Light induced material deposition by molecular immobilization

TECHNICAL FIELD

The present invention relates to light induced molecular immobilization. More specifically, the described subject matter relates to devices for and methods of material deposition by immobilization of molecules using light irradiation.

BACKGROUND

Micro-arrays of bioactive substances are becoming more and more important for medical diagnostics and drug research. The ability to carry out assays in parallel in a micro-array format is required for high-throughput analysis systems. The increasing interest in miniaturization of biological and chemical experiments or assays demands precise metering of the smallest amounts of reagents, e.g. on a planar substrate. Several techniques ranging from pin dispensing and piezoelectric injectors to laser direct write are successfully used to dispense tiny amounts of liquids in the form of either simple spots or more complicated micro-arrays (Strobl et al, 2004; Kuoni et al., 2004, Hsieh et al., 2004, Gutmann et al.,2004, Ringeison et al., 2002). Such micro-arrays, and the technology behind them, have become an important tool in genomic expression assays, proteomic applications, and even in the field of combinatory chemistry.

Micro-dispensers and micro-dispensing are typically applied in DNA and protein micro-array technology, biosensors, protein sensor arrays, printing of molecules, materials deposition printing, etc.

Uses of such technology enable e.g. LCD materials deposition and Polyimide (Pl) material coating and the ink jet printing of conductive patterns e.g. for RFID, electronics, PCBs and displays and the printing of silver or other materials on substrates like paper, PET, PEN, FR4, polyimide, display glass, ITO coated glass, silica (Si), etc.

Other uses comprise manufacturing of biosensor arrays, cell deposition for tissue engineering, crystallography, adhesive deposition, bar coding of clinical samples, biochemical and biological agent dispensation, drug delivery systems, protein binding, enzymatic analysis, micro-chemistry, micro-

pharmacology, micro-immunology, ink jetting of DNA plasmids and bacterial cells, etc.

In principle, any use involving deposition of material on a small scale in a controllable way.

Attempts are made for the downscaling of common fluid handling systems, to reducing the amount of analytes required, minimising fabrication and analysis costs, etc.

A well-known technology for micro-dispensing is e.g. the technology of Dimatix, Inc. and the Arraylt™ micro-array technology of TeleChem International, Inc. that uses printing techniques for material deposition involving the use of micro-fluid systems containing the material to be , deposited where small droplets are dispensed by micro-injection systems in order to create very tiny droplets that would immobilize on an appropriate surface. See e.g. www.dimatix.com and www.arrayit.com.

However, the deposition is limited by the size or volume of the material that can be 'printed' in a controllable way (e.g. relating to the viscosity of the material to be deposited and the drying of the printed material), where the droplets currently are currently ranging from about 333 pL to 1.1 nl_.

A further draw-back of such printing material deposition systems and similar systems are also their reliance on micro-injection systems (like pumps, valves, etc.) and that the material usage often is high, with an increased possibility of sample contamination. Additionally, they can easily get clogged and such systems are typically also relatively expensive.

The deposition of material using printing technology is shown and explained further in connection with Figures 4a, 4b and 4c.

Other immobilization methods, see e.g. Veilleux J (1996) IVD Technology, March p. 26 - 31 , use one or more thermo-chemical/chemical steps sometimes involving reagents having a degrading effect on the structure and/or function of the bound protein.

Other commonly used protein immobilization methods lead to a random orientation of the proteins immobilized on a carrier with a significant risk of causing a lower biological activity and/or raised detection limits.

In many instances, where proteins and other molecules have been immobilized onto glass surfaces, the modes of attachment are non-specific causing the molecules to be randomly oriented on the surface, thus impairing sensitivity of detection.

SUMMARY

It is an object to provide a device (and corresponding method) for material deposition by immobilization of molecules using light that at least to an extent alleviates some of the above mentioned shortcomings.

It is a further object to provide immobilization of molecules with a high degree of reproducibility.

An additional object is to provide immobilization of molecules with a high density.

Yet a further object is to provide relatively in-expensive immobilization.

An additional object according to an aspect is to enable secure marking or watermarking of a given object.

A further object is to enable very precise immobilization of molecules.

Yet an object is to enable a well-defined orientation of immobilized molecules.

Yet another object according to an aspect is to enable material deposition on an object or carrier in a single step or fewer steps, thereby increasing the processing speed.

This is achieved by a device for material deposition by immobilization of molecules using light comprising, a light source emitting light, during immobilization, to an object comprising, during immobilization, molecules to be immobilized, where said molecules to be immobilized are capable of receiving energy directly or in-directly by irradiation from said light source and where at least a part of said received energy upon release to said molecules to be immobilized causes the molecules to be immobilized.

In this way, molecules can be immobilized with a high degree of reproducibility and precision since the spatial dimension of the smallest individual immobilized area is substantially (e.g. except due to diffusion and scattering effects) defined by area of the spot of the light illuminating the object comprising the molecules to be immobilized instead of being defined by how small droplets of material that can be immobilized on a surface in a controllable way using standard material printing techniques.

Accordingly, the spatial dimension of an immobilized area or spot is limited to the focal area of the illuminating light, which e.g. for a diffraction limited beam of UV light can be less than one micrometer, i.e. in the magnitude of nanometers. This also allows for an extremely dense packing of identifiable and different molecules on a support surface and for immobilizing small patterns which could be useful e.g. for molecular electronics applications.

Furthermore, the pattern of immobilized molecules is not restricted to conventional array formats, since in principle any specific pattern can be obtained as long as the exposure of the light source irradiating the object comprising the molecules to be immobilized can be controlled according to a specific pattern, which is relatively easy using light.

In one embodiment, the molecules to be immobilized or linker molecules comprises photosensitizer molecules entering an excited energy state in response to being subjected to the emitted light and releasing said energy to said molecules to be immobilized.

As used herein, the term "linker" relates to a molecule to be provided to an element, e.g. to a molecule or a support, in order to provide to said element a

disulfide bridge or disulfide bridge-containing triad capable of being activated by irradiation to contain reactive thiol group(s) (-SH group(s)). When activated, the thiol group should preferably be available for coupling according to the invention. A linker comprising an appropriate disulfide bridge and/or disulfide bridge-containing triad may include, but is not limited to a linker comprised solely or partly by amino acids. Thus a linker may include other molecules than amino acids and may be comprised by one or more peptide groups and one or more groups of organic or non-organic materials, e.g. containing a peptide group and one or more carbohydrate groups, including small sugar molecules, oligosaccharides, large carbohydrate-based polymers. Inorganic part(s) of the linker may include e.g. metallic groups based on gold, silver, aluminium, silicon, and/or non-metallic groups based e.g. on ceramic.

Photosensitizer molecules have the capability of entering an excited energy state in response to being subjected to the excitation light and are capable of releasing the energy to the molecules to be immobilized. The molecules to be immobilized can also themselves be photosensitizer molecules. One example of a photosensitizer is e.g. a chromophor.

In one embodiment, the molecules to be immobilized or linker molecules comprise molecules capable of releasing energy in the form of electrons to said molecules to be immobilized. This can e.g. be done by using a photoelectric effect to release electrons from the surface or substrate (e.g. a metal layer). Alternatively, photo-ionization or photolysis could be used to release electrons from other molecules. Hydrolysis of water could also be used to supply such electrons.

In one embodiment, the molecules to be immobilized comprises at least one disulphide bridge and wherein the light source is adapted, during use, to emit light having a wavelength in an interval of 250 - 305 nm or to emit light having a longer wavelength that by means of non-linear processes and/or multiphoton excitation promotes the same electronic transitions as light in the wavelength interval of 250 - 305 nm, where said at least one disulphide

bridge is disrupted to form activated thiols when exposed to light of said light source.

This gives the added benefit that the immobilized molecules will be spatially oriented on the surface of the object and gives precise knowledge of the proteins' attachment point to the surface, while preserving the native structural and functional properties of the immobilized protein.

In one embodiment, linker molecules comprises at least one disulphide bridge and wherein said light source is adapted, during use, to emit light having a wavelength in an interval of 250 - 305 nm or to emit light having a longer wavelength that by means of non-linear processes and/or multiphoton excitation promotes the same electronic transitions as light in the wavelength interval of 250 - 305 nm, where said at least one disulphide bridge is disrupted to form activated thiols when exposed to light of said light source.

In one embodiment, the device further comprises a controller controlling, during use, an exposure of said emitted light from said light source to said object comprising molecules to be immobilized.

In this way, a flexible and adaptable way of controlling the immobilization process and/or obtaining even complex patterns of immobilized material is/are obtained.

In one embodiment, the controller controls said exposure of said emitted light from said light source according to a pre-determined or user-specified pattern.

In one embodiment, the device further comprises a light exposure controlling system influencing said light exposure, e.g. exposure time and/or power, of said object comprising molecules to be immobilized.

In one embodiment, the light exposure controlling system comprises one or more selected from the group of: - a linear and/or non-linear optical system,

- a fresnel lens,

- a galvanic mirror,

- a transparent sheet letting through only UV light,

- a transparent sheet letting through light according to a pre-determined pattern, - a mask pattern or template allowing light to pass through only in one or more areas,

- a unit a unit using the light source to immobilize said molecules to be immobilized with a focal spot of said light source, and

- a unit forming a diffraction pattern of the light emitted from the light source on the object comprising molecules to be immobilized.

