WO2017103007A1

WO2017103007A1 - Method for activating click reactions through laser induced forward transfer of molecules

Info

Publication number: WO2017103007A1
Application number: PCT/EP2016/081292
Authority: WO
Inventors: Ioanna Zergioti; Maria MASSAOUTI; Marianneza CHATZIPETROU; Anke Kristin SCHÜTZ-TRILLING; Luc Maria WILHELMUS SCHERES; Maarten Marinus JOHANNES SMULDERS; Johannes TEUNIS ZUILHOF
Original assignee: Institute Of Communication And Computer Systems (Iccs)- National Technical University Of Athens (Ntua)
Priority date: 2015-12-16
Filing date: 2016-12-15
Publication date: 2017-06-22
Also published as: GB2545443A; GB201522165D0

Abstract

The present invention involves a method and a device for transferring and activating click reaction reagents onto substrates by means of laser induced transfer of molecules onto surfaces. The method comprises the use of laser pulses for transferring molecules from a donor substrate and for activating where the molecules are capable of receiving energy and be covalently immobilized onto a receiving substrate through "click"chemistry reaction. The method and the device are of particular utility in producing biological and biochemical assay such as biosensors and microarray.

Description

METHOD FOR ACTIVATING CLICK REACTIONS THROUGH LASER INDUCED FORWARD TRANSFER OF MOLECULES.

FIELD OF THE INVENTION

[0001.] The present invention relates to a method of transferring and activating, in one single step, molecular "click" reaction reagents onto substrates for achieving site-specific immobilization of molecules/biomolecules onto substrates. More specifically, the described matter is directed to a method for immobilizing a chemical structure on a given pattern onto a section of the surface of a substrate, employing "click" reactions which are spatially and temporally controllable activated through direct laser induced forward transfer.

[0002.] This application claims the benefit of British (GB) Patent Application 1522165.8 filed 16 December 2015. BACKGROUND OF THE INVENTION

[0003.] The controlled immobilization of active molecules on surfaces and the rapid and direct creation of patterned arrays of molecules of controlled size and dimensions are an essential step in many fields and various technologies. For example, in biotechnology, such controllable immobilization of biomolecules is necessary for the development of certain biosensors or high-throughput analysis systems, which require spatially controlled attachment of ligands in the form of simple spots or more complicated micro-arrays. Other similar examples include surface patterning for some biological assays, micro-chemistry, drug delivery systems, protein binding and enzymatic analysis. The many requirements for an immobilization strategy include achieving stability of attached molecules, specificity of the immobilization chemistry, maintaining materials properties while having high resolution, control on size, spatial accuracy and simplicity of the process. During the last two decades, a large number of approaches has been developed for the immobilization of different biomolecular probes.

[0004.] Different immobilization strategies have been developed for attaching active molecules/biomolecules on surfaces, among which an elegant approach to immobilize the molecules with minimal loss of activity is to utilize "click" reactions, as ideal approach for coupling two molecules irreversibly and under mild conditions. The definition of "click" reactions was first introduced in 2001 by Sharpless and co-workers (H. C. Kolb et al., Angewandte Chemie International Edition 2001 : 2004-2021 ), stating as "click", reactions which are modular, wide in scope, give very high yields, generate only inoffensive by-products that can be removed by nonchromatographic methods, stereospecific and occurring under simple reaction conditions. During the past years, "click" reactions were ubiquitous and have been proven highly efficient in an ever increasing number of synthetic methodologies and applications, including biorthogonal conjugation (C. S. Mckay et al., Chemistry & Biology 2014: 1075-1 101 , A. H. El-Sagheer et al., Chemical Society Reviews 2010: 1388-1405), tailored material fabrication and polymer synthesis (W. Xi et al., Adv. Funct. Mater. 2014: 2572-2590) functionalizing surfaces and immobilizing molecules on surfaces (J. Escorihuela et al. Adv. Mat. Interfaces. 2015). However, for many applications, especially for applications featuring surface patterning, the specific features of "click" reactions are not sufficient, if not combined with processes that facilitate the complete or partial spatial and temporal control of the reaction.

