WO2003023402A1

WO2003023402A1 - Immobilisation method and surfaces produced using said method

Info

Publication number: WO2003023402A1
Application number: PCT/SE2002/001635
Authority: WO
Inventors: Sven Oscarsson; Arjan Quist; Elisabeth Pavlovic; Ove ÖHMAN
Original assignee: Sven Oscarsson; Arjan Quist; Elisabeth Pavlovic; Oehman Ove
Priority date: 2001-09-12
Filing date: 2002-09-12
Publication date: 2003-03-20
Also published as: SE0103021D0

Abstract

Objects, such as molecules, macormolecules, nanoparticles, cells and organelles are immobilised to a surface by covalent bonds and in a site-specific manner using an external source of energy, acting on the surface in the presence of said objects, when said surface and said objects have been chemically derivatised to present groups capable of forming reactive moieties when subjected to said source of energy. The exact location of the immobilisation is determined either by exposing the surface to said source of energy in a highly localised manner, or by creating site-specific defects or a pattern, to which groups capable of forming reactive moieties when subjected to said source of energy are first arranged. Said pattern may be in the form of electrodes, arranged on the surface.

Description

Immobilisation method and surfaces produced using said method

The present invention concerns a method for the site-specific immobilisation of objects, such as molecules, macromolecules, nanoparticles, cells and/or organelles on surfaces, as well as functional surfaces produced with this method and devices based on or including such surfaces.

Background

The recent interest in positioning and immobilising molecules, macromolecules and/or particles on surfaces on a nanometer scale brings up the need for surfaces displaying features of specific shape, size and chemical behaviour, as well as for methods for the production of such surfaces. Importantly, in most applications said features need to be site-specifically arranged on the surface, the arrangement of said features exhibiting high resolution.

Nanolithography methods, and in particular SPM-related lithography has attracted great attention because of its simplicity and possibilities of precise control. More recently, a "dip- pen" nanolithography method has been developed that uses an atomic force microscope (AFM) tip as a "nib" to directly deliver organic molecules onto suitable substrate surfaces.

Yan Li et al. (J. Am. Chem. Soc. 2001, 123, 2105-2106) describe a development of the above "dip-pen" nanolithography method wherein the tiny water meniscus filling the gap between the AFM-tip and the surface contains dissolved metal salts. These salts can then be electrochemically reduced into metals and deposited site-specifically on the surface. This would give the possibility to create metallic nanostructures with high resolution. Using dissolved platinum (H₂PtCl₆ in water) a Pt-line having a width of 30 nm was created on a Si surface.

DE 198 10 588 A1 describes a structure for storing information and for selectively binding molecules, said structure comprising a surface with aromatic nitro compounds or compounds which can be converted into nitro compounds arranged on a monolayer. By applying a voltage locally over the surface, these aromatic nitro compounds can be reduced to nitroso or hydroxy compounds, or further converted to amino compounds. Through oxidation, these compounds may then be returned to their original state. The structures according to DE 198 10 588 Al are I

intended to function as molecular memory structures, and the storing and reading is suggested to take place using a microelectrode tip.

ivka Maoz et al. (Adv. Mater. 1999, 11, No. 1, 55-60) have applied an electrical bias to a conducting AFM tip operated under normal ambient conditions, inducing electrochemical surface transformations affecting the outer exposed functions of certain to functionalised self- assembled monolayers (SAMs), with full preservation of the overall structural integrity of the SAM. Maoz et al. suggest that the local modifications thus produced could be used to induce site-selective self-assembly of a number of different materials. It is however noteworthy that the method according to Maoz et al. is based on adsorption phenomena, caused by alterations to the hydrophobic character of the surface.

Regardless of the extensive research in the field of nanolithography, the main part of the work is still experimental and the results speculative. Also established methods suffer from severe limitations. In particular, the immobilised molecules or particles are often randomly distributed and/or oriented, with small if any possibilities to predetermine their positioning at the molecular level. Also the possibilities to determine and verify the position and orientation of a particulate molecule or particle are limited. With the increasing interest for the production of e.g. protein arrays on chips for ultra fast and sensitive detection, and the creation of artificial biomimetic materials or enzyme catalysis, new techniques need to be developed.

One objective of the present invention is to make available a method, which allows flexibility in the choice of the objects to be positioned on a surface, regardless if these are nanoparticles, macromolecules or cells, to mention a few examples. It is also an objective to address the requirements with regard to controlling the exact location, orientation and time of positioning as well as time of release, of the positioned particles or molecules. It is also an objective of the present invention to make available a method applicable to a wide variety of surfaces, and consequently offering a large degree of freedom with regard to the combination of the objects to be positioned, and the surface they are to be positioned to. A truly flexible, but yet reliable and accurate method has hitherto not been available. Summary of the invention

The present inventors have surprisingly found that objects, such as molecules, macromolecules and/or nanoparticles including cells and organelles, can be immobilised to a surface in a site-specific manner, with high resolution, and bound to the surface with covalent bonds, by local activation. Both the surface and the objects to be immobilised are first derivatised to present groups capable of forming reactive moieties when activated, for example subjected to an external source of energy. The inventive methods and surfaces produced using said methods are characterised by the steps and features defined in the attached claims, incorporated herein by reference.

Short description of the drawings

The invention will be described in closer detail below, in the description, examples and claims, and with reference to the attached drawings in which

Fig. 1 shows a schematic representation of the set-up used in the working example to expose the protein solution on the 3-MPTMS surface to an electric current between the AFM tip and the surface;

Fig. 2 presents four AFM images showing (a) the reference, a clean 3-MPTMS-derivatised silicon surface, (b) no adsorption of SPDP-HSA molecules on the 3-MPTMS surface after 5 minutes incubation without current exposure, (c) adsorption of SPDP-HSA molecules on the 3-MPTMS surface after current exposure for 5 minutes, and (d) no immobilisation of native HSA on the 3-MPTMS surface after current exposure for 5 minutes;

Fig. 3 presents two AFM images showing (a) SPDP-HSA molecules still adsorbed on the 3- MPTMS surface after washing a 3-MPTMS surface reacted with SPDP-HSA with 1% SDS, (b) a clean 3-MPTMS surface after washing a 3-MPTMS surface reacted with SPDP-HSA with 50 mM DTT;

Fig. 4 showing possible reaction pathways of the HSA molecules with the surface disulphides (a), that can either be oxidised to thiosulfinates (b) and consequently react with reduced SPDP-HSA (c), or reduced to thiols (d) that will react with SPDP-HSA (e); Fig. 5 is an electron microscopy photograph, showing four pairs of electrodes, used in experiments evaluating one embodiment of the invention, where a surface has been prepared, where objects can be covalently bound, and released, at a desired time, and location;

Fig. 6 shows the principle behind one embodiment of the invention; and

Fig. 7 shows the principle behind another embodiment of the invention.