In one embodiment, the light exposure controlling system comprises a digital micro-mirror device comprising a number of hinge-mounted microscopic mirrors, where each microscopic mirror is controlled to reflect light or not towards the object comprising molecules to be immobilized in response to a control signal.

In this way, even very complex patterns of immobilized material may be obtained and an entire area (even the entire object) may be immobilized in one step, which speeds up processing time significantly.

In one embodiment, the controller is adapted to control said emitted light in order to generate a pattern of immobilized molecules where the generated pattern comprises areas of different-molecules.

In this way, multi-sensor bio-arrays or simply objects having different immobilized molecules may be obtained.

In one embodiment, the controller is adapted to control said emitted light in order to generate a three dimensional pattern of immobilized molecules where the generated pattern comprises layers of different molecules. Alternatively, the generated pattern may comprise layers of the same molecules.

In one embodiment, the controller is adapted to control said exposure of said emitted light to said object in order to generate a pattern of immobilized molecules functioning as a secure marking or watermarking of said object.

In one embodiment, the generated pattern of immobilized molecules immobilised according to is a light diffraction pattern.

In one embodiment, the molecules of said object comprises:

- DNA - protein (natural or genetically engineered),

- chimeric proteins,

- proteins tagged with a linker,

- chimeric DNA,

- DNA tagged with a linker, - any molecule that can be attached with a tag that allows light induced immobilisation,

- a thin film of sensor proteins,

- fluorescent protein,

- protein, - polypeptides,

- dyes (e.g. non-protein or non-peptide),

- peptides,

- immunoglobulins,

- Fab Fragments, - alkaline phosphatase,

- hydrolases,

- proteases

- Major Histocompatibility Complex (MHC) class I protein

- an electrically conductive material, - an electrically dielectric material,

- an electrically conductive material bound to another molecular material, and

- an electrically dielectrically material bound to a another molecular material.

In principle the molecules may be any type of molecules.

In one embodiment, the light source comprises one or more light sources selected from the group of:

- sun-light, - a lamp,

- an ultraviolet (UV) light source,

- a visible light source,

- an infra-red (IR) light source,

- a xenon arc light source, - a deuterium light source,

- a high pressure mercury light source,

- a plurality of light sources focused to form an interference pattern when exposed to the object comprising molecules to be immobilized, - a laser source,

- a single wavelength light source comprising a monochromator,

- a single photon excitation light source,

- a light emitting diode (LED),

- a solid state laser diode, - a multiple photon excitation light source,

- a high peak-power pulsed or continuous wavelength continuous wave laser,

- a mode-locked titanium-sapphire laser pumped by a high power solid state laser, and - a plurality of light sources where each light source emits light at different wavelengths.

It is to be understood that a light source can comprises several of a single light source, e.g. 2 lamps, etc. and combinations of various light sources.

In one embodiment, the device is adapted to generate fluorescence by receiving light at a small incident angle between an interface of a first medium and a second medium, where a refractive index of the second medium is greater than a refractive index of the first medium, the first medium is a solution comprising fluorescence molecules and where the received light exhibits total internal reflection at the interface between the first

and second medium and generates an evanescent wave with an electromagnetic field extending into first medium thus activating at least a part of said fluorescence molecules.

Furthermore, the invention also relate to a method of material deposition by immobilization of molecules using light comprising, emitting light, during immobilization, from a light source to an object comprising, during immobilization, molecules to be immobilized, where said molecules to be immobilized receives energy directly or in-directly by irradiation from said light source and where at least a part of said received energy upon release to said molecules to be immobilized causes the molecules to be immobilized.

The embodiments of the method correspond to the embodiments of the device and have the same advantages for the same reasons.

Other advantageous embodiments of the methods and devices are defined in the sub-claims and in the following.

Further, the invention also relates to a computer readable medium having stored thereon instructions for causing one or more processing units to execute the method according to the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 schematically illustrates a generalized aspect of material deposition by light induced molecular immobilization;

Figure 2 schematically illustrates a more detailed specific aspect of material deposition by light induced molecular immobilization;

Figure 3 schematically illustrates measurements of fluorescent emission of light from fluorescent proteins immobilized according to an embodiment of the present invention;

Figures 4a, 4b and 4c schematically illustrate measurements of fluorescence from fluorescent material immobilized by prior art micro-dispensing technology;

Figures 5a, 5b and 5c schematically illustrate measurements of fluorescence from fluorescent material deposited according to an aspect of the present invention;

Figures 6a and 6b schematically illustrate measurements by an atomic force microscope (AFM) of immobilized material;

Figures 7a - 7d illustrate immobilization of material that has been done in a wet environment;

Figures 8a - 8d illustrate immobilization of material that has been done in a dry environment;

Figure 9a schematically illustrates immobilized material, where the material has been immobilized according to a light diffraction pattern;

Figures 9b and 9c schematically illustrate another example of measured fluorescent emission from immobilized material, where the material has been immobilized according to a light diffraction pattern;

Figure 9d schematically illustrates yet another example of measured fluorescent emission from immobilized material, where the material has been immobilized according to a light diffraction pattern;

Figure 10 schematically illustrates one example of a light exposure controlling system comprising a DMD (digital micro-mirror device) used for DLP (digital light processing);

Figure 11 schematically illustrates an immobilisation system using a deuterium light source for immobilisation;

Figure 12 schematically illustrates an aspect where fluorescence can be generated very effectively from a solution;

Figure 13 schematically illustrates an immobilisation system using an optical fiber as part of a light exposure controlling system;

Figure 14 schematically illustrates an immobilisation system using an optical fiber bundle as part of a light exposure controlling system;

Figures 15a and 15b schematically illustrates a microarray of sensors and an expanded view showing individual sensor 'islands'.

Figure 16a schematically illustrates an immobilisation system with an expanded beam;

Figure 16b schematically illustrates an immobilisation system creating a using a diffraction pattern for immobilisation;

Figures 17a - 17c schematically illustrate measured fluorescent emission from immobilised material that has been immobilised by a system such as the one shown in Figure 16b;

Figure 18 schematically illustrates another example of measured fluorescent emission from immobilised material that has been immobilised by a system such as the one shown in Figure 16b;

Figure 19a schematically illustrates data corresponding to Figure 18 but where high frequency noise has been removed;

Figure 19b schematically illustrates the details of a single spot of Figure 19a;

Figures 20a and 20b schematically illustrate a 3D view of the data of Figure 19b and a line along which a horizontal intensity profile is shown;

Figures 21a and 21b schematically illustrate the data of Figure 19b and a line along which a vertical intensity profile is shown;

Figures 22a - 22c schematically illustrate measured fluorescent emission from immobilised material that has been immobilised according to a diffraction pattern;

Figures 23a and 23b illustrate an image of immobilized material formed from a bitmap file;.

Figure 24 schematically illustrates a 3D contour plot of 5x5 immobilized geometrical shapes;

Figure 25 schematically illustrates identification of spots in the array of Figure 24;

Figure 26 schematically illustrates another example of measured fluorescent emission from immobilised material that has been immobilised based on a bitmap file;

Figure 27 illustrates another image of immobilized material formed from a bitmap file;

Figure 28 illustrates the use of a dedicated microarray analysis package;

Figure 29 illustrate a probability plot allowing for a semi-quantitatively evaluation;

Figure 30a - d illustrate details of a 10x10 array of labeled cutinase created using UV-light induced immobilization;

Figure 31 discloses aspects of immobilising on wet or dry film;

Figure 32 illustrates another example of immobilised material immobilised based on a bitmap; and

Figures 33a and 33b illustrate a dense large array that is 100 micrometer x 100 micrometer with 5 micrometer spots and with 10 micrometers between spots (pitch value).

DETAILED DESCRIPTION

Figure 1 schematically illustrates a generalized aspect of material deposition by light induced molecular immobilization.

Shown is a device (100) for material deposition by immobilization of molecules using light, where the device (100) comprises a light source (102) (which does not need to be an integral part of the device) and a controller (101 ) like one or more personal computers, computational units or the like comprising appropriate software. Further shown is an object or carrier (103) (forth only denoted object) comprising molecules to be immobilized. The object (103) may e.g. be a film, quartz, glass, have a metal surface or in general be any type of suitable object.

Optionally, the device (100) may comprise a light exposure controlling system (104), e.g. like an optical system and/or another kind of light modifying system as explained in greater detail in the following.

The light exposure controlling system (104) may e.g. comprise one or more masks, templates, etc. that passes light through in certain places. In this way, the entire object (103) can be illuminated where the mask or template then defines which areas of the object (103) that receives the light and thereby causes an immobilization. This can be done in 'one go' for the entire object (103) or for a larger area of the object (103) than compared to illuminating the object (103) spot by spot. This greatly increases the production or immobilization speed as several areas are immobilized in a single step in a very simple way.

It is well established that one can place a mask in a focal point of an optical system and when the transmitted light then is refocused, the fourier transform of whatever pattern that the mask represented is seen at the image plane.

Another possibility could be to include a transparent object having embedded an optical grid according to a given pattern where light only will go through according to the pattern.

Such objects exists, also in the form of cards or sheets, .e.g. of plastic and usually of low-cost.

The exposure of larger areas may also be obtained by the use of a micro- lens array including an array of zone plate based lenses and/or by the use of a so-called digital micro-mirror device (DMD) e.g. from Texas Instruments and/or one or more galvanic mirrors, as explained in connection with Figure 10.