[0005.] In this aspect, new "click" reactions have been developed, which can be photo-activated at specific locations and time, by focusing photons onto a given area and by varying exposure time, wavelength, and intensity as needed. This level of control is not feasible with the conventional thermal activation approaches. Such light-induced "click" reactions include photo-initiated thiol- ene/thiol-yne coupling (C. E. Hoyle et al., Chemical Society Reviews 2010: 1355- 1387) the photo-induced 1 ,3-dipolar cycloaddition reaction of alkenes and nitrile imines ( Y. Wang et al., Angewandte Chemie 2009: 5434-5437), strain-promoted cycloaddition reactions of photochemically generated cycloalkynes and azides (S. V. Orski et al., Journal of the American Chemical Society 2010: 1 1024- 1 1026), photo-induced ester formation reactions of benzodioxinones with alcohols (V. Kumbaraci et al., Macromolecules 2006: 6031 -6035) and photo- induced Diels-Alder reactions (T. Pauloehrl et al., Angewandte Chemie International Edition 2012: 1071 -1074).

[0006.] An approach of light-induced creation of covalent surface patterns of proteins and small molecules, has been explored by P. Jonkheijm et al (P. Jonkheijm et al., Angewandte Chemie International Edition 2008: 4421 -4424), who immobilized alkene-functionalized biomolecules, such as biotin, on thiol- modified glass substrates. The local activation of the reaction is achieved by covering the thiol-modified surfaces with solution of the biotin-alkene conjugate and irradiation with a laser or through a photomask. [0007.] Similar approach is applied by J. Escorihuela et al. (J. Escorihuela et al., Bioconjugate Chemistry 2014: 618-627) for immobilizing DNA oligonucleotides on silicon-based substrates using a thiol-ene click reaction leading to the site-specific creation of DNA microarrays for the detection bacterial Escherichia coli. The covalent site-specific immobilization of the oligonucleotides on functionalized surfaces is achieved by dropcasting the biomolecules onto the surface and applying using masked photolithography (Continuous wave (cw) laser UV light).

[0008.] Wasserberg et al. present microcontact printing patterning of alkene- terminated perylenes onto thiolated surfaces using light-induced TEC reaction (D. Wasserberg et al., Journal of Materials Chemistry 2012: 16606-16610). For that, a perylene bisimide with an alkene group, has been prepared and was inked on polydimethylsiloxane (PDMS) stamps. By placing the stamps on the thiol-terminated silicon substrate and photo-inducing the TEC reaction through irradiation (365 nm) for 20 min, patterned surfaces have been constructed.

[0009.] Bowman et al., in US Patent Application Publication No. 2013/0323642 A1 , presents a method of immobilizing a chemical structure in a given pattern onto a section of the surface of a solid substrate, by photochemically controlling the reduction of Cu(ll) into Cu(l), and subsequently controlling the catalysis of the azide-alkyne cycloaddition (CuAAC) "click" reaction.

[0010.] In another approach, Popik et al., US Patent No. 2010/0210854 A1 discuss a method for linking two molecules triggered by the photochemical generation of cyclic alkynes (e.g., cyclooctynes) from corresponding cyclopropenones, without the need of using a catalyst. An example of this approach is implemented for the surface immobilization of cyclopropenones that undergo decarbonylation to yield dibenzocyclooctynes for catalyst-free cycloaddition with azides (S. V. Orski et al., Journal of the American Chemical Society 2010: 1 1024-1 1026).

[0011.] Alternatively to light-induced approaches for activating in a controllable way "click" reactions, chemomechanical and mechanical strategies have been also applied in the recent years, including dip-pen lithography (D. A. Long et al., Advanced Materials 2007: 4471 -4473, H.-Y. Chen et al., Journal of the American Chemical Society 2010: 18023-18025), noncontact piezoelectric plotting (S. Oberhansl et al., Small 2012: 541 -545) and microcontact printing (C. Wendeln et al., Chemical Science 2012: 2479-2484, H. Nandivada et al., Angewandte Chemie International Edition 2006: 3360-3363).