Description

In the present application, the vocabulary is intended to be that of a person skilled in the art, and technical and functional terms are used with the intention that they have the meaning as generally understood by a skilled person. For reasons of clarity, some examples are given below:

The term "nanoparticle" is used to describe particles having an average diameter of about 100 nm or less, regardless of their constitution or origin. Examples of nanoparticles include but are not limited to organic and inorganic particles, such as known bioactive substances to which the cellular surface adheres, e.g. hydroxyapatit.

The term "molecule" is used in its generally accepted form, as meaning the smallest independent mass of any given substance, and the term "macromolecule" is used to define molecules with a molecular weight of about 100 Dalton or more.

The term "cell" is used in its generally accepted form, encompassing both eukaryote and prokaryote cells, and the term "organelle" refers to functional sub-units of such cells or tissues, such as mitochondria, membranes, the endoplasmic reticulum, the Golgi apparatus, lysosymes, peroxisomes, parts of the cytoskeleton, chloroplasts and vacuoles (in plant cells).

The term "site-specific" is used to define the immobilisation method as being capable of producing exact and ordered patterns, or immobilising particles at exact, desired locations. In other words, the term "site-specific" is used to distinguish the present immobilisation method inter alia from prior art methods, capable only of random immobilisation or low-resolution immobilisation. The term "high resolution" is used in the present application to define patterns or arrangements of features with mutual distances between the individual features or immobilised objects of less than about 100 nm.

The word "pattern" used in the description, examples and claims is also used to define the immobilisation as being capable of more than random immobilisation, and effective to form desired two- or three-dimensional structures of immobilised particles on surfaces.

The term "functional" in the present context is used to define surfaces with immobilised particles and/or molecules having a desired and specific shape, size, or chemical behaviour, such as a specific affinity to particular compounds, an enzymatic, catalytic or other activity.

The present inventors have now surprisingly shown that objects, i.e. molecules, macromolecules, nanoparticles, cells and organelles, can immobilised to a surface by covalent bonds and in a site-specific manner using an external source of energy, acting on the surface in the presence of said objects, when both said surface and said objects have been chemically derivatised to present structures or groups capable of forming reactive moieties when subjected to an external source of energy.

Said external source of energy is either an electric current, site-specifically applied to the surface as in the attached experiments, or preferably a source of electromagnetic radiation which either as such is capable of activating the reactive moieties to form a covalent bond, or which acts via means generating an electric current on the surface at specific, desired locations.

The source of electromagnetic radiation is chosen so that the wavelength or other properties of said radiation matches the properties of the surface. For example, for polymer substrates, electromagnetic radiation having a wavelength from about 300 nm to about 1.5 μm is preferred. Depending on the application, a source of visible light, UV-light, X-rays, or a laser, can be used.

One embodiment of the present invention is a new method to achieve a spatially-controlled, site-specific, high resolution immobilisation of objects, such as molecules, macromolecules, nanoparticles, cells and organelles, in the present application exemplified by immobilising a N-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP)-modified protein, Human Serum Albumin (HSA) to a 3-mercaptopropyltrimetoxysilane (3-MPTMS) derivatised silicon surface using the SPM to supply a localised electric current. HSA is here used as a representative of macromolecules, and the derivatised silicon surface as a representative of inorganic surfaces.

Generally, the present invention makes available a novel method for the immobilisation of objects on surfaces, wherein both the objects and the surface are derived to present structures or groups capable of forming reactive moieties when subjected to an external source of energy; said objects are brought in contact with said surface; and the immobilisation effectuated by exposing the surface to said external source of energy in the presence of said objects under conditions at which said objects are covalently bound to said surface.

Said covalent bond can be a disulphide bond, and said groups capable of forming reactive moieties when subjected to an external source of energy, e.g. in the form of an electric current, electromagnetic radiation or the like, can be disulphide structures, such as thiol groups.

Thiol groups are very convenient for immobilisation of thiol-derivatised or thiol-containing molecules through disulphide bonds. The sulphur atom has a double behaviour of electron donor and acceptor that makes it suitable for electron transfer reactions, and allows switching of disulphide to thiol and vice-versa when exposed to an electron source.

According to the invention, said objects can be nanoparticles chosen among well known bioactive substances such as hydroxyapatit or other types of bioactive materials with desired properties; molecules or macromolecules such as amino acids, nucleic acids, ribonucleic acids, proteins, receptor structures, enzymes, biopolymers, fibres; cells such as cells of animal or plant origin, cells representing various tissues in the human or animal body; organelles such as mitochondria and chloroplasts, only to mention a few examples.

Said surface is a surface chosen among inorganic and organic surfaces, such as silicon, glass, metal, ceramic, polymeric and thermoplastic surfaces. According to one embodiment of the invention the surface is a bi- or multi-layered surface, where one or more layers are deposited on a carrier in a site-specific manner forming a desired pattern, e.g. an electric circuit or forming flow channels for sample fluids. Said layers can be conductive layers, arranged on a non-conductive carrier or vice-versa. Alternatively, according to another embodiment of the invention, the surface is a bi- or multi-layered surface, where site-specific defects are arranged in a desired pattern, exposing one or more underlying layers, forming e.g. electric circuits, flow channels for sample fluids, or specific locations for the immobilisation of particles, cells or macromolecules. In a three-dimensional structure, flow channels can be arranged within said structure and locations for immobilisation of objects defined within these flow channels, said locations being site-specifically activated either by directing a current to said location, or by directing external energy to said location, e.g. electromagnetic radiation which either is capable of activating the reactive moieties, or capable of generating an electric current at a specific location.