Furthermore, the light exposure controlling system (104) may e.g. comprises one or more selected from the group of: a linear and/or non-linear optical system, a fresnel lens, a galvanic mirror, a transparent sheet letting through only UV light, a transparent sheet letting through light according to a pre- determined pattern, a mask pattern or template allowing light to pass through i only in one or more areas, a unit using the light source (102) to form an image on the object (103) comprising molecules to be immobilized, and a unit forming a diffraction pattern of the light emitted from the light source (102) on the object (103) comprising molecules to be immobilized.

Different areas of a given object (103) may be immobilized with different molecules. One way to achieve this quite simply is to submerse the object in a solution comprising a first set of molecules and irradiate the appropriate areas of the object. Then rinse and submerse the object in a different solution comprising a second set of molecules and irradiating the appropriate areas (which may be overlapping the area of the first set of molecules in order to obtain a 3D structure or may not), and repeat as necessary.

This could e.g. be used to obtain a bio-sensor having various areas for sensing different things and a pattern for watermarking or secure labelling or marking. The large density obtainable by the described immobilization by light allows for a statistical sound results from the sensor molecules even if several areas of the object (103) are used for different things.

Different areas of different immobilized molecules may e.g. also be obtained by submersing the object (103) into a solution comprising a first and a

second type of molecules to be immobilized and using different light sources where one of the light sources only immobilizes one type of molecules and the other light source only immobilizes the other type of molecules.

Alternatively, a light exposure controlling system (104) could be used that splits and/or changes the emitted light in some respect (e.g. split into two light beams with different wavelengths, energy-level, 2-photon excitation, 3- photon excitation, etc.) where one property (e.g. one particular wave-length) only influences the immobilization of one type of molecules while the other property only influences the other type of molecules. It could also be that one type of molecules are immobilized by light having one property (e.g. wavelength, energy-level, etc.) and the other type of molecules are immobilized by the property of combined light.

Material may also be immobilized in 3D patterns/areas and could e.g. be used for fast prototyping or for making more complex structures. .

Material may e.g. also be conductive and/or dielectric material so that ^• electronic circuits, e.g. comprising bio-molecules, can be obtained.

One use of immobilized material could e.g. be to immobilize molecules at the tip of an optical fiber or at the tips of a bunch of optical fibers (see e.g. Figures 13 and 14 for examples of such setups) e.g. in a bio-sensor device or the like. In one embodiment, the immobilized molecules of the tip or bunch of tips of the optical fiber are molecules that in response to being brought in contact with a given substance and react in accordance with the function of the bio-sensor. The immobilized molecules may e.g. comprise a chemi- luminescent property which generates light in response to a given chemical reaction. In this way, light may be generated e.g. depending on whether a certain matter is present or not. By immobilizing such molecules at the tip of an optical fiber or bunch of tips in a bio-sensor, the optical fiber will very efficiently transmit the light (if any) from the immobilized molecules having a chemi-luminescent property to the other end of the fiber than the tip or bunch of tips. In this other end, the device could comprise light sensing and/or registering circuit for detecting and possible quantifying the light.

The tip could also be immobilized with different kinds of molecules.

Such a bio-sensor with an optical fiber tip or bunch of tips is useful e.g. for entering a body or another item for sampling, sensing, measurements, etc. since the fiber or fibers can be made very narrow and flexible.

The object (103) comprising the molecules to be immobilized may initially only comprise elements for binding with the molecules to be immobilized where the object (103) then is submersed in a solution of molecules to be immobilized and/or linker molecules and then is irradiated by the light source (102). The solution of molecules may be wet or alternatively dry when being irradiated by the light source (102) to cause immobilization, e.g. as explained in connection with Figures 7a - 7d and 8a - 8d.

According to one aspect, the molecules to be immobilized has the property of being able to absorb energy from being irradiated by the light from the light source (102) and then release at least some of this energy to cause a change in the molecules to be immobilized that allows the molecules to bind with the object (103) (e.g. via linker molecules) upon which material is to be deposited.

Alternatively, instead of the molecules to be immobilized having this property (absorption of energy and release to cause immobilization) the solution could comprise linker molecules that are able to absorb the energy from the light source (102) and release at least some of this energy to cause the change in the molecules to be immobilized that allows them to bind with the object (103).

The use of linker molecules and its indirect transfer of energy may have an advantage for some molecules to be immobilized if they cannot be subjected to the energy transfer from the light source (102) without being degraded in some way. Ensuring that only the linker molecules and not the molecules for being immobilized receives the energy can be obtained by choosing the irradiating light appropriately, e.g. by selecting a given wavelength, etc. as explained in the following.

The binding between the molecules to be immobilized and the object (102) may e.g. be done by a disulphide bridge (e.g. if the molecule comprises a disulphide bridge that upon light induced disruption leads to the formation of thiol group(s) and the object e.g. comprises thiol groups or disulphide bridges), a covalent binding or other types of bindings.

According to one aspect, the molecules to be immobilized contains, as mentioned, at least one disulphide bridge, which is an inherent property of e.g. proteins, where the disulphide bridge(s) protect molecules from photophysical and/or photochemical reactions when their aromatics are illuminated. When proteins are exposed to UV light, some disulphide bridges are disrupted to form activated thiols. Although disulphide bridges are commonly found in the structural core and near/on the surface of folded proteins, those located in close proximity to aromatic amino acids are the most susceptible to UV-induced disruption. During UV exposure of proteins, energy absorbed by side chains of aromatic amino acid residues is _* transferred to spatial neighbouring disulphide bridges, which function as - quenchers (Neves-Petersen MT., et al., 2002, Protein Science 11 : 588-600).

However, the flow of energy transferred to disulphide bridges and the likely formation of intermediate species such as electrons and solvated electrons and e.g. chemical species like radicals, ions, etc. formed upon light excitation of the object ultimately serves to trigger their disruption. The presence of a disulphide bridge with aromatics, aromatic residues, natural and/or artificial amino acids, and/or etc. as a close spatial neighbour in a protein occurs frequently in nature, indicating that photo-induced disulphide bridge disruption is a widespread phenomenon (Petersen MTN., et al., 1999, Protein Engineering 12: 535-548; Neves-Petersen MT et al., 2002, Protein Science 11 : 588-600; Vanhooren A et al. 2002, Biochemistry 10; 41 (36): 11035- 11043)."

An advantage with such bindings and selectively breaking of connections is that the orientation of the immobilized molecules will be the same.

The light source (102) emits light that is brought to influence the object (103) comprising the molecules to be immobilized. The light source (102) is

according to one aspect adapted, during use, to emit light having a wavelength in an interval of 250 - 305 nm or to emit light having a longer wavelength that by means of non-linear processes and/or multiphoton excitation promotes the same electronic transitions as light in the wavelength interval of 250 - 305 nm and thus immobilizes the molecules e.g. by breaking disulphide bridges in the molecules and forms activated thiols.

According to another aspect, photosensitizer molecules may be used, where the photosensitizer molecules enters an excited energy state in response to being subjected to the emitted light and can release the energy to the molecules to be immobilized.

As one alternative, the molecules to be immobilized or a solution comprising linker molecules could comprise molecules capable of releasing energy in the form of electrons to said molecules to be immobilized. This can e.g. be done by using a photo-electric effect to release electrons from the surface or substrate (e.g. a metal layer). Alternatively, photo-ionization or photolysis could be used to release electrons from other molecules. Hydrolysis of water could also be used to supply such electrons.

In general, coupling of two elements or immobilisation of molecules on an object is enabled, where the structural and functional properties of the coupled or immobilised elements or molecules can be preserved if desired, and the orientation of coupling can be controlled on a molecular scale.

As mentioned and as disclosed in WO 04/065928 by the same applicant, is the exploitation of appropriate naturally occurring triads for coupling of a molecule to an object or another molecule by thiol binding. Such a triad is present when a disulfide bridge is located in sufficient proximity (< 10 A) to an aromatic amino acid residue in the folded protein, to allow for light absorption by the aromatic residue followed by quenching of the excited state aromatic residue by the nearby disulphide bridge, that is disrupted in the process due to energy transfer to the disulphide bridge, e.g., in the form of electron transfer to the bridge. The thiol groups created in proteins in this way by light-induced disulfide bridge disruption may then used to immobilise the

protein to a support in an orientation-dependent and controlled manner. After excitation of the aromatic residues, several charged and/or radical species can be formed. For example, there is a chance that photo-induced ketyl radicals can propagate an electron/radical along the peptide chain across several residues from an aromatic to a disulfide bridge. If this happens, the aromatic residue does not need to be in close proximity (less than around 1 θA) from the disulphide bridge.

In certain situations the molecules to be immobilized and/or object comprising the molecules to be immobilized do not contain appropriate naturally occurring triad(s). In one case, the natural protein may not contain a triad or the triad(s) present is/are not accessible by light, or the created thiol(s) is/are not accessible for coupling because it is buried inside the ^■ protein or breakage of the disulfide bond will destroy the properties of the protein, such at binding capacity or enzymatic activity or the like. In such a case an appropriate disulfide bridge-containing triad may advantageously be added or providing to the protein. The same can be the case for peptides and polypeptides.