[0012.] Rozkiewicz et al. (D. I. Rozkiewicz et al., Angewandte Chemie International Edition 2006: 5292-5296) present that a Huisgen 1 ,3-dipolar cycloaddition reaction, a representative example of the Sharpless "click" chemistry, can be induced without the need of any catalyst and at a relatively fast reaction time (20 min), by microcontact printing (pCP) of acetylenes onto azido-terminated SAMs on silicon oxide substrates. Using a similar approach they prepare highly selective microarrays on silicon substrates for the detection of single-base-pair mismatches (I. Singh et al., Journal of the American Chemical Society 2013: 3449-3457). [0013.] Common for most of the described activation approaches of click reactions is the use of two or more steps for transferring and activating with spatial and temporal control the click reaction reagents, resulting in a time- consuming procedure. Photochemical lithography uses masked or focused light to irradiate a specific area on a surface where the molecules/biomolecules are pipetted or dropcasted. Dip-pen lithography is capable of direct and indirect writing of biological materials onto suitable surfaces to form highly structured arrays, but is limited to the creation of small images and features. Microcontact printing uses inexpensive elastomeric stamps, which are "inked" with the "click" reagents and as they are brought in conformal contact with the desirable substrate, images of the "inked" molecules are rapidly reproduced. However, the master for the stamps must be fabricated using another technique that is capable of directly writing the master, while in some cases, the vertical pressure applied on the stamp that is needed during printing for energetically favour the "click" reaction activation may deform the stamp, resulting to distorted printed features. Furthermore, not all the approaches are applicable for activating different "click" reactions.

[0014.] In view of the above, there is a need for more generic methods for activating "click" reactions that can be spatially and temporally controlled either through light and/or mechanical forces and where the structural and functional properties of the coupled molecules/biomolecules are preserved, the coupling can be spatially controlled to specific activated regions while the requirements of an activation method should include high spatial resolution, control of deposition size, maintenance of materials properties, special accuracy, speed and simplicity.

SUMMARY OF THE INVENTION

[0015.] The present invention provides an accurate method for the direct transfer of molecular "click" chemistry reagents onto substrates and activating in one single step their coupling/reaction with the surface, with spatial and temporal control.

[0016.] Generally, it is an object of the invention to provide a direct method for transferring molecules on specific sites and immobilizing them through photo- induced and/or pressure-induced "click" reaction activation processes using laser induced forward transfer.

[0017.] It is a further object of the present invention to provide a method for the direct immobilization of micro-dimensioned molecular patterns on substrate surfaces at high spatial resolution, wherein patterns include a plurality of single spaced-apart features forming arrays.

[0018.] It is a further object of the present invention to provide a method for creating biological and chemical sensing devices by direct immobilization of biomolecules onto sensing elements.

[0019.] The above and other objects are achieved by the present invention. Accordingly, the invention provides a method for transferring molecules/biomolecules modified with chemical moieties capable of participating in "click" reactions and activating their reaction/coupling onto a section of the surface of a substrate, in a single step process. The method comprises the step of: (i) providing a target substrate, comprising a laser-transparent target support and a transfer material comprising a (bio-)material with chemical moieties (reagent A) capable to "click" react with appropriate compounds upon photo- induced or chemomechanical processes; wherein the transfer material is provided in a liquid coating upon one surface of a laser-transparent target support. The target support may be covered on one surface by an assistant light- absorbing layer. The method further comprises the step of ii): providing a receiving substrate wherein at least a portion of the surface of the substrate is derivatized with a given compound (reagent B) capable of "click" reacting with the said reagent A of the transfer material upon photochemical or chemomechanical activation; wherein the receiving substrate is placed opposite to the source of laser energy. The method further comprises the step of iii): providing a laser source with a given wavelength and at a given energy whereby the said liquid coating of the transfer material can be energized by the absorbing portion of the said laser energy; positioning the target substrate at a defined location to the laser source and irradiating a portion of the said coating of the transfer material to the laser energy; wherein the laser beam has sufficient energy to eject/release and create high-speed droplets of the said liquid transfer material; portion of the said transfer material is transferred upon a defined section of the said surface of the receiving substrate and immobilization of the said transfer material onto the said section surface is achieved through the direct or indirect activation of the "click" coupling of the said reagent A of the transfer material with the said reagent B of the said surface of the receiving substrate. [0020.] In one embodiment, the said reagent A of the transfer material is capable of receiving energy directly or indirectly by irradiation from the laser source wherein at least a part of received laser energy upon release to the said material is absorbed by the said reagent A causing its photochemical reaction with the said reagent B of the receiving substrate upon the transfer and deposition of the transfer material onto the surface of the said receiving substrate.

[0021.] In this way, a surface "click" reaction can be activated with high precision and spatial and temporal control, since the spatial dimension of the smallest individually activated area is substantially defined by the size of the droplets of the said transferred material deposited.

[0022.] In another embodiment, the reagents B of the receiving substrate are capable of receiving energy directly or indirectly by irradiation from the said laser source; wherein at least a part of laser energy is used to release and transfer the said transfer material while a portion of the said laser energy is transmitted through the transfer substrate, irradiating a defined spatial site of the said surface, energizing the reagents B and thus creating reactive reagents B^* at the said site of the surface capable to bind the reagents A of the said transfer material upon the deposition of the said material onto the said surface of the receiving substrate.

