPE will be given with links to the original

publications (available free of charge for subscribers). The internal part contains all

reports and presentations from the BIOSCOPE meetings.

The project meetings, twice a year with all partners. All 7 meetings stipulated in the

contract have already taken place and was organised by the different partners to

maximize the exchange between the partners.


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Publication in scientific journals. So far 27 publications have been submitted with one of them awaiting decision and one is about to be submitted. The rest are in press or already published. 4 of the papers are reviews that try to put the outcome from BIOSCOPE in larger perspective. We expect many more papers (at least 20-25) to come out from the results generated within BIOSCOPE.

An open workshop, was held in the end, where the BIOSCOPE findings was

presented and discussed. The conference was entitled ”International Symposium on

Biomolecular Nanoscale Assemblies” and held January 24-26, 2007 at the Nano-

Science Center, University of Copenhagen, Denmark and also supported by Swedish

and Danish Research Councils. Thanks to this support we had the possibility to invite

some very prominent speakers: C. Dekker (University of Delft, The Netherlands), I.

Willner (Hebrew University, Jerusalem, Israel), D. Weitz (University of Harvard,

MA, USA), A. van Oijen (University of Harvard, MA, USA), L. Jaeger (UCSB, CA,

USA), B. Lounis (CNRS, Bordeaux, France), E. Delamarch (IBM, Zürich,

Switzerland) and from the BIOSCOPE: H. Gaub (LMU, Munich, Germany), J.

Hofkens (Katholieke Univeristeit Leuven, Belgium), A. Rowan (Radboud University

Nijmegen, The Netherlands), D. Stamou (University of Copenhagen, Denmark) and

M. Brust (University of Liverpool, UK). The conference attracted about 120

participant from most European countries who contributed with an additional 10 oral

presentations and 30 posters.

Home page will be kept open at least one year after the termination of the project.

The collaboration between the BIOSCOPE partners and with SME as well as other

research groups will continue after the termination of the project. The BIOSCOPE

partners have already established with SME:s like EKSPLA, Vilnius, Lithuania

(www.ekspla.com) and Camurus AB, Lund, Sweden (www.camurus.se).


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Annex – Plan for using and disseminating the knowledge Distribution of the results to interested partners within the European Union will be ensured in different ways: • The increased understanding of the generic scientific issues obtained during the course of

the project will be published regularly on the project home page, daily papers, media, general meetings etc. to reach the general public.

• The BIOSCOPE home page (www.BIOSCOPE.fkem1.lu.se).

• The project meetings that will take place twice a year with all partners will be one of the

ways to communicate the results to the different partners, apart from the informal day-to-

day contacts. These meeting will result in a written report. Publication of papers in

scientific journals.

• An open workshop, which was held in the end, where the BIOSCOPE findings, including

the novel BIOSCOPE nanomachine will be presented and discussed.

• The BISCOPE home page will be kept open at least one year after the termination of the

project.

• The collaboration between the BIOSCOPE partners and with SME as well as other

research groups will continue after the termination of the project.

1 Exploitable knowledge and its Use Previous year the lipase biosensor was presented as a spin-off from the development of the reporter molecules and the results from last years report are given below. This year no exploitable knowledge is yet ready for release to the market as we was during the second year assembling the different building blocks and further developing the methodology. The assembly of the BIOSCOPE nanomachine during the final year of BIOSCOPE is expect to trigger several products after project has formally ended. Overview table

Exploitable Knowledge (description)

Exploitable product(s) or measure(s)

Sector(s) of application

Timetable for commercial

use

Patents or other IPR protection

Owner & Other Partner(s) involved

Lipase activity can be monitored by electrochemical methods

Lipase sensor 1. Food 2. Biomedical

2008

None, results published

IBLT and Novozymes (owners)

1.1 Lipase sensor

1.1.1 Exploitable result

Two new electrochemical (amperometric) techniques for the enzyme activity characterization. The innovative interfacial and quasi-homogeneous electrochemical methods have been realized using reporter molecules and TLL. Contrasting other common methods for studying lipase activity, in the new interfacial electrochemical technique, the


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solid-supported substrates/electrodes have been developed which can be engineered in the form of the nanosensors. In the quasi-homogeneous electrochemical technique, a lipid-like synthetic compounds designed in WP1 have been employed as the micellar lipase substrates, possessing higher oxidation overvoltage compared to the reporting products of enzymatic reaction.

1.1.2 Partners involved

1. IBLT has contributed by synthesizing the novel self-assembling reporter molecule and by their knowledge on electrochemical methods and development of biosensor.

2. Novozymes has contributed with lipase and knowledge on lipolytic reactions and enzyme activity.

1.1.3 Exploitation potential

Both techniques, developed by IBLT in corporation with Novozymes, are practically insensitive to the presence of solid or/and liquid light-scattering microparticles in the solutions, and can be easily designed in the form of portable devices. A range of possible application can be imagined where there is a need to keep track of the lipase activity, e.g. in food industry, laundry detergent, and biomedical applications.

1.1.4 Additional research and development work needed

Additional research is needed on the structure of the reporting layer on the electrode. This will be aided collaborative work within BIOSCOPE on the single molecule enzyme activity as well as on the self-assembled substrate structure and morphology. To commercialize the product some technical development on the electrode design is needed.

1.1.5 Intellectual Property Rights protection measures

The basic findings have been published in: G. Valincius, I. Ignatjev, G. Niaura, M. Kazemekaite, Z. Talaikyte, V. Razumas, and A. Svendsen. Anal. Chem., 2005, 77, 2632-2636, and I. Ignatjev, G. Valincius, I. Svedaite, E. Gaidamauskas, V. Razumas and A. Svendsen. Anal. Biochem., 2005, 344, 275-277. Therefore it is open for commercialization. The development has not yet reached the stage where patent applications can be filed. The consortium agreement signed by the BIOSCOPE partners regulates the IPR.

1.1.6 Commercial contacts already taken

The developing team includes a commercial company, Novozymes, which will take care of commercialization if further test show that there is a potential. In addition frequent contacts with SME:s like EKSPLA, Vilnius, Lithuania (www.ekspla.com) and Camurus AB, Lund, Sweden (www.camurus.se).


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1.1.7 Other potential impact

A robust lipase sensor can have large impact as a tool to improve food quality as well as optimizing dosage of lipase in formulations. It can also be important tool in biomedicine and thereby it indirectly can have positive health aspects.

2 Dissemination of knowledge

2.1 Overview table Planned/

actual Dates

Type

Type of audience

Countries addressed

Size of

audience

Partner

responsible /involved

16 Nov

2005

Press release(press/radio/TV)

LTV2 channel in Program “Knowledge-based society”

General public

Lithuania

1 million

IBLT

2006-end Jan 2007

Science Theater play “Den Magiske Kugle” (The magic ball) (www.magiskekugle.sdu.dk)

General public Denmark 2000 SDU

2004 Seminar & Partnering Event British Embassy Copenhagen

Government, business Denmark,UK 100 UCPH

2005 Day of Research

Danish University of Pharmaceutical Sciences

General public Denmark 200 UCPH

Media briefing

Aug 2005

Conference SPIE Optics and Photonics 2005 – Program on Physical Chemistry of

Interfaces and Nanomaterials IV – San Diego, U.S.A.

Research

International

500

UNILIV

March 2006

ACS Spring Meeting in Atlanta Research International 200 ULUND/KUN

June 2005

Theodor Grotthuss Electrochemistry Conference, Vilnius

Research International 150 IBLT

July 2005 Materials Chemistry 7, Edinburgh Research International 150 UNILIV

Sept 2005

Bionanotechnology 2005 Research International 150 UNILIV

Oct 2005 19th Conference of the European Colloid and Interface Society – Geilo, Norway

Research International 150 ULUND

Nov 2005 Annual European Conference on Micro &

Nanoscale Technologies for the Biosciences (Nanotech) – Montreaux, Switzerland

Research International 300 ULUND

Dec 2005 Nanotechnology for Energy and Medicine, Beirut, Libanon

Research International 300 UNILIV

Nov 2005 18th FAOBMB Symposium - Lahore,

Pakistan

Research International 350 UNILIV, UCPH

Nov 2005 Royal Society of Chemistry “Chemistry of the Noble Metals” Award Lecture -

Warwick, U.K.,


2006 Conference on Statistical Physics, Soft

Matter and Biological Physics, NORDITA, Copenhagen

Research International 200 UCPH

2006 National Center Nanoscience and

Nanotechnology, Beijing, China


2006 Protein Engineering Conference, INPEC, Elsinore, Denmark


2006 Nano Ethics Workshop, Aarhus, Denmark Research International 100 UCPH

2006 Novo Nordisk TRAP Symposium

Copenhagen, Denmark


August 2005

Nathiagali Summer College - Nathiagali, Pakistan


Jan 2007 International Symposium on Biomolecular Nanoscale Assemblies”, University of Copenhagen

Research International 250 UNILIV, UCPH, PHYSLMU; KUN,KUL


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Planned/

actual Dates

Type

Type of audience

Countries addressed

Size of

audience

Partner

responsible /involved

Exhibition ”