As disclosed in a co-pending application by the same applicant filed on the same day, coupling of two objects or two molecules both lacking appropriate thiol-binding properties can also be provided, by utilizing the present principle of adding or providing appropriate disulfide bridge-containing triads. The principle can also be used to couple more than two elements. The principle may further be used to add the aromatic amino acid in a solution to the element(s) comprising an appropriate disulphide bridge.

An aspect relates to a method of providing an appropriate disulfide bridge- containing triad to an element A which lacks such appropriate triad for coupling element A to an element B via thiol binding. The invention also relates to a method of providing an appropriate disulfide bridge to element A and to methods of additional provision of appropriate disulfide bridge- containing triad or disulfide bridge to element B.

Thus, in an additional aspect, the present invention relates to a method of coupling two elements A and B, by

1) generating an element A* by providing an appropriate disulfide bridge or disulfide bridge-containing triad to element A, and if desired providing an appropriate disulfide bridge or disulfide bridge-containing triad to element B to generate element B ^*

(2) a) irradiating said element A* (and B*) to create an appropriate reactive thiol group, and b) incubating said irradiated element A* with said element B capable of binding a thiol group (or irradiated B ^* ), thereby obtaining a coupling between elements A and B; or a) incubating said element A* with said element B capable of binding a thiol group or said element B*, and

^' b) irradiating said element A* in the presence of said element B (or B*) to create a reactive thiol group by disulfide bridge disruption in said element A* (and B*), and thereby obtaining a coupling between element A and element B.

Elements according to the invention comprise molecules and object(s), thus providing great flexibility, allowing for coupling combinations of molecule + molecule, object + object, molecule + object and/or object + molecule. In case of coupling an object with object and molecule with molecule, different, similar or identical object(s) and/ molecule(s) can be coupled. In one example, two or more elements placed on the same object may be coupled in order to create a desired conformation of the object, which may be a object or a molecule such as a protein or polypeptide. The molecule may e.g. be a biomolecule, such as a peptide, a protein, a polynucleotide, a lipid, a sugar, a pharmaceutical, a cosmetical, a pro-drug and the like.

The usable molecules are not limited to proteins, but allow virtually any molecule(s) and object to be coupled, provided that an appropriate disulfide

bridge or disulfide bridge-containing triad can be provided, for example by covalent attachment and/or by genetic engineering. This includes virtually any object(s), provided that they can be coupled according to the invention, i.e. that an appropriate linker can be coupled to the object comprising the molecules to be immobilized and/or that the object comprising the molecules to be immobilized is capable of binding a thiol group. An object comprising the molecules to be immobilized according to the invention may comprise a soluble, semi-soluble or insoluble material to which an appropriate disulfide bridge or disulfide bridge-containing triad is capable of being attached. Such an object comprising the molecules to be immobilized may also comprise a thiol reactive surface or a surface that can be made thiol reactive, e.g. a surface comprising gold or quarts. An object comprising the molecules to be immobilized which is reactive for binding one or more molecule(s) is denoted "a carrier". A carrier may therefore be an object comprising the molecules to be immobilized which by nature is reactive for binding one or more molecule(s) containing a thiol group or an object which is made reactive for binding one or more molecule(s) via thiol binding.

Another aspect relates to a linker molecule comprising an appropriate disulfide bridge or disulfide bridge-containing triad, which may be provided to an element lacking an appropriate disulfide bridge or disulfide bridge- containing triad. Such an appropriate linker molecule may comprise one or more copies of a peptide comprising any of the formulas (I) X1 _m C X2 _n C X3 ₀ 0 X4 _P , (II) X1 _m C X2 _n 0 X3 ₀ C X4 _P and/or (III): X1 _m 0 X2 _n C X3 ₀ C X4 _P , wherein X1 _m , X2 _n , X3 ₀ and X4 _P represent the same or different peptides, each peptide respectively consisting of m, n, o, and p amino acids, where m, n, o and p are mutually independent numbers between 0 and 1000 (or 100, 25,or 10), and m + n + o + p < 1000 (or 100, 25, or 10), said amino acids being selected from all natural and synthetic amino acids, C is cysteine, and the two cysteines are covalently joined by a disulfide bridge, and 0 is an aromatic amino acid such as phenylalanine, tryptophane or tyrosine, or a peptide bond. The aromatic amino acid 0 may also be absent, or additional, similar or different aromatic amino acids 0 may be present.

In one aspect of the invention, the linker is provided to an element, object, molecule and/or protein, polypeptide or peptide through covalent binding of a linker molecule, e.g using NHS (N-hydroxysuccinimide), EDC (N-ethyl-N ¹ - (dimethylaminopropyl) carbodiimide hydrochloride), activated ester, maleimide, disulfide formation, streptavidin/biotin, activated alcohol, vinylsulfone, Schiff base formation and/or "click" chemistry and the like.

In a another aspect of the invention, a linker providing an appropriate disulfide bridge and/or disulfide bridge-containing triad is provided through genetic engineering techniques, comprising: (i) N-terminal extension, (ii) C- terminal extension, (iii) internal extension, (iv) amino acid substitution, (v) amino acid insertion, (vi) amino acid deletion or (vii) any combination or combinations of said methods (i-vi). Furthermore, genetic engineering may also result in a conformation change in a molecule, thereby bringing an aromatic amino acid and an appropriate disulphide bridge in the vicinity of each other.

In yet another aspect of the invention, coupling of two elements may be provided by adding a free aromatic amino acid (in solution), either alone or being part of a molecule, to the vicinity of one or more appropriate disulfide bridges.

The invention provides an irradiation step, comprising light of a wavelength that excites one or more aromatic amino acids. Such wavelength interval(s) comprises UV light in the wavelength interval of 250 to 305 nm (or 250 to

260nm, 270 to 280 nm and/or 290 to 300nm, or about 254, 275 or 295 nm), or with light having longer wavelengths that by means of non-linear processes and/or multiphoton excitation promotes the same electronic transitions as light in said wavelength interval of 250 to 305 nm.

An aspect of the invention provides a coupling between elements, resulting in an immobilization on an object comprising the molecules to be immobilized or

surface, which can be spatially controlled as disclosed in WO 04/065928. Such an object and/or surface may also be a derivatized object that is capable of binding a thiol group, such as an object and/or surface comprising a thiol group or a disulfide bridge. When appropriate, such a surface or object may comprise a spacer.

A further aspect of the present invention relates to a magnetic (nano)particle or a cantilever for atomic force microscopy, where the magnetic (nano)particles or a cantilever comprises a molecule coupled by irradiation of a disulfide bridge or disulfide containing triad.

Another aspect relates to dimers, such as homo- or heterodimers, consisting of two identical or different elements or subunits.

Yet another aspect relates to dendrimers, such as homo- or hetero dend rimers.

The controller (101 ) controls the exposure of said emitted light from said light source (102) to an object (103) comprising molecules to be immobilized. Depending on the specific embodiment, the controller (101 ) may control only the light exposure controlling system (104), only the light source (102) or both. The controller may also control a unit for moving the object (103) (in 2D or 3D) so that different parts of the object are exposed to the light from the

light source (102). Alternatively, the light source (102) may be moved or the light hitting the object may be moved or changed by use of an appropriate light exposure controlling system (104).

The light source (102) may generally be any source emitting light capable of immobilizing the molecules.

A variety of light sources are suitable for the irradiation at a range of wavelengths. As examples mention can be made of lamps e.g. a 75-W Xenon arc lamp from a research grade spectrometer such as a RTC PTI spectrometer, a deuterium lamp, a high pressure mercury lamp. Irradiation at a single wavelength can also be obtained by coupling the light source to a monochromator. As examples of a light source of single and multiple photon excitation are e.g. high peak-power pulsed or continuous wavelength CW laser.

Other usable lights sources are e.g. light emitting diodes (LEDs) and laser diodes. Alternatively, one or more pulsed lasers may be used that have ns, ps or fs lasting pulses. Yet another alternative is to use quantum dot lasers.

As a very specific example, the light source (102) may e.g. be a solid state diode-pumped mode-locked titanium-Sapphire (Ti-Sapphire) laser source as shown and explained in connection with Figure 2.

In one aspect, the light source is a fs laser (femtosecond laser). In this aspect, the energy per pulse may be in the interval of 1 picoJ to 30 milliJ. In a further aspect, the energy per pulse may be in the interval of 1 nanoJ to 10 milliJ and in yet a further aspect, the energy per pulse may be in the interval of 1 nanoJ to 1 milliJ.

In an aspect, the light source (102) irradiates light preferably having a wavelength of between about 250 nm and about 305 nm. In another aspect of the invention, the wavelength is between about 260 nm and about 300 nm. In yet a further aspect of the invention, the wavelength is between 270 nm and about 295 nm. In yet a further aspect of the invention, the wavelength is between 270 nm and 290 nm. In another aspect of the invention, the

wavelength is between 275 nm and about 285 nm. In a further aspect of the invention, the wavelength is about 280 nm.

As another alternative, the light source (102) may irradiate light with a wavelength of 250-305 nm such as UV light or visible light (e.g. via two- photon excitation) or infra-red light (e.g. via three-photon excitation). The use of infra-red light (via three-photon excitation) would facilitate light that is able to penetrate deeper into a given object than UV light. The multi-photon excitation principle allows larger penetration depths since the longer the wavelength of light (e.g. visible and IR light compared to UV light) the deeper light may penetrate into the material of the given object and the less light is scattered. Multi-photon excitation offers the additional advantage that excitation only takes place in the focal spot. Optical penetration may occur down to a depth of 5-7 cm, especially in the case of IR illumination, depending on the material being penetrated.