According to one embodiment of the invention, the current is applied to the surface in a site- specific manner using the surface as one electrode and at least one suitable electrode having the desired precision, such as a microelectrode or a scanning probe microscopy (SPM) tip, as the other electrode. Fig. 1 illustrates this embodiment.

A silicon tip can be used, but preferably a doped silicon tip is used. Alternatively, the SPM tip is made of an oxidation resistant metal, such as gold or platinum. In order to increase the resolution, to create specific patterns or a specific geometry of the immobilised locations, the geometry of the SPM tip is modified in accordance with the desired pattern or geometry of immobilisation. The tip can be given a specific cross-section, the shape and angle of the tip modified etc. Alternatively, several electrodes are used, e.g. an assembly of electrodes together forming a pattern or movable in order to form the desired pattern on the surface. In such an assembly of electrodes, the individual electrodes can be connected in parallel, in series or individually.

According to another embodiment of the invention, the external source of energy is applied to the entire surface or to macroscopic areas thereof, for example by exposing the entire surface or parts thereof to electromagnetic radiation, whereas the site-specific immobilisation with a nano-scale resolution is achieved by site-specifically localising groups, capable of forming reactive moieties when subjected to an external source of energy, such as electromagnetic radiation or an electric current, e.g. disulphide structures such as thiol groups, at surface defects created using known methods.

One method describing the formation of nano-scale defects with high resolution has been disclosed in WO 99/15895 by Oscarsson et al. The nano-scale defects which guide the immobilisation of the objects derivatised with groups capable of forming reactive moieties when exposed to an external source of energy, e.g. subjected to electromagnetic radiation or an electric current, can be defects of the following type: holes and/or rises in/on the surface, lines, dots etc, including localised deposition of metals such as indium, gallium, platinum, gold, silver or copper. The metals can be deposited on the surface for example by using the so called finely focused ion beam technique, the source of ions being chosen among the above metals. Of particular interest in this context are photoelectric materials, e.g. materials developed for the purpose of solar cells. A non-exhaustive list of suitable materials include silicon based materials, such as amorphic silicon, semiconductor materials, such as gallium arsenide (GaAs), copper indium diselenide (CuInSe₂). Such materials and methods for applying these materials to surfaces are known in the art.

Another method for creating the site-specific pattern, before exposing the surface to an external source of energy, e.g. applying the electric current, is the so called soft lithography method, which can be adapted to "printing" the surface with groups capable of forming reactive groups when subjected to an electric current, said groups being site-specifically localised.

Yet another method involves the creation of electrically conductive patterns, defects, rises or notches on an otherwise non-conductive surface, the groups capable of forming reactive groups when subjected to an electric current being activated only where in contact with these patterns, defects, rises or notches. This can be achieved e.g. by the deposition of electrically conductive material on an otherwise non-conductive or poorly conductive surface, or - when using a multi-layered surface - by damaging an upper, non-conductive layer thus exposing underlying conductive layers.

When a conductive pattern, e.g. a circuit is created on a surface, connecting at least one site on the surface to an electric current, the time and location of immobilisation can be controlled by choosing when and to which site to apply the electric current. Said pattern or circuit can connect site-specific locations or defects either individually, in parallel or in series, thus making it possible to accurately control when and at which site/-s the electro activation is to take place and thereby controlling when and where the immobilisation of objects is to be perfonned. One example of such a circuit is a number or electrodes on a surface, the electrodes forming a pattern defining electrode gaps distributed over said surface. Fig. 5 shows one example of such electrode pairs, forming electrode gaps which define specific sites for immobilisation of objects as desired.

Other methods applicable to creating the patterns guiding the localisation of the groups capable of forming reactive groups when subjected to an external source of energy, are e.g. microcontact printing and electron-beam lithography.

According to a preferred embodiment, a pattern of electrodes is provided on the surface, said pattern defining specific loci for the immobilisation of the desired objects, wherein said electrodes comprise a compound, e.g. a photoelectrical compound, which generates an electric current when exposed to an external source of energy, e.g. visible light. According to this embodiment, a pattern of electrodes is first created on the surface, each electrode or groups of electrodes being connected to a defined entity having the capability of generating an electric current when exposed to an external source of energy, and the immobilisation controlled by exposing said electrodes or groups of electrodes to said external source of energy.

One example will be given in order to illustrate this embodiment. The derivatisation of the surface and the objects to be immobilised is performed as described previously in the description. On a polymer substrate, a set of discrete points of an electrically conducting material is arranged, said discrete points evenly spaced over the entire substrate. At each point, a material capable of generating an electric current when exposed to an external source of energy, e.g. a photoelectric material, is arranged. A subset of said points are the exposed to an external source of energy, e.g. a scanning laser drawing a desired, pre-determined pattern on said surface, or a laser diffracted through an optical element projecting a desired, predetermined pattern on the surface. Thus an electric current will be generated only in a subset of the electrode gaps formed between said discrete points, and the immobilisation of a particular object or objects directed to this pattern.

Fig. 6 shows an example where a surface of a substrate 1 is provided with a pattern of discrete points 5 of a photoelectric material. A subset of this pattern, indicated as a square 2, is exposed to an external source of energy. This local exposure, either by light, radiation, electric current or otherwise, activates the reactive moieties. In the enlarged detail view 3, it is seen that derivatised objects 6 are immobilised to the points 5 only within the exposed area 4. Fig. 7 shows another example, where the substrate 1 has a three dimensional shape, defining a flow channel 7. Within this flow channel, discrete points 5, e.g. of a photoelectric material, are arranged in a predefined pattern. The square 4 indicates that only a subset of these points 5 are exposed to the external source of energy. This local exposure, either by light, radiation, electric current or otherwise, activates the reactive moieties. Thus the derivatised objects, supplied to the flow channel, are immobilised only to the points 5 within the exposed area 4.