[0023.] In some embodiments, the reaction activation and coupling of the said reagent A of the transfer material and reagent B of the surface of the receiving surface is achieved by chemomechanical processes due the high speed droplets deposition of the said transfer material onto the surface of the receiving substrate and the pressure-induced activation of the coupling between the reagent A and reagent B.

[0024.] In this way, transfer of a material/biomaterial and its coupling with a receiving substrate through "click" reaction occurs in one step process of transferring and activating of the "click" reagents reaction, with high spatial and temporal precision, fast and without damage. Typical laser energy densities range from about 10 mJ/cm² to 10000 mJ/ cm².

[0025.] One advantage of the present invention is the simplicity of the method wherein materials/biomaterials absorb the energy of the laser source and can be deposited on the substrates without the assistance of any transferring matrix material. Compared to the lithographic methods this method is a "clean", one- step process and it is not limited to oligomer structures only.

[0026.] The method and the apparatus are of particular utility in producing biological and biochemical assay systems such as biosensors and microarrays.

[0027.] Another advantage of the present invention is the versatility of the method that can be easily adapted and used for transferring, depositing with high spatial control a wide variety of "click" reaction reagents onto various receiving substrates, and activating their coupling in a single step process upon deposition. BRIEF DESCRIPTION OF THE DRAWINGS

[0028.] FIG. 1 is a schematic view of an apparatus according to an embodiment of the present invention.

[0029.] FIG. 2a and 2b are schematic representatives of two different types of target substrates.

[0030.] FIG. 3a and 3b and FIG. 3c are schematically illustrating the transfer molecules M, modified with chemical moieties A, which upon irradiation are deposited and immobilized through photochemical processes onto a defined spatial site of a surface of a receiving substrate.

[0031.] FIG. 4 is an illustrative immobilization of biomaterial using the invention for transferring thiol-modified biomaterials and induce their photochemical coupling with alkene-terminate surfaces.

DETAILED DESCRIPTION OF THE INVENTION

[0032.] The invention provides a method for spatial and temporal control of surface "click" reactions activation and site-specific immobilization of molecules/bionnolecules onto a section of a surface of a substrate. The transfer of the materials and the activation is achieved by employing laser light for direct, high speed transferring of the materials onto substrates.

[0033.] FIG. 1 schematically illustrates the apparatus used in the present invention. The apparatus includes: a laser source 10 that produces a laser beam 12; an optical controlling system 20 comprising a mirror 24 and an objective lens 25 for directing the laser beam towards the target substrate 13 and projecting the said beam onto a transfer material 16 coated on the upper surface of the laser transparent target support 14; a receiving substrate 18; an imaging system 26 comprising a CCD camera and a microscope imaging system can also be included for monitoring the deposition process. Optionally, the optical controlling system 20 may comprise an attenuator 21 for controlling and adjusting the energy of the laser beam 12; a beam homogenizer 22 for shaping the laser beam 12; and an aperture 23 for modulating the size of the laser beam 12. FIG. 2 schematically illustrates in detail the transfer substrate 13. The target substrate comprises a laser transparent target support 14 and a transfer material 16 comprising a molecular/biomolecular material with chemical moieties (reagent A) capable to "click" react with the compounds (reagent B) of the surface of the receiving substrate. Typically the said material to be transferred is provided in a liquid coating upon one surface of a laser transparent target support 14 facing the receiving substrate 18, as it is shown in FIG. 2a. The transfer of the said material to the facing surface of the receiving substrate 18 occurs due to photomechanical and/or photothermal phenomena arising from laser energy absorption by the said transfer material. In some embodiments, the said surface of the laser transparent target support 14 facing the receiving substrate 18 can be coated with a light absorbing layer 15, as it is shown in FIG. 2b, whereon the transfer material 16 is spread. In these embodiments, the transfer of the said material 16 to the receiving substrate 18 occurs due to photomechanical and/or photothermal phenomena arising from laser energy absorption by the light absorption layer 15. [0034.] The method of the invention for transfer and activation of molecular "click" reagents in a single step process, and in a spatially and temporally controllable way, comprises the steps of: i) providing the source of laser 10 that produces a laser beam 12; ii) providing the target substrate, comprising the laser transparent target support 14 and the transfer material 16; iii) providing the receiving substrate 18 iv) positioning the said transfer material 16 onto the laser transparent transfer support 14; v) positioning the receiving substrate 18; and vi) irradiating a defined spatial site of the said transfer material by projecting the laser beam 12 onto the said transfer material; and in some embodiments vii) repeating. In the step of irradiating the transfer material or the light absorbing layer with the laser beam, a large portion of the laser energy is absorbed inducing the release of the transfer material from the transfer support due to photomechanical and/or photothermal phenomena, resulting to high speed laser induced forward transferred material droplets. The residue portion of the energy of the irradiating laser beam can be either absorbed by the transfer material or transmitted through the released material, irradiating part of the defined spatial site of the surface of the receiving substrate towards which the said transfer material will be deposited.