Another possibility is to immobilise using visible light. This could e.g. be achieved due to second harmonic generation at an interface converting visible light into UV light at the interface.

Surface Second Harmonic Generation is a method for probing interfaces in atomic and molecular systems. In second harmonic generation (SHG), the light frequency is doubled, essentially converting two photons of the original beam of energy approximately E into a single photon of energy 2E as it interacts with non-centrosymmetric media. Using this and visible light, the light frequency is doubled thus creating UV light at the interface. Thus it is also possible to immobilise using visible light.

Surface second harmonic generation is a special case of SHG where the second beam is generated because of a break of symmetry caused by an interface. Surface SHG is possible even for materials which do not exhibit SHG in the bulk.

An exemplary use of immobilization at a depth of a given object may e.g. be the marking of objects (such as paper money, credit cards, luxury items, secure documents, etc.). Theses uses can also be done at the surface of the

object. Marking is explained and shown in greater detail in connection with Figures 9a - 9d, 15a, 15b, and 17a - 24b.

When IR photons are focused towards a particular target at a depth where scattering events does not lead to severe attenuation of intensity, multi- photon events may be induced, leading to excited states that normally would only occur if visual or UV light was used.

When using multi-photons, the beam can be expanded into multiple beams, and, e.g. using IR transparent optical fibres, these beams can be geometrically pointed at a single point and will enable multi-photon events. This can be advantageous if it is preferred to have the effect of the combined beams at as localised an area as possible.

Visible light can also be used. Two photon excitation would promote the same electronic transition that UV light can promote. Furthermore, far-IR/ Microwave sources could be used at the focal point to promote the same electronic transition that UV light can promote.

The light source (102) may e.g. be one or more of: sun-light, a lamp, an ultraviolet (UV) light source, a visible light source, an infra-red (IR) light source, a xenon arc light source, a deuterium light source, a high pressure mercury light source, a plurality of light sources focused to form an diffraction pattern when exposed to the object (103) comprising molecules to be immobilized, a laser source, a single wavelength light source comprising a monochromator, a single photon excitation light source, a multiple photon excitation light source, a high peak-power pulsed or continuous wavelength continuous wave laser, a mode-locked titanium-sapphire laser pumped by a high power solid state laser, and a plurality of light sources where each light source emits light at different wavelengths.

For a deuterium light source setup, please see Figure 11. For other types of setups please e.g. see Figures 13 - 14 and 16a and 16b.

Figure 2 schematically illustrates a more detailed specific aspect of material deposition by light induced molecular immobilization. Shown is a light source (102) in the form of a laser.

In one embodiment, the laser is a solid state diode-pumped mode-locked titanium-Sapphire (Ti-Sapphire) laser delivering e.g. 0.9 W of 840 nm near- infrared and approximately 80 femtosecond long laser pulses at a repetition rate of 80 MHz. One example of such a laser is e.g. the Tsunami 3960 laser by Spectra Physics, Mountain View, CA. The laser may e.g. be pumped by a high power (5W at 532 nm) solid state laser such as Millennia V by Spectra Physics.

In this exemplary embodiment, the laser pulses are passed through a pulse picker (110) reducing the repetition rate to 8 MHz before entering a doubler/tripler unit (111 ) (e.g. GWU from Spectra Physics) where the reduced

* laser pulses are used to generate the third harmonic of 840 nm to yield approximately 1 mW of approximately 200 femtosecond long 280nm UV pulses at 8 MHz. The pulse picker (110) is not necessary for immobilization but gives a simple way of controlling the power of the light.

The resulting UV pulse train is then passed to (e.g. using mirrors, optical fibres and/or components, and/or the like (113)) a light exposure controlling system (104) comprising in this particular embodiment a shutter (112), being controlled by the controller (101), receiving the UV pulse train and passing the UV pulse train through (when the shutter is controlled to be open) to a beam expander (114) (or an optical lens) expanding the beam, an iris diaphragm (115) and a finally a focusing lens (116) focusing the beam into a spot (or an area) of an appropriate size (e.g. about 25 micrometers but via an appropriate light source (102) and/or light exposure controlling system (104) even down to 1 micrometer and below in the nanometer range).

Please note, that the shutter is not needed for embodiments using a mask, a template, a micro-lens array and/or by the use of a so-called digital micro- mirror device (DMD), etc. where the object or a larger portion (than a spot) of the object is illuminated by the light source.

The object (103) comprising molecules to be immobilized is, in this particular embodiment, mounted on a 2D or 3D translation stage (117) (controlled by controller (101 )) with the surface of the object comprising molecules to be immobilized in substantially the focal plane (at least when light is to immobilize the molecules on the object (103)) of the UV-light).

The translation stage may also be used to bring the surface out of focus, i.e. bring the object out of the focal plane, e.g. if an immobilization pattern is to be produced due to diffraction as explained for one example in greater detail in connection with Figures 9a - 9d, 15a, 15b, and 17a - 24b.

Figure 3 schematically illustrates measurements of fluorescent emission of light from fluorescent proteins immobilized according to an embodiment of the present invention. Shown is a 3D graph illustrating measured amount of fluorescent emission in the z-axis for immobilized fluorescent proteins distributed on an object (in the x- and y-axis plane).

In this particular example, a film comprising fluorescent proteins (cutinase) was irradiated at each spot as described earlier thereby immobilizing the fluorescent proteins immobilised cutinase on a film. In this particular example, a film of 1mL (1 mM) protein was deposited on a slide e.g. on the surface of a 2D or 3D translation stage. The arrays were made on a wet film and at the time of immobilisation a focused laser beam illuminated for about 100 ms per spot. After illumination, the slides were washed with double- distilled water, 16PBS buffer, 16PBS plus 0.1% Tween 20 detergent and water. The slides were then scanned with a Tecan LS 200 scanner green laser (excitation 532 nm, cy3 filter) and processed in a visualization tool BNIP Pro. The fluorescence emission was then measured and the measured values were visualized. In this particular example, each spot has a diameter of about 20 - 30 micrometers and their centres are spaced apart approximately 75 micrometers, but this may vary and even be smaller depending on the focus of the spot used.

As can be seen, the proteins can be immobilized at a high resolution with a large density by well defined and localized spots with a large uniformity. This enables large reproducibility and a high signal to noise ratio.

A large density or resolution is desirably, since more sensor molecules in a bio-sensor can be packed on a limited space thereby increasing the statistical certainty of the results from the sensor molecules. Alternatively, a more compact bio-sensor can be obtained having the same statistical properties as a larger bio-sensor produced according to prior art methods.

Figures 4a, 4b and 4c schematically illustrate measurements of fluorescence of fluorescent material immobilized by prior art micro-dispensing technology.

The results illustrated in Figures 4a, 4b and 4c are measured fluorescence of immobilized fluorescent protein. The results have been obtained by using conventional micro-dispensing technology like the SpotBot® Personal Microarray Robot from TeleChem's Arraylt™ with an average spot deposition of 1.1 nl_ and 140 micrometers. Similar results are obtained by other dispensing techniques.

Figure 4a shows a 2D plot of measured fluorescence of the traditional technology while Figure 4b shows the measured fluorescence along a profile line going through a line of spots in Figure 4a. As can be seen, e.g. from the spot profile of Figure 4b, the individual spot peaks are not well-defined spots and each peak of the spot exhibit a 'crater-like' profile, which can be caused by contact during printing of the pin delivering the material fluid or ink with the object that is being deposited with material and/or by localised drying of the spot.

Figure 4c shows a close-up 3D view of a single spot deposition and its corresponding 2D overview.

The spots produced by the traditional technology spot has a diameter of about 140 micrometers and their centres are spaced apart approximately 300 micrometers.

Figures 5a, 5b and 5c schematically illustrate measurements of fluorescence of fluorescent material deposited according to an embodiment of the present invention.

The results illustrated in Figures 5a, 5b and 5c are measured fluorescence of immobilized fluorescent protein immobilized according to an aspect of the present invention.

Figure 5a shows a 2D plot of measured fluorescence from fluorescent material immobilized with light according to an aspect of the present invention while Figure 5b shows the measured fluorescence intensity profile along a profile line going through a line of spots in Figure 5a.

Figure 5c shows a close-up 3D view of a single spot deposition and its corresponding 2D overview.

The spots produced in this example has a diameter of about 20 micrometers and their centres are spaced apart approximately 75 micrometers.

The spot size and/or the distance between the individual spots can easily be reduced by using a more focused light source like shown in Figures 33a and 33b since each immobilized spot is defined by size of the light hitting the object and not by the physical size of dispensed droplets.

As can be seen, e.g. from the spot profile of Figure 5b, the individual spot peaks are well-defined and uniformly distributed on the object and the density is greater than the peaks in Figures 4a - 4c.

In addition to a relatively larger minimum spot size, such printing or liquid dispensing systems have disadvantages like relatively high cost, their reliance on micro-injection systems (like pumps, valves, etc.) that easily can get clogged and that the material usage is often high. Furthermore, they do also typically require the use of a humid chamber or similar.