The present invention also makes available a surface with covalently bound nanoparticles and/or macromolecules immobilised to said surface, produced using the novel method described above. Such surfaces can also constitute functional surfaces, based on the immobilisation of particles imparting specific chemical, biochemical, electrochemical or physicochemical functionality to said surfaces. Further, the invention makes available a device having chemical, biochemical, electrochemical or physiochemical function, built on such a surface. Such devices include, but are not limited to surfaces and devices having a desired biocompatibility or tissue-compatibility, biomolecular memories, artificial membranes, nano- and/or microarrays for the screening of drags, biocatalytical surfaces etc.

The present invention also makes available a reusable surface, i.e. a surface which can be regenerated, as well as the corresponding method, comprising the step of chemically breaking the covalent bonds, removing the previously immobilised objects, e.g. by rinsing with a reducing agent or other agent, capable of releasing the immobilised objects without removing the groups capable of forming reactive moieties when subjected to an external source of energy, which are present on the surface. After removing the previously immobilised objects, a new batch of objects to be immobilised, derivatised to present groups capable of forming reactive moieties when subjected to an external source of energy, are brought in contact with the surface, whereupon the surface is exposed to said external source of energy. This embodiment has surprising advantages in that expensive substrates, such as tailor-made silicon chips can be reused, and adapted to various use, simply by removing the previously immobilised objects, and replacing them with others, or simply by altering the spatial arrangement of said immobilised objects.

One embodiment of the invention encompasses such a reusable or adaptable surface, on which a number of electrodes have been arranged. The electrodes are arranged on the surface in such manner, that discrete positions can be defined. When one particular pair of electrodes is activated, e.g. by exposing a photochemical region of said electrode to an external source of energy, a current is generated and immobilisation of an object to this particular location takes place. This embodiment of the invention comprises the possibility of producing substrates, e.g. silicon substrates or polymeric substrates, with a number of electrodes defining electrode gaps on their surface, e.g. produced by nanolithographic methods, said electrodes / electrode gaps defining a pattern, e.g. a grid. By activation of specific electrodes, the immobilisation of objects on the surface can be accurately controlled. For example, on a surface having a set of electrodes, defining four locations, by activating one electrode gap at the time, four different objects can be immobilised, each to a specific location. It is immediately evident that this embodiment also encompasses surfaces having a very large number of such electrodes / electrode gaps, and that numerous possible variations fall within the scope of the present invention. Again, it becomes possible to mass produce surfaces, e.g. a silicon or a polymeric surface, having such electrodes defining a pattern, and then adapt these to different use and function, simply by activating said electrodes one by one, or in groups, and sequentially immobilise different objects, with respect to their location and orientation, according to the desired end use of the surface.

Examples

Experiments have been performed in order to prove the concept of site-specific reduction on the one hand, and site-specific electro oxidation on the other hand.

AFM images were taken before and after the exposure to proteins with as well as without current to look for the presence of the protein molecules on the surfaces. It was surprisingly found that SPDP -modified HSA molecules only bind to the 3-MPTMS surface when exposed to an electric current. Neither exposure to SPDP -modified proteins without current, nor exposure to native HSA with/without current, resulted in the immobilisation of proteins on the surface.

The nature of the bond between the HSA molecules and the surface was investigated by detergent washing with sodium dodecyl sulphate (SDS) and disulphide reduction with dithiotreitol (DTT), to assess if the molecules were merely adsorbed on the surface or covalently bound through disulphide bonds. Site-specific reduction of thiols:

Materials and methods

N-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP)-derivatised HSA preparation and SPDP reduction were performed according to SPDP manufacturer's instructions (Pharmacia Fine Chemicals AB, Uppsala, Sweden). The obtained thiol content was 7 moles of SH groups per mole of HSA.

Solutions of 0.17 mg/ml SPDP-HSA, reduced SPDP-HSA and native HSA in MilliQ water were prepared.

Derivatised silicon was prepared by evaporating the 3-MPTMS molecules (ABCR GmbH & Co., Karlsruhe, Germany) on silicon surfaces (Silchem™ Marketing Inc., Germany) previously cleaned with a piranha solution (30% H₂O₂/H₂SO₄ 1 :2 v/v) according to established praxis.

The surfaces were imaged in tapping mode with a Nanoscope Ilia AFM (Digital Instruments, Santa Barbara CA) before and after exposure to protein solution with as well as without electric current between the tip and the samples. After the desorption step, surfaces were imaged as well using the same instrument.

A PicoSPM AFM (Molecular Imaging Co., Phoenix, AZ) was used to perform the electric current exposure, with a silicon tip as negative electrode and the surface as positive electrode spaced by 40 μm. A 30 μl droplet of protein solution was placed on the surface as described in Fig.1. A fixed 5 V bias voltage between tip and sample was applied for 5 minutes using a laboratory DC power supply GPC-3020 (Good Will Instrument Co., Taiwan), resulting in an electric current.

The current between tip and sample and the resistance of the solution were measured using a Keithley 2400 source meter (Keithley Instruments, Inc., Cleveland, OH ) to be respectively 4.6 μA and 1.1 MΩ . In-situ temperature measurements were performed using a 10 kΩ needle pin thermistor placed in the solution close to the tip. To determine the pH of the solution during the reaction, a 20 μl volume of solution was pipetted from the surface and placed in a pHboy-P2 pH meter (Shindengen Electric MFG. CO., Saitama, Japan). A detergent solution of 1% (w/v) SDS (Sigma, St-Louis, MO) and a disulphide reducing agent solution of 50 mM DTT (Pharmacia Biotech, Uppsala, Sweden) were prepared in MilliQ water. 50 μl of each solution was placed on a protein-reacted surface for 30 minutes, then rinsed three times with 1 ml MilliQ water and dried with a nitrogen flow.