[0035.] In some embodiments, where the activation of the "click" reaction and the binding of the said material to be transferred to a section of the surface of the receiving substrate is achieved though photochemical processes, the laser induce transfer provides a method for transferring the said transfer material and activating its reaction with the said surface in a single step process. This is done by comprising: [0036.] (a) a material modified with chemical moieties (reagent A, e.g. a thiol- group), able to absorb portion of laser energy from the projected laser beam 12 and due to photomechanical and/or photothermal phenomena arising from laser energy absorption by the said transfer material, to be ejected/released from the transfer support and transferred towards the said surface of the receiving substrate; wherein at least a portion of the residue laser energy transmitted through the target support is absorbed directly or indirectly by the reagents A and create appropriate reactive elements A^* (e.g. thiol-radicals) capable to "click" react with the compounds (reagents B, e.g. alkenes/-ynes) of the surface of the said receiving substrate.

[0037.] (b) a receiving substrate wherein at least a portion of the surface of the substrate is derivatized with a given compound (reagent B, e.g. alkenes/-ynes) capable of binding the said reactive elements A^* of the transfer material.

[0038.] FIG. 3 is a schematic illustration of the effect of exposing to the laser beam, a transfer material 16 comprising a material M modified with chemical moieties (reagent A) wherein after irradiation (FIG. 3b) the said material is released from the laser transparent support 14 while the reagent A is transformed, by absorbing the residue laser energy, to a reactive element A^* and upon transfer and deposition of the said material to a defined spatial site of the surface of the receiving substrate is immobilized onto the said surface through the photochemical reaction of click reagents A^* and B (FIG. 3c).

[0039.] In some embodiments, the reagents A may be compounds of the surface of the said receiving substrate and react upon absorption of the said residue laser energy, transmitted through the target support, with the said reagents B of the transferred material.

[0040.] In other embodiments where the molecules of the said transfer material cannot be exposed directly to laser energy, the transfer support 14 is coated with a light absorbing layer 15, where upon the said transfer material 16 is deposited. The photon energized interlayer causes the transfer of the material upon being irradiated with the laser energy. At the same time it permits a small portion of the laser energy to be transmitted through it and absorbed by the said reagent A, photo-activating the binding of the transfer material 16 with the surface of the receiving substrate 18 upon its transfer and deposition on the said surface. The interlayer can be made of any inorganic or organic material that absorbs the laser energy. A non-limited example of a suitable range for the thickness of the interlayer may be 1 nm to 10 μιτι.

[0041.] Alternatively, in cases where the transfer material can not directly absorb the transfer laser energy, linker molecules can be included to the transfer material 16. The linker molecules are able to absorb the energy from the laser beam 12 and release at least some of this energy to cause the photomechanical and/or photothermal ejection/release of the said material while transfer a portion of the absorbed laser energy to the chemical moieties of the said material, enabling its photochemical reaction with the compounds of the surface of the said receiving substrate upon deposition.

[0042.] Another aspect of the method is that it can be used to transfer "click" reagents and activate their reaction to a defined spatial site of the surface of a receiving substrate through chemomechanical induced processes. This can be done after the step of irradiation of the transfer material and its release and deposition in high speed onto the said site of the surface. Upon deposition onto the surface of the receiving substrate, the pressures arising by laser induced transfer may be, by way of a non-limited example in the range of about 0.01 MPa to 20 MPa. This range is comparable to the mechanical pressures employed at conventional methods such as microcontact printing used for the activation of click reactions.