Furthermore, it has been shown that the object (103) comprising molecules to be immobilized can be both wet and dry with satisfactory results as described in greater detail in connection with Figures 7a - 7d and 8a - 8d.

Figures 6a and 6b schematically illustrate measurements by an atomic force microscope (AFM) of immobilized material. It can be seen that the molecules are immobilised as monolayers. Single molecules immobilised by means of our technique can be observed.

Figure 6a shows a 3D AFM visualization of a light immobilized protein being immobilized as explained earlier, where the height is measured (along the z- axis) for immobilized proteins distributed on an object (in the x- and y-axis plane).

Figure 6b shows a line profile of single cutinase molecules taken along a line of the 3D AFM image and showing the measured height. The height matches the hydrodynamic radius of the protein reported in literature by means of X- ray diffraction studies.

As can be seen, a uniform distribution of single molecules with very little conglomeration is obtained. Furthermore, the height of the deposited material is in the range of individual molecules.

Figures 7a - 7d illustrate immobilization of material that has been done in a wet environment.

Figure 7a illustrates immobilization of molecules according to an aspect of the present invention where the molecules have been immobilized as described earlier but in a wet environment. A wet environment is e.g. where a solution or film comprising the molecules to be immobilized (and possibly any activation molecules) has been applied to the surface of an object to which the molecules are to be immobilized. The molecules were immobilized by illumination while the solution or film comprising the molecules to be immobilized was still wet.

Shown in Figure 7a is a 3D graph illustrating measured amount of fluorescence (or in principal any other activity) in the z-axis for immobilized fluorescent molecules distributed on a surface of an object (in the x- and y- axis plane).

Figure 7b shows a 2D view and Figure 7c shows a profile along an axis going through a row of spots of the corresponding data in Figure 7a.

Figure 7d shows the mean distribution, the integral distribution, and the signal-to-noise ratio of a micro-array spots. As can be seen the mean distribution and the integral distribution have a normal distribution with very little skew signifying that the spots exhibits uniformity with relatively low variance, even if the molecules have been immobilized in a wet environment, The mean samples are obtained by taking the mean of each spot and the integral samples are obtained by taking the area of each spot. Furthermore, a relatively high signal-to-noise ratio is obtained.

As a note, the technology enables printing arrays or other patterns without the use of humid chambers.

Figures 8a - 8d illustrate immobilization of material that has been done in the same way as was done for Figures 7a - 7d but where the data has been obtained for molecules immobilized in a dry environment instead of a wet environment. A dry environment is e.g. obtained by letting the wet solution comprising the molecules to be immobilized to evaporate to a certain extent, e.g. simply by letting the object dry for a bit of time. No active drying is necessary (although it could be used). There will still be an amount of water content, e.g. of 20%, in the dried environment depending on the specific molecules.

Shown in Figure 8a is a 3D graph illustrating measured amount of fluorescence (or other activity) in the z-axis for immobilized fluorescent molecules distributed on a surface of an object (in the x- and y-axis plane).

Figure 8b shows a 2D view and Figure 8c shows a fluorescence intensity profile along an axis going through a row of spots of the corresponding data in Figure 8a.

Figure 8d shows the mean distribution, the integral distribution, and the signal-to-noise ratio of a number of spots. As can be seen the mean

distribution and the integral distribution have a normal distribution with very little skew signifying that the spots exhibits uniformity with relatively low variance, even if the molecules have been immobilized in a wet environment. The mean samples are obtained by taking the mean of each spot and the integral samples are obtained by taking the area of each spot. Furthermore, a relatively high signal-to-noise ratio is obtained.

Comparing Figures 7a - 7d with 8a - 8d it can be seen that good results (uniformity, high density, low size, etc.) are obtained according to both environments signifying that the described method works well both in dry and in wet environments. For immobilization in the dry environment there is increased signal intensity and a better signal-to-noise ratio.

An advantage of using a dry environment is that specialised equipment and/or conditions, e.g. like a humidity chamber, is not needed. An additional advantage of using a dry environment when immobilizing with light is that light will not be diffracted in the wet solution thereby causing immobilization other places than intended.

Figure 9a schematically illustrates immobilized material, where the material has been immobilized according to a light diffraction pattern.

In this particular example, the light diffraction pattern has been created by focusing down the diffraction pattern from a circular aperture and positioning the object in a plane slightly different from the focal plane.

Shown is an image obtained by a confocal microscope of a spot having a diameter of approximately 20 micrometers within an immobilized protein array where the material of the shown spot has been immobilized using light as described above but where a diffraction pattern of the light immobilizing the material has emerged. The width of the inner rings is 1.2 micrometer.

The diffraction pattern will have regions of differing light intensities and will immobilize molecules according to sufficient intensities.

Thus it is possible to 'copy' a diffraction pattern onto an object comprising molecules to be immobilized, where molecules will only be immobilized in the presence of light in the diffraction pattern. This enables complex patterns in a simple way.

Immobilisation in this way, also has the advantage that the immobilized area is done in 'one go'.

The diffraction pattern may e.g. be obtained by a suitable light exposure controlling system and/or optical system. Examples of such systems are e.g. systems comprising several light sources causing a diffraction pattern of light, one or more lenses that cause the irradiation light to be slightly out of focus, a movement of the object to be outside the focal plane of the light source, diffraction from a sharp edge (e.g. like the system shown in Figure 16b) where a razor blade is used to block one half of the light beam, diffraction from a circular aperture (i.e. a 'pinhole'), diffraction of one or more parallel slits etc. or combinations of the above.

In this way, it is possible to make quite complex patterns of immobilized molecules in an easy way. As mentioned earlier, it is also possible to obtain patterns using masks, templates, specific control of where the light is exposed to the object, etc.

Such patterns or markings may e.g. be used for watermarking or secure labelling or marking purposes of luxury goods, documents, money, credit- cards, or in principle any object where it is desirable to have a mark for authenticity or another purpose that is hard to copy or imitate.

One use could be e.g. to immobilize a specific pattern of molecules where the molecules are only visible in UV light, 'black' light, etc. and/or it could be so tiny (e.g. made of spots of 1 micrometer or even nanometer-size) that it would be hard to locate if prior knowledge of its location is not given.

Immobilization according to a diffraction pattern is also suited for this purpose as there can readily be made quite unique and as even though the pattern might be obtained it would be very hard, if not impossible, to re-create exactly

the same (or substantially the same pattern within a given threshold) diffraction pattern thereby making them hard to fake as this would require substantially the same physical setup that was used during creation, e.g. the object exactly out of focus in the same way or exactly the same light sources with the same wave-lengths interfering in creating the specific diffraction pattern.

Specific immobilization patterns and/or masks, templates, optical components etc. used during immobilization are also well-suited for this purpose as it would require the exact same patterns, etc. used under the same conditions to falsify a mark.

Immobilization of fluorophores (attached to a molecule, e.g. protein) with different excitation and/or emission profiles will give a mark with different colours of light.

When authentication is to be made, a scanning unit or the like could scan the object to obtain the mark and compare it digitally with a pre-stored version of the mark (which could be stored at the inspection site or stored at a remote location for even further security), and if the two digital images compares within a given threshold it would be determined to be authentic. Instead of working with the entire mark appropriate profiles, e.g. as shown in Figures 17b, 20b, 21b, and 22c, and/or combinations thereof could be used. Alternatively, other well known authentication methods could be used.

Figures 9b and 9c schematically illustrate another example of measured fluorescent emission from immobilized material, where the material has been immobilized according to a light diffraction pattern. Shown is an image of fluorescence from protein molecules immobilised onto a quartz surface according to an UV diffraction pattern. The shown inner rings are about 1 μm apart and the image is about 10 μm across.

Figure 9d schematically illustrates yet another example of measured fluorescent emission from immobilized material, where the material has been immobilized according to a light diffraction pattern. In this example, the

diffraction pattern has been obtained by passing light through a pinhole prior to immobilisation.

Figure 10 schematically illustrates one example of a light exposure controlling system comprising a DMD (digital micro-mirror device) used for DLP (digital light processor).

A DMD is a device comprising a semiconductor-based "light switch" array of a large number (thousands or even millions) of individually addressable, hinge-mounted and tiltable, microscopic mirrors that can tilt either toward a light source (ON) or away from the light source (OFF) thereby creating a light or a dark pixel or spot on the projection surface. A bit-streamed signal direct each mirror to switch either 'on' and 'off up to several thousand times per second. Such micro-mirrors measure less than one-fifth the width of a human hair and are mass-produced by Texas Instruments.

According to an aspect, a light exposure controlling system (104) comprises, in this particular example, a collimating lens (802), a bandpassfilter (803), an aperture (804), a shutter (112), a large number of DMD mirrors (806) and an objective lens (807).

The light exposure controlling system (104) receives light from a light source (102) where the light is passed through the collimating lens (802), through the bandpassfilter (803), through the aperture (804) and through the shutter (112) to the DMD mirrors (806) where each mirror reflects light or not towards the object (103) comprising molecules to be immobilized depending on a signal received from a controller (not shown; see e.g. 101 in Figures 1 and 2). The mirrors that are 'on' (as controlled by the controller) will pass light to the objective lens (807) focusing the light on the object (103) comprising molecules to be immobilized.

The objective lens (807) may focus or direct the light on only a part of the object or may focus or direct the light on the entire or substantially the entire surface of the object having material to be immobilized, thereby immobilizing molecules over a large area in 'one go' instead of immobilizing one spot at a time.