Publications

2004 Faraday Discussions Research International 5000 UCPH,UNILIV

2005 Materials Today Research International 5000 ULUND

2005 Anal. Biochem. Research International 5000 IBLT,

Novozymes

2005 Anal. Chem. Research International 5000 IBLT, Novozymes

2006 J. Electroanal. Chem.. Research International 5000 IBLT

2006 J. Phys. Cond. Matter Research International 5000 SDU

2005 BBA- Rapid Report Research International 5000 SDU

2007 J. Am. Chem. Soc. Research International 5000 IBLT, Novozymes

2007 BBA-Biomembranes Research International 5000 UCPH, Novozymes

2006 Langmuir Research International 5000 UNILIV, ULUND

2006 Chem. Phys. Lett. Research International 5000 KUN, KUL

2006 Bioconjugate Chem. Research International 5000 UNILIV, KUN,

IBLT, Novozymes

2006 Chem. Com. Research International 5000 KUN

2006 Chem. Com. Research International 5000 KUN, KUL

2006 Chem. Com. Research International 5000 KUN, KUL, Novozymes

2006 Chem. Biochem. Research International 5000 PHYSLMU

2007 SPIE, Biomedical optics Research International 5000 KUL

2006 J. Colloid Interface Sci. Research International 5000 SDU

2006 J. Biotech Research International 5000 PHYSLMU

2006 Chem. Lett Research International 5000 ULUND

2007 Langmuir Research International 5000 ULUND

Aug 2004

Project web-site www.BIOSCOPE.fkem1.lu.se

General public International - ULUND

Nov 2004

Nov 2004 26-27 April

2006 sept 2006

Posters

International Conference in Honour of David Schiffrin – University of Liverpool, U.K.

Bionanotechnology 2004 – London, U.K. The Lithuanian Conference “Chemistry and Chemical Technology”, Kaunas

University of Technology, Kaunas, Lithuania International conference MAF 9, Lisbon

Portugal

Research

Research Research

Research

International

International INational

International

100

150 150

200

UNILIV

UNILIV IBLT

KUL

Autumn

2006

Courses

Physics an Chemistry of Nanostructures

Higher education International 50 UCPH, ULUND

2005 7th Course: "Nanotechnologies for Drug Delivery and medical Applications",

International School of Advanced Material Science and Technology, Ancona, Italy

Students, Higher education

International 100 UCPH

Flyers

Direct e-mailing

Film/video

2.2 Press release(press/radio/TV)

9th of January 2005, Vilnius – On the first national TV channel, the Lithuanian National Radio and Television (LRT) broadcasted the information about the “BIOSCOPE” project during the most popular news program “Panorama”. In the telecast, prof. Valdemaras Razumas (IBLT) briefly introduced the project. The program is available on the website of


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LRT (select 15:36 min from the news start): http://www.lrt.lt/dayarchive.php?d=2005-01=09# .

2.3 Other forms of communication results to general public.

SDU has been very engaged in science play in Danish for the general public entitle “Den Magiske Kugle” (The magic ball) (www.magiskekugle.sdu.dk) which presents some of the BIOSCOPE topics. The actors are scientists including Ole G. Mouritsen and Adam Cohen Simonsen who are in the BIOSCOPE team. Thomas Bjørnholm (UCPH) gave invited talks at

Seminar & Partnering Event: Bio-nanotechnology, British Embassy Copenhagen "Bio-

nanotechnology research at The Nano-Science Center", 2004 for an audience of government

official and business representative. He also gave a popular science lecture at the Day of

Research at the Danish University of Pharmaceutical Sciences "Nanoscience", 2005.

2.4 Conference

Dr. Gintaras Valincius, IBLT, delivered an oral presentation on BIOSCOPE electrochemical systems of lipase analysis at

• International Theodor Grotthuss Electrochemistry Conference - Institute of Chemistry, Vilnius, Lithuania, June 5-8, 2005, to an international audience of approx. 150 people.

Dr. Jennifer Brennan, UNILIV, delivered oral presentations on BIOSCOPE enzyme-nanoparticle hybrids at:

• Materials Chemistry 7 - Edinburgh, U.K., July 2005, to an international audience of approx. 300 people.

• Bionanotechnology 2005 - Brighton, U.K., September 2005, to an international audience of approx. 150 people.

Prof. Alan Rowan, KUN, gave an invited talk at: • American Chemical Society spring meeting in Atlanta, March 2006 to an

international audience of at least 200 people on supramolecular chemistry and nano-assemblies used in BIOSCOPE.

Dr. Tommy Nylander, ULUND, gave two invited talks at: • American Chemical Society spring meeting in Atlanta, March 2006 to an

international audience of at least 200 people on DNA compaction and nanoconstructs as well as on polymer-surfactant interactions at interfaces. Both talks presenting BIOSCOPE work.

Dr. Justas Barauskas, ULUND, delivered oral presentations on BIOSCOPE self-assembled lipid structures and their interaction with lipase at: • 19th Conference of the European Colloid and Interface Society – Geilo, Norway,

October 2005, to an international audience of approx 150 people • Annual European Conference on Micro & Nanoscale Technologies for the

Biosciences (Nanotech) – Montreaux, Switzerland, November 2005, to an international audience of approx. 300 people.

• Fourth Scandinavian Workshop on Scattering from Soft Matter – Lund, Sweden, February 2007, to an international audience of approx. 60 people

Dr. Mathias Brust, UNILIV delivered various lectures incorporating sections on BIOSCOPE enzyme-nanoparticle hybrids at:


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• Materials Chemistry 7 - Edinburgh, U.K., July 2005, to an international audience of approx. 400 people (keynote lecture).

• Pakistan Academy of Sciences - Islamabad, Pakistan, July 2005, to a Pakistani audience of approx. 200 people (invited address).

• SPIE Optics and Photonics 2005, Program on Physical Chemistry of Interfaces and Nanomaterials IV - San Diego U.S.A., July 2005 - to an international audience of approx. 500 people (invited lecture).

• Nathiagali Summer College - Nathiagali, Pakistan, August 2005, to an international audience of approx. 250 people (invited lecture).

• Royal Society of Chemistry “Chemistry of the Noble Metals” Award Lecture - Warwick, U.K., November 2005, to a mostly U.K. audience of approx. 150 people.

• 18th FAOBMB Symposium - Lahore, Pakistan, November 2005, to an international audience of approx. 350 people (invited lecture).

• Conference on Nanotechnology for Energy and Medicine - Beirut, Lebanon, December 2005, to an international audience of approx. 300 people (invited lecture).

Thomas Bjørnholm (UCPH) Gave invited talks at • 18th FAOMBM Symposium, Lahore, Pakistan: "Bionanotechnology and

nanomedicine", 2005 • Conference on Statistical Physics, Soft Matter and Biological Physics, NORDITA,

Copenhagen "Single molecule, single electronic transistors and self-assembled molecular electronic circuitry", 2006

• Protein Engineering Conference, INPEC, Elsinore, Denmark "Bionanotechnology and nanomedicine", 2006

• National Center Nanoscience and Nanotechnology, Beijing, China:"Bionanotechnology and nanomedicine", 2006

• Nano Ethics Workshop, Aarhus, Denmark: "Bionanotechnology - Drug Delivery for Cancer Treatment", 2006

• Novo Nordisk TRAP Symposium, Copenhagen: "Nanotechnology in Life Science" 2006

• Seminar, University of Lund: “Bionanotechnology and Nanomedicine”, 2006.

H. Gaub (LMU, Munich, Germany), J. Hofkens (Katholieke Univeristeit Leuven, Belgium),

A. Rowan (Radboud University Nijmegen, The Netherlands), D. Stamou (University of

Copenhagen, Denmark) and M. Brust (University of Liverpool, UK) from the BIOSCOPE

team presented the highlights from BIOSCOPE at the open BIOSCOPE symposium

”International Symposium on Biomolecular Nanoscale Assemblies” and held January 24-26,

2007 at the Nano-Science Center, University of Copenhagen. The conference attracted about

120 participants from most European countries who contributed with an additional 10 oral

presentations and 30 posters.

2.5 Exhibition

No exhibition


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2.6 Publications

T. Nylander “Self-reporting biological nano-systems to study and control biomolecular acion on the single molecule level (BIOSCOPE)” Materials Technology 20 (2005), 32-35 G. Valincius, I. Ignatjev, G. Niaura, M. Ka em kait , Z. Talaikyt , V. Razumas and A. Svendsen. A new electrochemical method for the detection of lipase activity. Anal. Chem. 77 (2005), 2632-2636. I. Ignatjev, G. Valincius, I. vedait , E. Gaidamauskas, M. Ka emekait , V. Razumas and Allan Svendsen. Direct amperometric determination of lipase activity. Anal. Biochem. 344 (2005), 275-277. J. L. Brennan, N. S. Hatzakis, T. R. Tshikhudo, N. Dirvianskyt , V. Razumas, S. Patkar, J. Vind, A. Swendsen, R. J. M. Nolte, A. E. Rowan, M. Brust. Bioconjugation via click chemistry: The creation of functional hybrids of lipases and gold nanoparticles. Bioconjugate Chem. 17(2006), 1373-1375. A. Bulovas, N. Dirvianskyt , Z. Talaikyt , G. Niaura, S. Valentukonyt , E. Butkus and V. Razumas. Electrochemical and structural properties of self-assembled monolayers of 2-methyl-3-( -mercaptoalkyl)-1,4-naphthoquinones on gold. J. Electroanal. Chem., 591 (2006), 175-188 M. Cardenas, J. Barauskas, K. Schillen, J.L. Brennan, M. Brust, T. Nylander. Thiol-Specific and Non-Specific Interactions between DNA and Gold Nanoparticles, Langmuir 2006, 22, 3294-3299.. J. Barauskas, M. Johnsson, T. Nylander, F. Tiberg. Hexagonal Liquid-Crystalline Nanoparticles in Aqueous Mixtures of Glyceryl Monooleyl Ether and Pluronic F127, Chem. Lett. 2006, 35, 830-831. G. Popescu, J. Barauskas, T. Nylander, F. Tiberg. Liquid Crystalline Phases and their Dispersionsin Aqueous Mixtures of Glycerol Monooleate and Glyceryl Monooleyl Ether, Langmuir 2007, 23, 496-503. G. Niaura, Z. Kuprionis, I. Ignatjev, M. Kazemekaite, G. Valincius, Z. Talaikyt , V. Razumas, and Allan Svendsen. Probing of lipase activity at air/water interface by Sum-Frequency Generation Spectroscopy. J. Am. Chem. Soc., 2007, submitted W. Verheijen, H. Uji-i, C. Flors, J. Hotta, J. Hofkens, “Probing dynamics of individual bio molecules by single molecule spectroscopy” In Ultrasensitive and Single-Molecule Detection Technologies (Eds. Jörg Enderlein, Zygmunt K Gryczuznski) Proc. SPIE, Vol 6092, 2006, In press. Wendy Verheijen, Davey Loos, Frans C. De Schryver, Johan Hofkens, Book chapter in Springer Series in Biophysics, Volume: Single Molecules and Nanotechnology, 2007


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J. (Ton) Dirks, Sander S. van Berkel, Nikos S. Hatzakis, Joost A. Opsteen, Floris L. van Delft, Jeroen J. L. M. Cornelissen, Alan E. Rowan, Jan C. M. van Hest, Floris P. J. T. Rutjes and Roeland J. M. Nolte, “Preparation of biohybrid amphiphiles via the copper catalysed Huisgen [3+2] dipolar cycloaddition reaction.” Chem. Commun. 2005, 4172-4174. O. G. Mouritsen, T. L. Andresen, A. Halperin, P. L. Hansen, A. F., Jakobsen, U. B. Jensen, M. Ø. Jensen, K. Jørgensen, T. Kaasgaard, C. Leidy, A. C. Simonsen, G. H. Peters and M. Weiss "Activation of interfacial enzymes at membrane surfaces” (Review) Journal of Physics: Condensed Matter, 18, (2006), S1293–S1304 U. B. Jensen, A. C. Simonsen “Shape relaxations in a fluid supported membrane during hydrolysis by phospholipase A2” BBA - Rapid Report 1715 (2005) 1 – 5 A. C. Simonsen, L. A. Bagatolli “Structure of spin-coated lipid films and domain formation in supported membranes formed by hydration”. Langmuir, 20, 9720-9728. A. C. Simonsen, U. B. Jensen, P. L. Hansen “Hydrolysis of fluid supported membrane islands by phospholipase A(2): Time-lapse imaging and kinetic analysis” J. Colloid Interface Sci. 2006, 107-115. K. Velonia, O. Flomenbom, D. Loos, S. Masuo, M. Cotlet, Y. Engelborghs, J. Hofkens, A. E. Rowan, J. Klafter, R. J. M. Nolte, F. C. Schryver, “Single-enzyme kinetics of CalB-catalyzed hydrolyzes” Angew. Chem. Int. Ed. 2005, 44, 560-564, O. Flomenbom, K. Velonia, D. Loos, S., Masuo¶ M. Cotlet, Y. Engelborghs, J. Hofkens, A. E. Rowan, R. J. M. Nolte, M. Auweraer, F. C. Schryver, J. Klafter, “Stretched exponential decay and correlations in the catalytic activity of fluctuating single lipase molecules” PNAS, 2005, 102, 2368-2372. O. Flomenbom, J. Hofkens, K. Velonia, F. C. Schryver, A. E. Rowan, R. J. M. Nolte, J. Klafter, R. J. Silbey. “Correctly validating results from single molecule data: The case of stretched exponential decay in the catalytical activity of single lipase B molecules” Chem. Phys. Lett. 432 (2006), 371-374. Hans Engelkamp, Nikos S. Hatzakis, Johan Hofkens, Frans C. de Schryver, Roeland J. M. Nolte and Alan E. Rowan, “Do enzymes sleep and work?”Chem. Commun. 2006, 935-940 Nikos S. Hatzakis, Hans Engelkamp, Kelly Velonia, Johan Hofkens, Peter C. M. Christianen, Allan Svendsen, Shamkantr A. Patkar, Jesper Vind, Jan C. Maan, Alan E. Rowan and Roeland J. M. Nolte, “Synthesis and single enzyme activity of a clicked lipase–BSA hetero-dimer.”Chem. Commun. (2006) 2012-2014.

Nørgaard, K., Weygand, M.J., Kjaer, K.,Brust, M., Bjørnholm, T. Adaptive chemistry of

bifunctional gold nanoparticles at the air/water interface. A synchrotron X-ray study of giant

amphiphiles. Faraday Discussions 125, 221-233 (2004).


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Microscope Visualization of Lipid Bilayer Degradation due to Action of Phospholipase A2

and Humicola Lanuginosa Lipase Biochemica et Biophysics Acta – Biomenbranes, 1768, 90-

99, (Jan 2007) K. Blank, J. Morfill, H. Gumpp and H. E. Gaub. Functional Expression of Candida antarctica Lipase B in Eschericha coli. Journal of Biotechnology, 125 , 2006, 474-483. K. Blank, J. Morfill, H. E. Gaub. Site-Specific Immobilization of Genitcally Engineered Variants of Candida Antarctica Lipase B. (2006) ChemBioChem 7: 1349-1351 M Cardenás and T. Nylander “Adsorption of DNA and DNA-surfactant complexes at solid surfaces” In Interaction of DNA with Surfactants and Polymers (Eds. B. Lindman and R. Dias), 2007, Blackwell Publishing In Press J. Barauskas, A. Kanaras, J. L. Brennan, A. Svendsen, M. Brust, T. Nylander. Direct

visualization of Lipase-Functionalized Gold Nanoparticle Activity on Self-Assembled Lipid

Superstructures, 2007, manuscript.

2.7 Project web-site

A BIOSCOPE homepage (www.BIOSCOPE.fkem1.lu.se) has been established which in the public domain contains a description of the project and links to the partners.

2.8 Poster

UNILIV - poster presentation on BIOSCOPE enzyme nanoparticle design presented by Dr. Jennifer Brennan at two conferences: • Royal Society of Chemistry Chemical Nanoscience Meeting - Nottingham, U.K., March

2005, to an international audience of approx. 150 people. • “Nanotechnology, small subject, big potential” - Liverpool, U.K., April 2005, to a local

chemical industry audience (Northwest England) of approximately 100 people. IBLT - one poster presentations on the BIOSCOPE spectroelectrochemical systems of lipase analysis and structural characterization by the PhD-students Sandra Valentukonyte and Audrius Misiunas at the Lithuanian Conference “Chemistry and Chemical Technology”, Kaunas University of Technology, Kaunas, Lithuania, April 26-27, 2006, to an audience of approx. 150 people. KUL – poster presentation by Wendy Verheijen at the MAF9 (9th international conference on Methods and Applications of Fluoresence in Lisbon, Portugal, 4-7 sept 2005 All the BIOSCOPE partners presented their findings at the open BIOSCOPE symposium

”International Symposium on Biomolecular Nanoscale Assemblies” and held January 24-26,

2007 at the Nano-Science Center, University of Copenhagen.


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2.9 Courses

No dedicated course was organized by the net-work within the BIOSCOPE research area. However, UCPH and ULUND are organizing a yearly masters/PhD course, The Physics and Chemistry of Nanostrucutres, which attracts 40-50 students. Thomas Bjørnholm gave lecture

at 7th Course: "Nanotechnologies for Drug Delivery and medical Applications", International

School of Advanced Material Science and Technology, Ancona, Italy ‚”Supramolecular

nanocontainers for drug delivery” and “Boron neutron capture therapy based on

nanoparticles", 2005 for students.

3 Publishable results The main focus on the third year of BIOSCOPE was to assemble the building blocks provided during the first years of BIOSCOPE as a the tool-box to study and control biomolecular action on the single molecular scale that was, i.e. the BIOSCOPE nanomachine. Publishable results have been obtained in several areas including

1. Computer modelling of the influence on the linker length the activity of the hetero-dimer on a surface.

2. Biconjugation of several enzymes to a gold nanoparticle. 3. Liquid crystalline lipid based nanoparticles as a well-defined and natural substrate

for study of the lipase activity 4. The hydrolysis of phospholipid layer studied by means of fluorescence

microscopy and simultaneously track single molecules of enzyme (single molecule resolution) acting on phospholipid layers and to monitor the changes in the phospholipids layers (not single molecule resolution)..

5. Direct visualization of enzymatic processes at the air/water interface with Single molecule resolution and in Real time.

6. Why phospholipase only attack one side of the lipid substrate domain at the air/water interface

7. Correlate the turnover of CalB with an external force, which is applied to the single enzyme molecule.

8. Manipulating the enzymatic activity of CalB by external force. 9. Review of the BIOSCOPE work in Chem. Soc. Rev.

The knowledge promises a potential viable commercialization of the results, although no exploitation has taken place during the project.

3.1 Publications (IBLT and Novozymes)

G. Valincius, I. Ignatjev, G. Niaura, M. Ka em kait , Z. Talaikyt , V. Razumas and A. Svendsen. A new electrochemical method for the detection of lipase activity. Anal. Chem. 77 (2005), 2632-2636. I. Ignatjev, G. Valincius, I. vedait , E. Gaidamauskas, M. Ka emekait , V. Razumas and Allan Svendsen. Direct amperometric determination of lipase activity. Anal. Biochem. 344 (2005), 275-277.


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G. Niaura, Z. Kuprionis, I. Ignatjev, M. Kazemekaite, G. Valincius, Z. Talaikyt , V. Razumas, and Allan Svendsen. J. Am. Chem. Soc., 2007, submitted

3.2 Publications (IBLT, UNILIV, KUN and Novozymes)

J. L. Brennan, N. S. Hatzakis, T. R. Tshikhudo, N. Dirvianskyt , V. Razumas, S. Patkar, J. Vind, A. Swendsen, R. J. M. Nolte, A. E. Rowan, M. Brust. Bioconjugation via click chemistry: The creation of functional hybrids of lipases and gold nanoparticles. Bioconjugate Chem. 17(2006), 1373-1375.

3.3 Publications (KUN, KUL and Novozymes)

Nikos S. Hatzakis, Hans Engelkamp, Kelly Velonia, Johan Hofkens, Peter C. M. Christianen, Allan Svendsen, Shamkantr A. Patkar, Jesper Vind, Jan C. Maan, Alan E. Rowan and Roeland J. M. Nolte, “Synthesis and single enzyme activity of a clicked lipase–BSA hetero-dimer.”Chem. Commun. (2006) 2012-2014.

3.4 Publications ULUND, UNILIV and Novozymes

J. Barauskas, A. Kanaras, J. L. Brennan, A. Svendsen, M. Brust, T. Nylander. Direct

visualization of Lipase-Functionalized Gold Nanoparticle Activity on Self-Assembled Lipid

Superstructures, 2007, manuscript

3.5 Publications ULUND and UNILIV

M. Cardenas, J. Barauskas, K. Schillen, J.L. Brennan, M. Brust, T. Nylander. Thiol-Specific and Non-Specific Interactions between DNA and Gold Nanoparticles, Langmuir 2006, 22, 3294-3299.

3.6 Publications UCPH and UNILIV

Nørgaard, K., Weygand, M.J., Kjaer, K.,Brust, M., Bjørnholm, T. Adaptive chemistry of

bifunctional gold nanoparticles at the air/water interface. A synchrotron X-ray study of giant

amphiphiles. Faraday Discussions 125, 221-233 (2004).

3.7 Publications UCPH and Novozymes


Microscope Visualization of Lipid Bilayer Degradation due to Action of Phospholipase A2

and Humicola Lanuginosa Lipase Biochemica et Biophysics Acta – Biomenbranes, 1768, 90-

99, (Jan 2007)


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3.8 Publications (KUN and KUL)

K. Velonia, O. Flomenbom, D. Loos, S. Masuo, M. Cotlet, Y. Engelborghs, J. Hofkens, A. E. Rowan, J. Klafter, R. J. M. Nolte, F. C. Schryver, “Single-enzyme kinetics of CalB-catalyzed hydrolyzes” Angew. Chem. Int. Ed. 2005, 44, 560-564, O. Flomenbom, K. Velonia, D. Loos, S., Masuo¶ M. Cotlet, Y. Engelborghs, J. Hofkens, A. E. Rowan, R. J. M. Nolte, M. Auweraer, F. C. Schryver, J. Klafter, “Stretched exponential decay and correlations in the catalytic activity of fluctuating single lipase molecules” PNAS, 2005, 102, 2368-2372. O. Flomenbom, J. Hofkens, K. Velonia, F. C. Schryver, A. E. Rowan, R. J. M. Nolte, J. Klafter, R. J. Silbey. “Correctly validating results from single molecule data: The case of stretched exponential decay in the catalytical activity of single lipase B molecules” Chem. Phys. Lett. 432 (2006), 371-374. Hans Engelkamp, Nikos S. Hatzakis, Johan Hofkens, Frans C. de Schryver, Roeland J. M. Nolte and Alan E. Rowan, “Do enzymes sleep and work?”Chem. Commun. 2006, 935-940

3.9 Publications (IBLT)

A. Bulovas, N. Dirvianskyt , Z. Talaikyt , G. Niaura, S. Valentukonyt , E. Butkus and V. Razumas. Electrochemical and structural properties of self-assembled monolayers of 2-methyl-3-( -mercaptoalkyl)-1,4-naphthoquinones on gold. J. Electroanal. Chem., 591 (2006), 175-188

3.10 Publications ULUND

J. Barauskas, M. Johnsson, T. Nylander, F. Tiberg. Hexagonal Liquid-Crystalline Nanoparticles in Aqueous Mixtures of Glyceryl Monooleyl Ether and Pluronic F127, Chem. Lett. 2006, 35, 830-831. G. Popescu, J. Barauskas, T. Nylander, F. Tiberg. Liquid Crystalline Phases and their Dispersionsin Aqueous Mixtures of Glycerol Monooleate and Glyceryl Monooleyl Ether, Langmuir 2007, 23, 496-503. M Cardenás and T. Nylander “Adsorption of DNA and DNA-surfactant complexes at solid surfaces” In Interaction of DNA with Surfactants and Polymers (Eds. B. Lindman and R. Dias), 2007, Blackwell Publishing In Press


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3.11 Publications PHYSLMU

K. Blank, J. Morfill, H. Gumpp and H. E. Gaub. Functional Expression of Candida antarctica Lipase B in Eschericha coli. Journal of Biotechnology, 125 , 2006, 474-483. K. Blank, J. Morfill, H. E. Gaub (2006) ChemBioChem 7: 1349-1351

3.12 Publications KUL

W. Verheijen, H. Uji-i, C. Flors, J. Hotta, J. Hofkens, “Probing dynamics of individual bio molecules by single molecule spectroscopy” In Ultrasensitive and Single-Molecule Detection Technologies (Eds. Jörg Enderlein, Zygmunt K Gryczuznski) Proc. SPIE, Vol 6092, 2006, In press. Wendy Verheijen, Davey Loos, Frans C. De Schryver, Johan Hofkens, Book chapter in Springer Series in Biophysics, Volume: Single Molecules and Nanotechnology, 2007

3.13 Publications KUN

J. (Ton) Dirks, Sander S. van Berkel, Nikos S. Hatzakis, Joost A. Opsteen, Floris L. van Delft, Jeroen J. L. M. Cornelissen, Alan E. Rowan, Jan C. M. van Hest, Floris P. J. T. Rutjes and Roeland J. M. Nolte, “Preparation of biohybrid amphiphiles via the copper catalysed Huisgen [3+2] dipolar cycloaddition reaction.” Chem. Commun. 2005, 4172-4174.

3.14 Publications SDU

O. G. Mouritsen, T. L. Andresen, A. Halperin, P. L. Hansen, A. F., Jakobsen, U. B. Jensen, M. Ø. Jensen, K. Jørgensen, T. Kaasgaard, C. Leidy, A. C. Simonsen, G. H. Peters and M. Weiss "Activation of interfacial enzymes at membrane surfaces” (Review) Journal of Physics: Condensed Matter, 18, (2006), S1293–S1304 U. B. Jensen, A. C. Simonsen “Shape relaxations in a fluid supported membrane during hydrolysis by phospholipase A2” BBA - Rapid Report 1715 (2005) 1 – 5 A. C. Simonsen, L. A. Bagatolli “Structure of spin-coated lipid films and domain formation in supported membranes formed by hydration”. Langmuir, 20, 9720-9728. A. C. Simonsen, U. B. Jensen, P. L. Hansen “Hydrolysis of fluid supported membrane islands by phospholipase A(2): Time-lapse imaging and kinetic analysis” J. Colloid Interface Sci. 2006, 107-115.


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