[0043.] An important asset of the method is that it can be applied to a variety of materials, including, but not limited to, polymers, polypeptides and proteins such as enzymes, antibodies, antigens, protein A, hormones, receptors, lectins, avidin, oligopeptides, nucleic acids such as DNA, RNA, oligonucleotides, polysaccharides, glycoproteins, proteoglycans, glycolipids, lipids, obtained from either biological sources or by chemical synthesis, derivatives thereof, and artificial counterparts such as the peptide nucleic acids, capable to be modified with reagents A (or B). Prior to the step of irradiation, the target material is spread onto the laser transparent support into a form of liquid solution coating. The spread can be achieved by applying various techniques, including but not limited to spin coating, spray coating, drop casting, dispensing, roller coating, and doctor blading. In embodiment where the transfer material is biomaterial, the liquid solution (biomaterial solution) can be made with a variety of compounds selected from the following functional groups: [0044.] Buffers such as carbonate, formate, acetate, citrate, phosphate, borate, dimethylarsinate, ethanolamine, triethanolamine, trimethylamine, triethylamine, imidazole, histidine, pyridine, collidine tris(hydroxymethyl)aminomethane, N-2- hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 2-(N-morpholino) ethane- sulphonic acid, 1 ,3-bis[tris (hydroxymethyl) methylamino] propane, 3-(N- morpholino) - 2- hydroxypropane-sulfonic acid, 1 ,4-piperazinediethanesulfonic acid, at concentrations of 10-100 mM, to maintain the pH of solution. Detergents such as dodecyl sultfate, lauroyl sarcosine, deoxycholate, subsalicylate, cetyldimethylethylammonium bromide, 3-[3- cholamidopropyl9- dimethylammonio]-1 -propane sulfonate, alkyl-glucosides, alkyl-thioglucosides, alkyl-maltosides, alkyl-thiomaltosides, polyoxyethylene esters, polyoxyethylene ethers, polyoxyethelenesorbitan esters, alkyl-N-hydroxy-ethylglucamides, at 0.1 - 10.0 % weight per volume, to solubilize insoluble biomaterials. Chaotropic agents such trichloroacetic acid, perchlorate, urea, guanidine hydrochloride, guanidine thiocyanate, formamide, glyoxal, used at concentrations 0.5-5.0 M, when the biomaterials are intended denatured. Reductants such as dithiothreitol, dithioerythritol, 2-mercaptoethanol, at a concentration range 5.0-50.0 mM, to prevent oxidation of thiols. Chelating agents such as ethylenediamine tetraacetic acid, ethyleneglycobis (-aminoethyl)ether tetraacetic, at 1 .0-20.0 mM acid to bind unwanted bivalent metals. Stabilizers such as glycerol and other polyols, glucose, N-acetyl glucosamine, sorbitol, ascorbic acid, sucrose, trehalose, at concentration range 5-20 % weight per volume, to maintain biological activity and conformation of the biomaterials. Polymers able to imbibe or retain water, generally referred to as hydrogels, such as agarose, alginates, dextran, poly(ethyleneglycol), polyethylenimine, polyacrylic acid, polyacrylamide, poly(1 - vinyl-2-pyrrolidon), poly (hydroxyethyl - methacrylate), poly {[tris(hydroxymethyl) - methyl]acrylate}W. Xi et al., Advanced Functional Materials 2014: 2572-2590, usually at a concentration range 1 -10% weight per volume, to provide an aqueous microenvironment to biomolecules. Inorganic salts such as sodium chloride, ammonium sulfate, and organic compounds and solvents such as dimethylsulphoxide, to modulate the ionic strength of the solution and the solubility of the biopolymers. Specific enzyme inhibitors such as (4- amidinophenyl)methanesulfonyl fluoride, leupeptin,3,4-dichloroisocoumarin, N- [N-(L-3-trans-carboxirane-2-carbonyl) -I- leucyl]-agmatine, phenylmethylsulfonyl fluoride, N-ethylmaleimide, benzamidine for proteases, ethylenediamine tetraacetic acid for deoxynucleases, typically at concentrations 0.1 -1 .0 mM, to protect enzyme degradable biomaterials. Preservatives such as sodium azide, methylisothiazone, 2- [(ethylmercurio)thio] benzoic acid, bromonitrodioxane, at concentrations 0.01 -0.1 % weight per volume, to protect biodegradable biomaterials. Dyes such as 1 -anilinonaphthalene-8-sulfonic acid, 3-hydroxy-4-[2- sulfo-4-(4-sulfophenylazo) phenylazo-2,7-naphthalenedisulfonic acid, 2,7- diamino-10-ethyl- phenyl-phenanthridinium bromide, to facilitate the monitoring of the coating and deposition process.

[0045.] Typically, the above compounds are selected with regard to the function they serve and their compatibility with, the transferring material, the surface of the receiving substrate and the click reagents participating to the reaction. [0046.] The target support should be of high optical and surface quality and composed of a material that does not absorb at the wavelength in use. Materials such as fused silica, sapphire, magnesium and calcium fluoride can be used in a wide range of wavelengths from UV to IR.

[0047.] Generally, the efficiency of the laser light coupling into the materials depends on the materials optical properties, the wavelength, the time duration of the incident light and in some embodiments on the thickness of the light absorbing layer. Particularly, the laser source wavelength is selected with regard to the absorption spectrum of the molecular/biomolecule material to be transferred and in some embodiments with respect to the photon energy needed to photo-initiate "click" reactions which is typically in the UV-visible spectral range.

[0048.] A variety of pulsed laser sources are available in the full spectral range from UV to IR and can be utilized in this method including, but not limited to, excimer lasers at wavelengths 248 nm (KrF) and 308 nm (XeCI) with pulse duration down to 0.5 ps and pulse repetition frequency up to 10 Hz, DPSS lasers at wavelengths 1064 nm, 800 nm, 532 nm, 400 nm and 266 nm, with pulse duration up to 105 ns and pulse repetition frequency up to 100 KHz, femtosecond laser systems with wavelengths up to 1070 nm, repetition rates up to 80 MHz, and pulse widths up to 900 fs and pulsed high power diode lasers at 1064 nm and 532 nm, with pulse duration of 500 ps and pulse repetition frequency up to 1 KHz. [0049.] The pulsed laser energy can be adjusted by means of an attenuator 21 in order to obtain high laser energies sufficient to transfer the material and photoactivate the reaction or induce high velocity transfer of the said material onto the surface of the receiving substrate. Typical laser fluences can range from about 10 mJ/cm² to 10000 mJ/cm². The pulsed laser beam shape and dimensions onto the target material can be adjusted by means of a variable aperture 22 of any special shape such as, rectangular or triangular or circular etc. in order to expose the material or the absorbing layer in an area so that, a precise and defined portion of the said transfer material is transferred and deposited onto a defined spatial site of the surface of the receiving substrate. The spatial resolution of the deposited material can be ranged between 1 to 1000 μιτι using an optical system to project the aperture on a large-reduction basis onto the transfer material. In some embodiments, the irradiation of the transfer material can be done directly by using the laser beam unmodified in terms of shape and size that allows for faster large area deposition and activation.

[0050.] The receiving substrate should be placed in in close proximity with the target substrate. Typically, the distance between the upper side of the transfer material and the lower side surface of the receiving substrate can be varied from 0.5 μιτι to 500 μιτι.

[0051.] The laser beam, the transfer material and the receiving substrate can be positioned in relation to each other and can be controlled and moved with respect to each other by means of translation stages and computer controlled translation stage drivers. This is a well-known technology in the field of laser micromachining, i.e. laser cutting, drilling, etc. More specifically, the laser beam is directed onto the donor substrate and irradiates the material or the absorbing layer with sufficient energy to transfer and immobilize a selected portion of the material onto the rough receiving substrate. Repeating the transfer process at different target and rough receiving substrate position i.e. pixel by pixel step and repeat operation by means of a computer and the translation stages driver results in the production of patterns such as a plurality of single spaced apart features, forming arrays, and repeat of adjacent features, forming localized coatings.

[0052.] The transfer method can be used to activate various photo-induced "click" reactions. The reagents A may include but are not limited to thiol-groups, azide, tetrazole, azirine, naphthoquinone methide, o-quinodimethane, o- nitrobenzyl acetal, benzodioxinone, perfuorophenylazide which upon absorption of UV light-visible light form reactive intermediates (such as free radicals, electronically excited states, or carbocations and others) or reactive elements A^* capable to "click" react with reagents B, such as alkynes, alkenes, cyclopropenone, vinyl ether, maleimides, dithioether, naphthoquinone methide, alkoxyamines, alcohols, alkane, upon found together.

[0053.] The present invention will be further explained with reference to the following non-limiting examples. EXAMPLE 1

Creation of microarray of aptamers onto silicon nitride substrates using thiol-ene click chemistry

[0054.] In this specific application, thiol-modified aptameric sequences are transferred and immobilized at specific spatial sites onto an alkene-terminated surface of a silicon nitride substrate.

A liquid solution of aptamers against Ochratoxin A, modified at their 5-'end with a thiol-group, was prepared by dissolving the aptamers in 10 mM Phosphate Buffer pH 7, containing 10 mM KCI and 5 mM MgC^. The liquid solution of 5 μΙ was spread onto a titanium-coated (40 nm) surface of the laser-transparent support made of quartz (25.4 mm diameter and 1 mm thick), to form a film 5 μιτι in thickness. The laser used to irradiate the light-absorbing layer of titanium was a pulsed Nd:YAG laser emitting 10 ns laser pulses at photon energy 355 nm. The laser energy density ranged from 150 mJ/cm² to 350 mJ/cm² and the laser beam spot size projected onto the titanium layer was 50x50 μιτι². The receiving substrate was functionalized so as to be terminated with alkene moieties. The liquid solution spread onto the transfer support was released by irradiating the absorbing layer of titanium quartz and transferred and deposited onto the alkene-terminated surface of the receiving substrate resulting to the immobilization of the aptamers onto a defined spatial site of 100 μιτι diameter size of the surface. The transfer process was repeated at different target and receiving substrate locations by means of the computer-controlled translation stages in order to form a microarray of aptamers. For validating the transfer and immobilization the aptamers were tagged at their 3'-end with Cy5-streptavidin and the fluorescence was observed from the array by using a Leica fluorescent microscope. Prior recording the fluorescence, shown in FIG. 4, the resulting receiving substrates with microarrays of aptamers were washed.

REFERENCES

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Claims

1 . A method for transferring and activating, in a single step, molecular click reagents onto substrates, the method comprising:

a) providing a donor substrate comprising a transfer material wherein the transfer material is a material (reagent A) with chemical moieties capable of participating in "click" reactions.

b) providing a receiving substrate wherein a given compound (reagent B) capable of forming bond with the said reagents A of the transfer material and wherein a gap exists between the receiving substrate and the donor substrate.

c) providing a laser beam source.

d) directing the laser beam through the donor substrate to the material, at a defined location, exposing a portion of a coating material to laser energy sufficient to remove, transfer, react, activate and immobilize the said portion on the receiving substrate.

2. The method according to claim 1 , wherein the material/biomaterial coating is applied on a donor substrate composed of laser transparent support.

3. The method according to claim 1 , wherein the material/biomaterial coating is applied on a donor substrate composed of laser transparent support and a radiation absorbing layer.

4. The method according to claim 1 , wherein the material/biomaterial is selected from the group consisting of polymers, proteins, polypeptides, enzymes, antibodies, antigens, nucleic acids (DNA & RNA) by chemical synthesis derivatives thereof, and artificial counterparts such as the peptide nucleic acids (PNAs), Molecular Imprinted Polymers (MIPs, Carbohydrates and others.

5. The method according to claim 1 and claim 4, wherein the materials/biomaterials are selected from a group of materials having, but not limited to thiol-groups, azide, tetrazole, azirine, naphthoquinone methide, o- quinodimethane, o-nitrobenzyl acetal, benzodioxinone, perfuorophenylazide.

6. The method according to claim 1 where the substrate is modified with alkenes, alkynes, cyclopropenone, vinyl ether, maleimides, dithioether, naphthoquinone methide, alkoxyamines, alcohols, alkane.

7. The method according to claim 1 , where the receiving substrate is the sensing area of a biosensor or a chemical sensor, or any other sensing device or a patterned surface within microstructures used to direct flow, separate or combine liquid phases or emulsion components.

8. The method according to anyone of claims 1 to 7 wherein the deposition step is further repeated at different locations on the target coating and the substrate is moved so as successive deposits of the material are at overlapping, or adjacent, or space-apart, spatial relation on the receiving surface. An array of discrete reagent regions obtainable by the method of claim 5.

9. A biochemical assay comprising a sensing region as obtained by the method of any one of the claims 1 to 8.

10. A biochemical assay comprising a matrix region as obtained by the method of any one of the claims 1 to 8.

1 1 . The method according to claim 1 , wherein the energy density of the laser is between 0.01 J/cm² and 10 J/cm²

12. A device for producing patterns of biomaterials on solid substrates comprising:

(a) A laser source,

(b) an optical delivery system,

(c) a holder for target supports,

(d) translation stage moving target holder,

(e) a receiving substrate platform,

(f) translation stage moving substrate platform,

(g) microcomputer system controlling the translation stages and the laser. The device configured to transfer and activate in a single transferring and activating, in a single step, molecular click reagents onto substrates according to the method of claims 1 -8.