The object (103) may e.g. be mounted on a 2D or 3D translation stage (117) for moving the object.

This gives a very high degree of control and precision of where light irradiates the object and thereby enables very complex 'images' of immobilized molecules.

Figure 11 schematically illustrates an immobilisation system using a deuterium light source for immobilisation. Shown are a deuterium light source or lamp (102) emitting light to be received by a light exposure controlling system (104) before the light is subjected to an object (103) comprising molecules to be immobilized. The object (103) may e.g. be a film, have a metal surface or in general be any type of suitable object.

According to an aspect, the light exposure controlling system (104) comprises, in this particular example, a beam reducer (120), a shutter (112), a filter (806) and a 2D beam deflector (121 ). The purpose of the beam reducer (120) is to focus the light from the light source (102), if needed. The purpose of the 2D beam deflector (121 ) is to direct the light towards the object (103) so that the exposing light can scan the object, which avoid the need for moving the object (e.g. by a translation stage). The use of a beam deflector (121 ) allows for fast and accurate positioning of the beam without the requirement for the shutter to close between individual spots in the array. The beam deflector (121 ) may e.g. be realised by using two mirrors mounted on galvanometer scanners.

Furthermore, a controller (101 ), like one or more a personal computers, computational units or the like comprising appropriate software, is connected to and controlling the shutter (112) and can thereby control the exposure of the light of the object (103).

The light exposure controlling system (104) receives light from the deuterium light source (102) where the light is passed through the beam reducer (120), through the shutter (112), through the filter (803) to the 2D beam deflector

(121 ) towards the object (103) comprising molecules to be immobilized depending on a signal received from the controller (101 ).

The object (103) may e.g. be mounted on a 2D or 3D translation stage (117) for moving the object.

Alternatively, the system may be implemented without the beam reducer (120).

The use of such a light source and associated system compared to the laser light source and associated system as shown in Figure 2 provided a much cheaper system.

Figure 12 schematically illustrates an aspect where fluorescence can be generated very effectively from a solution.

Shown in Figure 12 is a first medium in the form of a liquid sample (128) of solution located on a second medium in the form of an object (103), in this particular example a quartz slide. UV light is transmitted through the quartz slide at a small incident angle between the interface of the solution and the quartz so that total internal reflection will take place. This happens when the incident angle is greater than the so-called critical angle being dependent on the used solution and the object and when the refractive index of the object is greater than the refractive index of the solution.

Under such circumstances the electro-magnetic wave of the UV light will propagate into the solution as a so-called evanescent wave and an electromagnetic field will thus extend into the medium (i.e. the solution) with the lower refractive index. The evanescent depth will be about the wavelength of the UV light and the intensity of the field will decrease exponentially.

Thus an evanescent field will be generated into the solution. If the solution comprises fluorescent molecules e.g. provided by a flow of solution flowing along the object or quartz the fluorescent molecules will be activated by the evanescent field. There will be maximum fluorescence at the near the

surface of the object or quartz, i.e. near the interface, since the evanescent field or wave will only propagate a small distance (about the wavelength of the used light) into the solution as the intensity decreases exponentially. Additionally, a prism for guiding the light to the object and a flow chamber for recycling the solution comprising to fluorescent molecules may be added.

In this way, it is possible to create strong fluorescence at the location(s) where UV light is brought under total internal reflection as described above, where the location(s) can be provided with a great amount of control.

A CCD element or the like could be placed above the object or quartz and could thus register the amount of fluorescence e.g. to be used in a sensor arrangement.

When the light is stopped the fluorescence will quickly fade away and stop. Thus fluorescent light can be created in response to UV light.

Additional aspects and examples in relation to this total internal reflection fluorescence is described in connection with Figures 34a - 34f.

Figure 13 schematically illustrates an immobilisation system using an optical fiber as part of a light exposure controlling system (104). Shown is a light exposure controlling system (104) receiving UV light from an appropriate light source (not shown) as described earlier. The light is subjected to an object (103) comprising molecules to be immobilized where the object (103) e.g. is located on a 2D or 3D translation stage (117) for moving the object. The object (103) may e.g. be a film, have a metal surface or in general be any type of suitable object.

The received UV light is passed through a shutter (112), through a focusing lens (116) focusing the beam into a spot (or an area) of an appropriate size (e.g. about 25 micrometers and even down to 1 micrometer and below in the nanometer range) which is received by an optical fiber (122) at one end where the other end of the fiber is used to expose the object (103) with light for exposure.

Furthermore, a controller (101 ), like one or more a personal computers, computational units or the like comprising appropriate software, is connected to and controlling the shutter (112) and the translation stage (117) and can thereby control the exposure of the light of the object (103).

A blow-up illustrates the tip (123) of the fiber (122) and the object (103) to be immobilised.

Figure 14 schematically illustrates an immobilisation system using an optical fiber bundle as part of a light exposure controlling system (104). The shown system corresponds to the one shown in Figure 13 except that instead of a single fiber a fiber bundle (124) is used. A blow-up also illustrates the tips or end (125) of the fiber bundle (124).

By using a tapered fiber tip, near-field effects can be exploited allowing irradiation of areas smaller than the diffraction limit and thereby allowing for smaller features or patterns to be immobilized.

Furthermore, using a bundle of fibers allows for example to print/immobilise arrays of molecules in one single step. With only a single fiber there is a need for scanning the stage with the material to be immobilised or moving the fiber for illuminating different parts of the surface where we want the array to be created.

Figures 15a and 15b schematically illustrates a microarray (e.g. protein or DNA) of sensors and an expanded view showing individual sensor 'islands'. Shown in Figure 15a is a microarray of a large number of sensors. In Figure 15b a blow-up of the part of Figure 15a defined by the red square is shown. As can be seen each sensor or sensor 'island' is readily identifiable. In this particular example, each sensor has been immobilised according to a diffraction pattern.

Shown is a setup comprising a shutter (112), a beam expander (114) (or an optical lens) expanding the beam, an iris diaphragm (115) and a mirror (113) for reflecting light towards an object (103) comprising molecules to be

immobilized. The object (103) may e.g. be a film, quartz, glass, have a metal surface or in general be any type of suitable object.

The function of the system corresponds largely to the one described in connection with Figure 2 with one single difference. After the beam of UV pulses was passed through the controlled shutter, a beam expander to expand the beam to about 4mm diameter, and an iris diaphragm and turned 90 degrees by a mirror, it was not focused onto the sample with a 25mm focal length quarts lens.

Figure 16b schematically illustrates an immobilisation system creating a using a diffraction pattern for immobilisation. Shown is a light exposure controlling system (104) receiving light from a suitable light source (not shown) as explained elsewhere before the light is subjected to an object (103) comprising molecules to be immobilized. The object (103) may e.g. be a film, have a metal surface or in general be any type of suitable object.

The light exposure controlling system (104) comprises in this particular example a shutter (112) receiving light from the light source, a beam expander (114) (or an optical lens) expanding the beam, an iris diaphragm

(115), a mirror (113) an a diffraction generating component (126) comprising in this example a razor blade (127) or the like blocking one half of the light beam, as also can see from the expanded view.

Furthermore, a controller (not shown), like one or more a personal computers, computational units or the like comprising appropriate software, is connected to and controlling the shutter (112) and can thereby control the exposure of the light of the object (103).

The light exposure controlling system (104) receives light from the light source where the light is passed through the shutter (112), through the beam expander (114), through the iris diaphragm (115) and being reflected on the mirror (113) towards the object (103) comprising molecules to be immobilized passing by the diffraction generating component (126).

The object (103) may e.g. be mounted on a 2D or 3D translation stage (117) for moving the object.

The resulting immobilised material may be as shown and explained in connection with Figures 17a - 17c.

The resolution of the immobilised protein is depending on the used optical components. With the system of Figure 16b using a particular setup and components, the diameter of the first dark ring around the central peak (from the center of the the top to the center of the first dark ring in the circular part of the diffraction pattern) can be estimated to be roughly 1 micrometer, which fits exactly with the expected value of !4 micrometer for the radius being half the diameter. The expected value can be derived as the following. The diffraction limited resolution of the setup is given' by d = lambda * 0.61 / NA, where lambda is the used wavelength (280 nm) for the light source and NA is the numerical aperture of the focusing lens. The used lens, in this particular , example, has a NA of 1/3 which yields d = 512 nm, i.e. just about half a micron, i.e. the above estimated radius.

As can readily be seen from the following images, the observed pattern of immobilised proteins reproduces the diffraction pattern of light used to induce molecular immobilisation. The flexibility of this technology allows creating basically any pattern of molecules, with micrometer resolution, thus being of relevance for present and future nano-technological bioelectronics applications. Furthermore, as the number of immobilised proteins is correlated with the light intensity, this technology allows mapping electric field intensities present on a surface.

Figures 17a - 17c schematically illustrate measured fluorescent emission from immobilised material that has been immobilised by a system such as the one shown in Figure 16b.

Shown in Figure 17a is the measured fluorescence of immobilised material done according to Figure 16b.

For these images the protein was immobilised according to a diffraction pattern. The used protein is Bovise Serum Albumine BSA labelled with FITC (fluorescent probe) where the ratio dye/protein was 2 (2 molecules of FITC per molecule of BSA) and with a concentration of 50 mM)

As can be seen a very distinct diffraction pattern has been obtained, which may be used e.g. for secure labelling, marking, etc. as explained elsewhere or for other uses. Varying certain parameters can ensure that the immobilised material is immobilised according to a unique pattern.