Results

The AFM image taken after the electric current was applied through a SPDP-HSA solution in contact with a 3-MPTMS surface shows protein molecules on the surface (Fig. 2C), compared to a clean 3-MPTMS derivatised surface as reference (Fig. 2A). The same result was obtained when reduced. SPDP-HSA molecules are present in the solution. Native HSA contains 17 disulphide bonds, involved in the stabilisation of the three-dimensional structure, and one free thiol from Cysteine 34, protected from the solvent. Nevertheless, when native HSA was used, no protein was observed on the surface (Fig. 2D). This shows that the interaction between the HSA molecules and the surface is not due to the sulphurs present within the native HSA structure but indeed requires SPDP derivatisation of the HSA molecules. In addition, there was no SPDP-HSA or native HSA molecules on bare silicon surfaces exposed to electric current, indicating the 3-MPTMS molecules on the surface play a role in the immobilisation of HSA molecules to the surfaces.

When SPDP-HSA and native HSA solutions were placed on 3-MPTMS surfaces without electric current exposure, there was no protein immobilisation on the surfaces (Fig. 2B). Indeed, the concentration of the protein solution used for this experiment is 10 times lower than the concentration that gives the same surface coverage of native HSA molecules on a bare silicon surface without application of an electric current, by a non-covalent spontaneous adsorption process (unpublished results). This was confirmed by placing native HSA and SPDP-HSA solutions on bare silicon surfaces, which showed no protein immobilisation. These results correlate the presence of the proteins on the surface to the exposure to electric current.

The present inventors also investigated the possible physico-chemical effects the electric current might have on the solution, for instance by denaturation of the proteins with a subsequent binding of the molecules to the surface. A high temperature in the protein solution caused by dissipated energy from the current could cause the proteins to precipitate on the surface. The measurements of the current showed a value of 4.6 μA at the start, and a following decrease during the 5 minutes, with a final value of 2.5 μA. The resistance measurements showed an increase in the resistance from 1.1 MΩ to 2.0 MΩ. This is consistent with the growth of the silicon oxide layer on the surface during exposure to the current.

Knowing the current, it is possible to calculate the heating of the solution caused by the dissipated energy. The heating was calculated to be 0.2x10^"3 degrees Celsius for the 30 μl droplet exposed to the current for 5 minutes.

Moreover, the temperature measurements showed that the solution kept a temperature within the interval of 21.7°C±0.1 during the current exposure, indicating that no change in the temperature plays a role in the observed effects.

A dramatic change in pH due to the fact that no buffer was used in the protein solution could also cause protein precipitation. Therefore, the pH of the solution was also measured and found to be 6.3+0.5. The changes in pH were not significant to account for protein precipitation and thus seem unlikely to be involved in the presence of the proteins on the surface.

These observations imply that the immobilisation of the HSA molecules requires the presence of the 3-MPTMS on the surface and SPDP or reduced SPDP on the HSA molecules, as well as a current and that the interaction between the protein molecules and the surfaces is not due to non-covalent spontaneous adsorption.

To investigate if the bond between the HSA molecules and the surface was covalent and possibly a disulphide, or if they were just adsorbed, the present inventors also tested two different ways of desorption. The HSA molecules were released from the surface by DTT, a disulphide reducing agent (Fig. 3 a), but remained on the surface when SDS, a surfactant commonly used for desorbing non-covalently bound proteins from surfaces, was used (Fig. 3b). This is a strong indication for a covalent bonding of HSA molecules through a disulphide to the surface.

The effect of the current on the thiol groups has not yet been fully investigated. The thiols on the 3-MPTMS surface were shown to be in their oxidised form before exposure to electric current (Fig. 4a). They could therefore be reduced to SH groups with a very short lifetime (Fig. 4b), that will react with the SPDP group on the protein to form a disulphide bond (Fig. 4c). This hypothesis is not consistent with the immobilisation of reduced SPDP-HSA molecules since the reaction rate between two thiol groups is low. Further oxidation to thiosulfinates, much more stable than SH groups, can also be considered (Fig. 4c) and will require the SPDP group on the HSA molecules to be reduced to SH in order to form a disulphide bond (Fig. 4d). Nevertheless, the exposure of the thiol surface to current in water with a subsequent exposure to the SPDP- and reduced SPDP-HSA solutions does not result in any immobilisation, which is in contradiction with the oxidation of the thiols.

A site-specific localisation of the modification has also been verified since HSA molecules are only immobilised on an area with a diameter of approximately 1 mm under the tip. This indicates that the modification happens at a higher rate than the diffusion rate in the solution, since the immobilisation of the molecules does not happen on the total area in contact with the solution. The exact width of the current beam is not known but it can be expected that the electrons will not diffuse in the solution because of the high resistivity of the MilliQ water (18.2 MΩ.cm) since it does not contain any electrolytes.

This method of controlled covalent bonding of molecules to surfaces has potential interest in applications employing protein attachment in general, as well as more specific applications such as nanometer scale surface modifications using AFM, where proteins are required to link to spatially organised positions on the surface. The size of the modifications can be reduced to a nanometer scale by using for instance doped silicon or noble metals that are not susceptible to oxidation such as gold or platinum. The geometry of the tip is one of the major parameters to define the modified area as well as the nature of the solvent used to solubilise the molecules to react with the surface.

The above study of 3-MPTMS surfaces in contact with a SPDP-HSA solution exposed to electric current showed the presence of HSA molecules on the surface. The presence of these molecules depends on the presence of 3-MPTMS on the surface and SPDP on the HSA molecules, and an exposure to current. Desorption experiments verify that the molecules are covalently bound to the surface through a disulphide bond, thus directly involving the thiols groups on both the surface and the protein molecules. The reaction taking place has not yet being fully assessed and a deeper investigation is needed. The spatial control of the reaction seems to be related to the size of the current beam, therefore an optimisation of the set-up would allow nanometer-scale modifications. Alternatively, the immobilisation can be guided with high resolution by the arrangement of site-specific defects and/or suitable patterns on the surface.

Further experiments relating to site-specific electro oxidation of thiols on silicon surfaces were performed during the priority year, and reversible immobilisation of peptides was shown.

Site-specific electro oxidation:

Materials and methods

3-MPTMS was purchased from ABCR (Karlsruhe, Germany). n-Doped <100> silicon wafers (Silchem, Germany) were used to prepare thiolated surfaces as previously described. The surfaces were washed 4 times using in alternation a "piranha" solution (H₂SO₄:H₂O₂ 30% (v/v) 2: 1) for 5 minutes and a rinse in ultra pure water (18 MΩ, low organic content) and were finally dried in an argon flow inside the reaction chamber. Despite this extensive washing, it is still possible that a little contamination remains on the surface. 20 μl of reagent was deposited at the bottom of the chamber next to the argon flow inlet. The reaction was allowed to take place for 60 min. The surfaces were subsequently sonicated for 10 minutes in ethanol, and 10 minutes in ultra pure water, and finally dried using an argon flow.

3-MPTMS surfaces were freshly prepared before each experiment. A droplet of 100 μl of phosphate buffer (10 mM, pH 7.0), was placed between the surface and a platinum electrode. Biases of 0.5, 0.8, 1.0, 1.2 or 2.0 volts were applied for 1 minute using a Keithley 2400 source meter (Keithley Instruments Inc., Cleveland, OH), with the silicon surface as the anode and the platinum electrode as the cathode. After the electro activation, the surfaces were rinsed and sonicated for 10 minutes in ultra pure water.

A short peptide (Interactiva, Ulm, Germany) composed of 2 valines and one cysteine was used to react with the activated disulfides. A solution was prepared to the peptide concentration of 0.1 mM, in phosphate buffer (10 mM, pH 7.0), immediately before use. To perform covalent binding of peptides, a surface electro activated using 1.0 V, was incubated in 5 ml of the peptide solution under agitation for 5, 15 and 40 minutes, consecutively. After each reaction time, the surface was sonicated for 10 minutes in ethanol (Kemetyl AB, 99.5%), and 10 minutes in ultra pure water, before being dried with argon.

To release the bound tripeptides, surfaces were then incubated under agitation in 50 mM DTT (Sigma, St. Louis, MO) solution in phosphate buffer (0.1 M, pH 8.0), for 1 to 3 hours, then sonicated for 10 minutes in ethanol, and 10 minutes in ultra pure water.

The same surface was then electro activated using 1.0 V as described above, rinsed and sonicated for 10 minutes in ultra pure water to test the possibility of repetitive use of a surface.

X-ray photoelectron spectroscopy for chemical analysis (ESCA) was prefonned after every step of surface modification. ESCA spectra at 10° grazing angle were acquired in a Scienta- 300 instrument (Scienta, Uppsala, Sweden) using monochromized Al Kα X-ray radiation with an energy of 1487 eV. Curve fitting was realized using the ESCA 300 Data Analysis program on the non-oxidized sulfurs and oxidized sulfurs relative areas.

The cyclic voltammetry study was performed using equipment designed at the laboratory. A copper counter-electrode and a reference platinum wire electrode were set inside the phosphate buffer droplet (0.1 M, pH 7.0) placed on a 3-MPTMS derivatised silicon wafer. The voltage was swept in the 0 to 2.0 V range. The contact potential between the solution and the silicon surface was measured using the Keithley 2400 source meter by introducing a platinum electrode in the solution.

Results

Oxidation of thiols to disulfides and reactive thiolsulfinates or thiolsulfonates using chemical oxidants such as H₂O₂ or magnesium monoperoxyphtalate is known in the art. In this work, the present inventors have investigated and analysed the effect of applying a positive bias to a 3-MPTMS modified surface by use of cyclic voltammetry and XPS.

Scheme 1 describes the first oxidation steps possibly undergone by thiols in a 3-MPTMS monolayer, subsequent reaction with free thiols and reduction of disulfides by DTT. Free thiols are oxidized to disulfide (1). Further oxidation of the disulfide results in the formation of thiolsulfinates (2) or thiolsulfonates (3), which can both react with free thiol groups. This results in a new disulfide bond in both cases, with respectively an unstable sulfinic acid or a stable sulfonic acid (4-5). Reduction with DTT results in free thiol groups (6), and free thiol groups and sulfonic acid (7).

Scheme 1. Oxidation pathway undergone by the surface thiols, and reaction of the activated disulfides with free thiols from the solution.

It was not possible to distinguish between disulfides and free thiols on the sample surface with the ESCA instrument used for this work since the chemical shift between the two peaks is too small to result in separated peaks. Therefore, the presence of disulfides even on freshly prepared surfaces can not be completely excluded. Free thiols display also a very low reactivity toward those disulfides at neutral pH. Nevertheless, it was possible to test the reactivity of freshly made 3-MPTMS surfaces with 2,2-dithiodipyridine, which contains a disulfide extremely reactive to free thiols. XPS data showed that most of the thiol groups on those surfaces had reacted and consequently, were still in their reduced form. The oxidation rate of thiols into disulfides depends on the concentration of oxygen present in the environment. However, even when the surface is exposed to air, disulfides form at a very slow rate. This oxidation rate is even slower in water due to the low solubility of oxygen. Further oxidation can occur as well at a very slow rate. Indeed, no oxidized sulfur peaks were detected with the ESCA before electro activation. It should be noted that the very weak structure at 168 eV binding energy (not shown) is the first plasmon-loss peak corresponding to the silicon 2s bulk line at 151 eV.

A double peak at 168.5 eV binding energy was detected in all experiments after electro oxidation, and its intensity increased with increasing voltage (Not shown). The proximity of the thiols in the 3-MPTMS monolayer suggests oxidation to disulfides as the first oxidation step undergone by the sulfurs. Therefore the sulfur 2p peak mainly corresponds to thiolsulfonates. However, the presence of a few percent of sulfonates can not be excluded. All the spectra show a decrease of the intensity of the non-oxidized sulfur double peak at 164 eV matching the increase of oxidized sulfur. After a bias of 0.8 V, and even more pronounced at 1.0 V, an additional sulfur 2p peak corresponding to thiolsulfinates appeared at about 166.5 eV (Not shown). Sulfinates are very unstable and should therefore not be detectable.

Electrochemical evaluation of the contact potential between the solution and the silicon surface was measured to be -0.5V using a platinum electrode. Therefore this voltage has to be added to the applied voltage. Cyclic voltammetry experiments showed that silicon oxide growth does start from higher voltages (above 2.0 V). After a 2.0 V bias, the growth of the silicon oxide is indeed observed on the XPS spectrum.

The cyclic voltammetry results show the voltage dependence of the oxidation process. No anodic wave corresponding to thiol oxidation to disulfides was observed on the cyclic voltammogram in the 0 to 2.0 V range, showing that in this system, this process is not detectable. The anodic wave of water oxidation (eq 3) can indeed be observed from 0.8 V on the cyclic voltammogram. This indicates that the oxidation of thiols at the anode is most probably due to the oxygen resulting from anodic water oxidation.

H₂O -» 2e Z+ 2H⁺ + "O₂ (3)

The potential use of electro activated surfaces for covalent immobilization of biomolecules was investigated. It was demonstrated that the peptide molecules do not spontaneously bind to the non-activated thiol derivatised silicon surface by performing a reference experiment in which a freshly prepared 3-MPTMS surface (without electro activation) was incubated for 1 hour under agitation in the peptide solution. If covalent bonding of the peptide had occurred, the peptide would still remain on the surface after the sonication and rinsing process. The presence of the peptide molecules would be revealed by the appearance of a nitrogen Is signal from the three nitrogen atoms in the peptide. However, the results show that no such detectable nitrogen Is signal appears on the non-activated surfaces. Therefore, this clearly points out the need to activate the thiols to achieve binding of the peptide.

The present inventors subsequently demonstrated that the electro activated surface effectively immobilizes the thiol-containing peptide. As showed above, the electro activation converts the thiol terminated 3-MPTMS surface to a thiolsulfinate/thiolsulfonate rich surface. The maximum conversion of all thiols to such thiolsulfinates/thiolsulfonates would result in that 50% of all the sulphur atoms have been oxidized to sulfmates/sulfonates. Curve fitting indicated that at least 40 % of the total sulphurs have been oxidized either to thiolsulfinates or to thiolsulfonates after applying a 1.0 V bias. This corresponds to at least 80% of the maximum surface activation. Therefore 1.0 V was chosen as the standard activating voltage. A peak corresponding to the peptide nitrogens was observed after the activated surface was incubated in a peptide solution. The signal increased with reaction time and reached saturation after 20 minutes.

Even though the sulfur signal was attenuated by the added peptide, the amount of reacted sulfurs can be calculated by comparing the sulfur signal before electro activation and the nitrogen signal at saturation from the peptide molecules bound to the surface. The amount of nitrogen contamination present on the surface before peptide reaction was subtracted to the amount of nitrogen after peptide reaction. The amount of sulfurs reacted with peptide molecules was evaluated to be approximately 5% of the total sulfur amount after 5 minutes of reaction with the peptide, and 10 % after 15 additional minutes, which represents 25 % of the thiolsulfinates/thiolsulfonates. This saturation of the reaction is likely to be a result of the comparatively large three dimensional structure of the peptide, which hinders further peptide molecules from reaching the surface and react with the activated disulfides. Therefore, full coverage of the surface by the peptide has probably been achieved.

The visible changes in the sulfur 2p spectra due to the reaction with the peptide are small. Taking into account that the total amount of sulfurs increased by 5% after 5 minutes, and 10 % after 15 minutes of reaction with the peptide, results from curve fitting allowed to calculate the non-oxidized and oxidized sulfur signal change (Table lc and d), due to peptide reaction. The non-oxidized sulfur amount increased 8% after 5 minutes, and 15% after 15 minutes of reaction with the peptide. Since the amount of oxidized sulfur does not change significantly, the peptide only reacted with the thiolsulfonates. Reaction with thiolsulfinates would have resulted in a higher increase in non-oxidized sulfurs, as seen from Scheme 1. Table 1. Change (in %) in the amount of non-oxidized sulfur and oxidized sulfur obtained by curve fitting, relative to the amounts on the electro activated surface before peptide reaction.

^*The letters correspond to the sulfur 2p XPS spectra from figure 3.

In order to investigate the reversibility of the immobilization which is of importance for future applications where surfaces must be reusable, the peptide derivatised surfaces were treated with a disulfide reducing agent, DTT. XPS data show that the nitrogen signal decreased 40% after 1 hour in DTT solution (Not shown). It was not possible to remove the remaining peptide molecules with longer incubation times in DTT solution, neither with sonication in ethanol, nor with a detergent, sodium dodecyl sulfate. A similar phenomenon was observed by Batista- Viera et al. (J. Applied Chemistry and Biotechnology, 1991, 31, 175-195), in a similar study using chemical oxidation. They excluded the possibility of amino groups reacting with thiolsulfonates and explained the irreversible binding by unspecific non-covalent interactions. The peptides can also remain covalently bound to the surface if the disulfide bond is not accessible to DTT. This steric hindrance effect can be caused by the dense packing of the 3- MPTMS monolayer obtained with the silanization method used to prepare the thiolated surfaces in this work.

After DTT treatment, a decrease of the thiolsulfinates can be observed and the non-oxidized sulfur peak has gained intensity (Not shown), which is consistent with scheme 1, since thiolsulfinates will be reduced to unstable sulfinic acid groups, which are spontaneously reduced to thiols. In addition, the flattening of the thiolsulfonate peak indicates a decrease of thiolsulfonates, which are reduced by DTT to sulfonic acid and thiols. Curve fitting showed that after DTT exposure, 30% of total sulfurs remained oxidized. Taking into account that 60% of the peptides are left on the surface, calculation shows an increase in non-oxidized sulfurs of 25%, and a matching decrease of oxidized sulfurs of 21% (Table If), compared to the electro activated surface before peptide reaction. The reduction process is most probably not complete, since the spectra indicate that some thiolsulfinates/thiolsulfonates remain present, probably due to their inaccessibility to DTT. Even if not all the immobilized peptide molecules are released by DTT, it is still possible to reoxidize the same surface in order to obtain reactive tHolsulfinates/thiolsulfonates. To demonstrate this, a second oxidation using 1.0 V was performed on the surface, which has previously been oxidized at the same voltage and subsequently treated with DTT. The subsequent ESCA analysis of this sample showed that the non-oxidized sulfur peak had decreased and that more thiolsulfinates and thiolsulfonates were formed (Not shown). Curve fitting showed that the oxidized sulfurs increased from 30% after DTT treatment to 54% after the second electro activation.

It is of prime interest to be able to release all peptide molecules from the surfaces and to control the electro activation process in order to form only thiolsulfinates. This will allow a complete regeneration of the surfaces. The optimization of the peptide release and oxidation processes are currently under investigation. The densely packed 3-MPTMS monolayer could cause steric hindrance, therefore less dense monolayers will be investigated.

This activation method has been designed to achieve spatially controlled immobilization of biomolecules on surfaces. The present study to investigate the surface chemistry has been performed at a large scale allowing the use of XPS as analytical technique. This method opens up possibilities of reaching controlled activation of a thiol monolayer at the nanometer size, by using nanoelectrodes or an SPM tip as counter-electrode, experiments which are currently in progress.

Oxidation of free thiols from a 3-MPTMS monolayer on silicon oxide was performed by applying a positive bias to the surfaces. It was found that the oxidation of the sulfurs increases along with the voltage, and about 80% of maximum activation was obtained at 1.0 V. Subsequent use of a free thiol-containing peptide solution allowed to covalently bind the peptides to the surface through disulfide bonds. After DTT treatment, the same surface was reactivated using the same method.

Using a bias voltage has proved to be a fast and efficient way of activating disulfides or thiols. It also opens up possibilities of spatially controlled positioning of molecules on surfaces using nanosized electrodes. Although the invention has been described with regard to its preferred embodiments, which constitute the best mode presently known to the inventors, it should be understood that various changes and modifications as would be obvious to one having the ordinary skill in this art may be made without departing from the scope of the invention as set forth in the claims appended hereto.

Claims

1. A method for site-specific immobilisation of nanoparticles and/or macromolecules at one or more specific locus/loci on a surface, characterised in that

the nanoparticles and/or macromolecules chosen to be immobilised are derived to present groups capable of forming reactive moieties;

the surface area on which said nanoparticles and/or macromolecules are to be immobilised is derived to present groups capable of forming reactive moieties at said specific loci;

said nanoparticles and/or macromolecules are brought in contact with said surface, and

the immobilisation is effectuated by activation of said at least one specific locus/loci simultaneously in the presence of said nanoparticles and/or macromolecules or followed by the addition of the same, under conditions at which said nanoparticles and/or macromolecules are covalently bound to said surface at said locus/loci.

2. The method according to claim 1, wherein said reactive moieties are capable of spontaneous formation of covalent bonds upon activation.

3. The method according to claim 2, wherein said activation is performed by exposing the reactive moieties to electromagnetic radiation.

4. The method according to claim 2, wherein said activation is performed by generating a local current in the locus/loci.

5. The method according to claim 1, wherein electrodes are arranged on said surface in a pattern defining the place and distribution of said locus/loci, and the activation in at least one specific locus/loci by externally activating at least one electrode or at least one pair of electrodes, the external activation being performed by exposing the electrode to an external source of energy.

6. The method according to claim 5, wherein said external source of energy is a source of electromagnetic radiation.

7. The method according to claim 1, wherein a current is generated in at least one specific locus/loci using the surface as a first electrode and at least one separate electrode as the second or further electrode, at least one of said electrodes being movable in relation to the other.

8. The method according to claim 7, wherein a scanning probe microscopy (SPM) tip is used as one of said electrode/-s.

9. The method according to claim 8, wherein a silicon tip is used.

10. The method according to claim 8, wherein the SPM tip is a doped silicon tip.

11. The method according to claim 8, characterised in that the SPM tip is made of an oxidation resistant metal, such as gold or platinum.

12. The method according to claim 8, wherein the geometry of the SPM tip is modified in accordance with the desired pattern of immobilisation.

13. The method according to claim 1 , wherein the locations at which the derivatised nanoparticles and/or macromolecules are immobilised are predetermined as follows:

a site-specific pattern of surface defects is first created on the surface;

the area at or immediately around said surface defects is derivatised to present groups capable of forming reactive groups; and

- the surface is subjected to an external source of energy in the presence of the nanoparticles and/or macromolecules to be immobilised.

14. The method according to claim 13, wherein said external source of energy is a source of electromagnetic radiation.

15. The method according to claim 13, wherein a conductive pattern is created on a surface, connecting at least one locus/loci on the surface, thus controlling the time and location of immobilisation by choosing when and to which site to apply the electric current.

16. The method according to claim 1, wherein said current is applied to the surface in a site- specific manner using electrodes arranged on the surface.

17. The method according to claim 16, wherein said electrodes are arranged on the surface by means of lithography, etching or the like.

18. The method according to claim 2, wherein said reactive moieties are disulphide structures and that said covalent bond is a disulphide bond.

19. The method according to claim 2, wherein said reactive moieties are thiosulfinate or thiosulfonate structures and that said covalent bond is a disulphide bond.

20. The method according to claim 1, wherein said objects are chosen among molecules, macromolecules, nanoparticles, cells and organelles.

21. The method according to claim 1, wherein said surface is a surface chosen among inorganic and organic surfaces, such as silicon, glass, metal, ceramic, polymeric and thermoplastic surfaces.

22. The method according to claim 1, wherein said surface comprises at least one conductive and one non-conductive or poorly conductive layer.

23. A surface with covalently bound nanoparticles and/or macromolecules immobilised to said surface, produced using the method according to any one of the previous claims.

24. A reusable surface with covalently bound nanoparticles and/or macromolecules immobilised to said surface, produced using the method according to any one of the previous claims

25. A device having biochemical function, built on a surface according to claim 23 or 24.

26. A device having electrochemical function, built on a surface according to claim 23 or 24.

27. A device having physiochemical function, built on a surface according to claim 23 or 24.