Figure 17b illustrates a horizontal fluorescence intensity profile along a line of Figure 17a and Figure 17c illustrates a 3D linear segment of Figure 17a along with a corresponding intensity map.

Figure 18 schematically illustrate another example of measured fluorescent emission from immobilised material that has been immobilised by a system such as the one shown in Figure 16b. Here a 4x4 array produced by the system of Figure 16b is shown. As can be seen four distinct patterns are obtained each corresponding to the pattern shown in Figure 17a.

Figures 19a and 19b schematically illustrate data corresponding to Figure 18 but where high frequency noise has been removed. Figure 19a corresponds to Figure 18 while Figure 19b illustrate the details of a single spot or immobilised material immobilised according to a diffraction pattern.

Figures 20a and 20b schematically illustrate a 3D view of the data of Figure 19b and a line along which a horizontal intensity profile is shown. The horizontal profile is shown in Figure 20b.

Figures 21a and 21 b schematically illustrate the data of Figure 19b and a line along which a vertical intensity profile is shown. The vertical profile is shown in Figure 21 b.

Figures 22a - 22c schematically illustrate measured fluorescent emission from immobilised material that has been immobilised according to a diffraction pattern. Figure 22a shows the measured fluorescence of material

immobilised according to a specific diffraction pattern. As can be seen the material reproduces the diffraction pattern. Figure 22b shows a segment of Figure 22a and a line along which Figure 22c illustrate the profile.

Figure 23a illustrates an image of immobilized material formed from a bitmap file. Such images can be obtained by controlling the exposure of the light source(s) appropriately, e.g. by moving the light source(s), by moving the object comprising the molecules to be immobilized, by controlling where the light emitted from the light source hits the object comprising the molecules to be immobilized (e.g. by having an appropriate light exposure controlling system) and combinations thereof.

This specific exemplary image has been done by reading a bitmap file where the controller for each pixel has made an immobilized spot of approximately 20 micrometers and the entire image is 1 mm across. If the spot is focused to be e.g. 5 times smaller then the image will be about 200 micrometer across. Each pixel may also be made by immobilizing several spots.

Such markings could e.g. be used for tagging objects, e.g. bio-arrays, for easy identification, for engraving various patterns, for secure watermarking, secure labelling or marking, etc. Colour could also be obtained by immobilizing e.g. three different molecules emitting light in the three different primary colours red, green, and blue or according to another colour-system.

The entire image could be immobilized (for one colour) in one go if the above described DMDs are used where a micro-mirror (or a group of micro-mirrors) simply should be 'on' for each pixel that is 'on ¹ . A colour image could be immobilized in e.g. three immobilizations procedures, e.g. one for each primary colour.

Figure 23b illustrates the image of immobilized material formed from a bitmap file of Figure 23a with an expanded section where additional details can be seen. The area of the small dots making each letter or pattern is equivalent to the area of the focus beam of light.

Figure 24 schematically illustrates a 3D contour plot of 5x5 immobilized geometrical shapes, which e.g. can be used to test capabilities of the system and software package.

Figure 25 schematically illustrates identification of spots in the array of Figure 24. Shown is a 2D plot of the same array after appropriate software has been used to identify the spots in the array. Notice that even complex shapes like the doughnut shaped spots in the left column of the array can be identified correctly. This is an important step for correct image sectioning when analyzing microarrays (i.e. to quantify the intensity of the signal from each spot in the array, one needs to first correctly identify the spots in the array).

Figure 26 schematically illustrates another example of measured fluorescent emission from immobilised material that has been immobilised based on a bitmap file. Shown is a drawing of a DNA double helix printed with labeled cutinase on a thiol derivatized glass slide using the UV assisted immobilization technique. The bitmap used for the image consists of 43x81 pixels and the distance between pixels were 15 μm, with a total image size of 645μm x 1215μm. In principle, any bitmap can be immobilized this way as long as the total size is within the scan range of the instrument that moves the sample (or the beam).

Figure 27 illustrates another image of immobilized material formed from a bitmap file. Illustrated is that patterns of immobilized molecules on the surface are not restricted to conventional array formats. Any pattern that can be created by the UV illumination source (or other sources) can be immobilised onto the derivatised surface. In the Figure, a logo has been written with the enzyme cutinase onto a derivatised quartz surface. The image covers an area of 1 mm2. What is observed is the fluorescence from the biomolecules immobilised with light.

Figure 28 illustrates the use of a dedicated microarray analysis package. The dedicated microarray analysis package, allows the user to identify the array and carefully processed it, with the aim of obtaining the most precise information with the highest possible S/N. Among the processing steps used, is an interactive base plane correction. Following this step, the array image is

segmented into two images one containing only the array spots, and one containing only the background behind the array. A threshold value has to be selected for making this possible. Based on these two images the S/N level obtained per spot can be calculated. For each spot, the following parameters are extracted: max intensity, mean intensity, integrated intensity, median intensity, area of spot, variance across spot, and the elliptic long and short axis (a bad beam profile or bad focusing will make the spots deviate from perfect circular spots).

Figure 29 illustrate a probability plot allowing for a semi-quantitatively evaluation if the array spot property distribution is consistent with e.g. the normal distribution as is shown in the figure. If the observed points follow the curve, the normal distribution is a valid model - if the tails of the distribution diverts significantly from the ideal normal distribution, another model should be considered.

Figure 30a - d illustrate details of a 10x10 array of labeled cutinase created using UV-light induced immobilization. Each spot in the array consists of 3x3 spots separated by 20 μm, which corresponds to the FWHM of the focused UV laser beam in the focal plane. The distance between the centers of two neighboring squares is 120 μm. The protein immobilized was cutinase labeled with Alexa Fluor 488, concentration 1 μM.

Figure 31 discloses aspects of immobilising on wet or dry film. The minimum size of the spots of molecules we can immobilize will in principle only be limited by the diffraction limit of the light, but in reality, there are several other factors to consider before one can attempt to reach this limit. For sensor applications there is no reason to focus down to smaller spots sizes than the spatial resolution of the system to be used for reading the sensor arrays. In addition, to the focussing of the UV beam and the resolution of the detector, there is one more important factor that one has to consider. Differences in the density of immobilized molecules are observed, dependent on whether we immobilize on a wet or dry film, as illustrated in Figure 31. A 5x5 array of immobilized Fab fragments with 250 μm pitch. The spots in the array were written one column at a time and the delay between writing the five columns has been chosen such that the left column was written right after the film was

applied and the column to the right was written when the film had just dried out. There is a clear increase in signal from the left to the right.

Figure 32 illustrates another example of immobilised material immobilised based on a bitmap. As shown, in addition to creating regular arrays of spots, we can also immobilize molecules in more complicated patterns. Creating a bitmap with the pattern and we move the focal spot though the bitmap pixel by pixel, thus exposing each pixel for a time proportional to the pixel value. Figure 32, shows an image of the a logo written with labeled cutinase immobilized onto a thiol derivatized glass slide. The bitmap used for the image consists of 50x50 pixels and the distance between pixels were 20 μm, with a total image size of 1 mm2. The image appears less sharp at the top than at the bottom due to the drying effect. One can avoid this by letting the film dry before exposing it to UV light.

Figures 33a and 33b illustrate a large array that is 100 micrometer x 100 micrometer with a high density. The distance between spots is 10 micrometer. The peaks full width half maximum is 2.5 micrometer. Figure 33b shows a small 3D image that is a zoom into the first array of Figure 33a.

To summarize the following is achieved. Diffraction limited molecular immobilisation is achieved, i.e., our resolution is only limited by the limits of optics. There is not limitation to immobilising molecules according to conventional patterns like microarrays and molecules can now be immobilised on a surface according to any arbitrary pattern, including immobilisation of biomolecules according to diffraction patterns of light. The observed pattern of immobilised proteins reproduces the diffraction pattern of light used to induce molecular immobilisation. The flexibility of this new technology allows creating any patterns of molecules, with micrometer resolution, thus being of relevance for present and future nanotechnological applications. Molecular immobilisation with a diffraction limited resolution is achieved. Furthermore, since the number of immobilised proteins is correlated with the light intensity, this technology allows us to map electric field intensities present on a surface.

This technology is ideal for the creation of protein/DNA microarrays and can potentially replace present micro-dispensing arraying technologies and is ideal as a molecular imprinting technology. This is obtained without the need for humidity chambers.

Furthermore, high density sensor arrays is obtainable e.g. as shown in Figure 33a and 33b.

This new technology produces photonics based microarray sensing technology and watermarking and has potential for biomedical, bioelectronic, surface chemistry, security markers production, nanotechnology and therapeutical applications and for for present and future bioelectronics developments (coupling biomolecules into electronic circuits).

In the claims, any reference signs placed between parentheses shall not be constructed as limiting the claim. The word "comprising" does not exclude the presence of elements or steps other than those listed in a claim. The word "a" or "an" preceding an element does not exclude the presence of a plurality of such elements.

The invention can be implemented by means of hardware comprising several distinct elements, and by means of a suitably programmed computer. In the device claim enumerating several means, several of these means can be embodied by one and the same item of hardware. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage.