IMPROVED METHODS FOR FORMING MOLECULAR IMPRINTS AND MOLECULAR IMPRINTS FORMED BY THE METHOD

[001] This application claims the benefit of US Provisional Patent Application No. 60/766,857, filed February 15, 2006, and US Provisional Patent Application No. 60/767,302, filed March 16, 2006.

[002] The present invention relates to molecular imprints and methods for forming molecular imprints. More particularly, the invention relates to methods for forming identical molecular imprints from a template made as a duplicate of a print molecule and to uses of a molecular imprint formed by the method of the invention.

BACKGROUND OF THE INVENTION

[003] Molecular imprinting is a well known process for forming polymer structures that are selective for a particular molecule, called the print molecule or analyte molecule. A typical imprinting synthesis reaction involves the introduction of the molecule to be imprinted into a solution of polymeric precursors. Polymerization reactions within the solution are then initiated and either by chemically binding the print molecule to a precursor or simply by inclusion during polymerization, a polymer matrix is formed around the print molecule. The final step of such a reaction is to remove the print molecule from the newly formed polymer matrix by washing or selective dissolution. The resulting structure is then an organic or inorganic matrix with physical imprints, i.e., complementary negative image imprints, of the print molecule that will attract print (analyte) molecules if exposed to them. Because the matrix is in fact a high fidelity mirror image of the source print molecule, there is an extraordinary degree of regio and stereo specificity creating a favorable environment for idiotypic reaction between the imprint and the print molecule. Molecular imprints have found use in such areas, among others, as sensor systems as described in US Patent No. 6,223,589 to Dickert et al., in synthesis of compounds having complementary structure to a print molecule, described in US Patent No. 6,127,154 to Mosbach et al., and for the preparation of artificial anti-idiotypic imprints described in US Patent No. 6,489,418 to Mosbach.

[004] The drawbacks to known synthetic techniques are substantial. Print molecules can be sensitive to harsh conditions, which limits the number and types of polymerization reactions that can be used for imprinting. Further, print molecules must be cultured or harvested each

[010] Major advantages of this technique, which, will become apparent as the invention is described include the ability to use the permanent template many times, reducing and possibly eliminating the need to repeatedly handle the biological sample, the ability to form the imprint of the template in a large number of materials, the ability to provide localized functionality by enabling the use of a wide variety of materials, and the ability to place a molecular template with localized functionality very precisely onto other functional structures and devices.

BRIEF DESCRIPTION OF THE DRAWINGS

[011] The present invention will be better understood by reference to the following discussion and when read in conjunction with the appropriate drawings in which like reference characters refer to like parts throughout the views, in which:

[012] Figure 1 illustrates schematically a structure formed during a method according to the invention in which a print molecule layer is deposited on a substrate, with a planarized conformal coating layer covers the print molecule layer and a carrier substrate is attached to the planarized conformal coating layer;

[013] Figure 2 illustrates a step of applying pressure to the print molecule layer to form imprints in the material on the substrate;

[014] Figure 3 illustrates a step of orienting the deposited print molecules with an electric field induced between a metal layer and the deposition solution by electrodes

[015] Figure 4 illustrates a negative image layer formed according to a method of the invention on a carrier substrate;

[016] Figure 5 illustrates a template or imprinting tool made according to the invention which contains a surface bearing an image identical to the layer of print molecules;

[017] Figure 6 illustrates a positive image template stamping molecular imprints into a polymer layer; and,

[018] Figure 7 illustrates a method for forming an imprint on a device, such as a SAWR.

DETAILED DESCRIPTION OF THE INVENTION

[019] The invention is described here in terms of preferred embodiments, structures, and

time a new imprint is to be formed, which is often difficult and time consuming. If the print molecules constitute a health hazard, as in the case of virulent pathogens, there is a need to provide isolation between the reaction and the environment when the print molecule is handled. This need for isolation significantly adds to the difficulty and, commensurately, the expense of forming the imprint.

[005] Given the potential of imprinting to greatly advance the art of chemical separation and molecular identification, there is a need to improve the techniques for molecular imprinting.

SUMMARY OF THE INVENTION

[006] The present invention addresses deficiencies in current imprinting processes by providing a method for forming multiple identical molecular imprints (complementary images of print molecules) from molecular templates (surfaces having images identical to at least a portion of the print molecule). The manufacturing complexity is thus greatly reduced, and a greater degree of chemical functionality becomes available by overcoming the requirement for in-situ polymerization to form the imprint.

[007] The present invention includes techniques to fabricate chemically sensitive surfaces and structures which broadly fall into the class of imprinted idiotypic, antiidiotypic, and/or immunoassay templates, where it is understood that an immunoassay template is one in which an imprinted negative or mirror image of a molecule has been transferred and with which the molecule may subsequently react in an idiotypic fashion. This invention will allow one who is skilled in the art to realize very useful molecular templates and will provide those skilled in the art with a novel array of additional functionalities and devices which can now be realized.

[008] The invention provides a method by which an imprinting tool is formed as the direct and permanent positive image(s) of the print molecule. This duplicate print molecule tool, or a copy or copies of the duplicate template tool, replaces the print molecule for the subsequent formation of molecular imprints.

[009] According to the invention, an imprint of a print molecule is formed on a surface of a substrate by any suitable means. The imprinted surface is conformally coated. The imprinted substrate and conformal coating layer are separated and a surface of the conformal coating layer which bears a positive image of the print molecules is revealed, which positive image forms a tool which can be used to form additional imprints on substrates.

method steps, however, it will be understood that substitutions and equivalents as will be apparent to those of ordinary skill in the art may be used without departing from the scope of the invention as defined by the appended claims.

[020] According to the invention, a permanent imprinting template, having an identical image of the print molecule or at least a portion of the print molecule, is formed from the original print molecule and is used to form any desired number of imprints having negative images of the print molecule. The method begins with the preparation of a molecular imprint of the print molecule. The molecular imprint is conformally coated with a suitable material, which is then provided with a backing layer. The molecular imprint and coating layer (with its backing) are separated, and the resulting positive image of the molecular imprint formed in the conformal coating layer becomes a template for forming additional molecular imprints. A print molecule as used here encompasses molecules, proteins, enzymes, hormones, biological materials such as cells and viruses, or anything capable of forming a molecular imprint.

[021] The original molecular imprint may be formed by any of the methods known in the art. Preferably, the direct molecular imprint is formed on a surface of a polymer or other material according to a process known as two-dimensional imprinting (2D MIP). Alternatively, bulk molecular imprinting may be used, and the method will then include a step of removing an amount of the polymer sufficient to expose an imprinted surface.

[022] As is known, in forming a molecular imprint on a surface, a layer of the print molecules is first formed. The template fabrication techniques used to form the layers are well known to those skilled in the art of semi-conductor and MEMS process development and are incorporated herein without further detailed discussion. A 2D MIP process for forming an imprint of a biological molecule is described here as an exemplary embodiment. The example here describes a layer that is formed from single macro-molecules. It should be understood, however, that layers formed from Langmuir Blodgett (LB) films of the print molecule, as well as crystallized versions of the print molecules, or in fact any suitable means to form a suitable layer, may also be used.

[023] A solution is prepared containing a mixture of molecules and/or macromolecules which may hereafter be referred to as print molecules or components. This solution may be purified with respect to a particular component. The purified liquid solution is applied to a solid

substrate using any suitable means such as spin-coating, immersion, spraying, dripping, and/or painting. The liquid phase of the solution is chosen as the most advantageous for applying the solution to a substrate, and may be a suspension, emulsion, dissolution or other phase. The substrate materials may comprise glass, metal, polymer, ceramic, composites of such materials, or mixed surfaces of such materials. It will be recognized by those skilled in the art that a great variety of application techniques and a great variety of substrates may be utilized.

[024] After deposition, the now formed template layer has an areal concentration depending upon both the concentration of print molecules in the solution and the number of applications of the solution to the substrate. It may be necessary to prepare the chemistry of the surface so that it bonds to particular molecular functionality. The resulting layer of deposited molecules may be used in a variety of states: dry, wet, frozen, lyophilized, or in any form deemed suitable by those skilled in the art and which satisfy the requirements of the invention.

[025] According to the exemplary embodiment, the component layer is next coated with a conformal layer. An example of one such suitable conformal material is vapor polymerized Parylene. To prevent water from being evaporated during a vacuum deposition of this form of Parylene, the samples may be frozen or lyophilized and held at a temperature sufficiently low so as to prevent evaporation. Such techniques, as well as freezing techniques, are well known to those skilled in the art, as described in US Patent No. 4,447,374 to Tanaka. Alternatively, the substrate may be chosen and/or have its surface modified in such a way so as to prevent evaporation under moderate vacuum. One of the advantages of Parylene is that it will grow conformally at very low temperatures. As alternatives to Parylene, one may select any number of polymers formed from monomers such as ethylene, propylene, styrene, benzene, toluene, xylene, chlorobenzene, 1,2,4-trichlorobenzene, ferrocene, ethlybenzene, or any other monomer known by those skilled in the art to produce a polymer film using vapor or liquid phase deposition techniques. Examples of such coating techniques are cited in the aforementioned U.S. Patent No. 4,447,374, and have demonstrated the ability to produce complementary images of sufficient resolution to be used in the present method.

[026] An alternative to Parylene and a vacuum deposition process is to use a liquid phase conformal coating such as bis-benzocyclobutene (BCB). Another alternative is to form the complementary image by reacting a water coordinated biological molecule with A1(CH ₃ ) ₃ in an ALD (atomic layer deposition) process. In this manner a direct mirror image is formed in the

growing AI ₂ O ₃ layer. Irrespective of the choice of material used to form the conformal layer over the biological template, it should be recognized that any suitable conformal layer (that is, one with conformal resolution at least 2 times better than the smallest critical dimension of the print molecule) deposited onto the molecular layer by any deposition technique deemed suitable by one skilled in the art can be chosen without fundamentally affecting the intent of this invention.

[027] After formation of the conformal layer, the conformal layer may optionally be planarized. Planarization may be accomplished by polishing, by reflowing a suitable low-temperature material, such as the wax used in Si wafer polishing, over the deposited layer, by a suitable choice of a planarizing adhesive, or the cold flow planarization of In or In-based solders, or any other technique known to those skilled in the art. Next, a carrier substrate is adhered to the conformal built up layer or planarized layer. The carrier substrate provides a handle to separate the conformal layer from the print molecules. The resulting structure is shown schematically in Figure 1, illustrating the component layer 10 deposited on the substrate 12, with a planarized conformal coating layer 14 and the carrier substrate 16.

[028] The carrier substrate 16, conformal layer 14 and component layer 10 are then separated as a unit from the substrate 12. The imprint, that is, the negative images of the print molecules, are exposed when the print molecules are extracted, as further described below.

[029] An alternative to forming an image of the template layer by conformal coating is to apply and press the print molecules into a substrate containing a thin layer of polymer, as described in "Chemical Sensors - from Molecules, Complex Mixtures to Cells - Supramolecular Imprinting Strategies," Dickert et al., Sensors (2003, Vol. 3, pps. 381-392). According to this method, a print molecule layer is deposited on a molecularly thin layer of polyurethane and force is applied to the print molecule layer to press it into the polymer layer, producing a negative image of the print molecules in the polyurethane.

[030] According to the present invention, an improved method of applying pressure to the print molecule layer, shown in Figure 2, includes placing a first plate 20 on a print molecule layer 10 which has been deposited on a polymer layer 22 and applying force to the first plate with a second plate 24 acted on by a force producing device 26. Between the first and second plates is an engineered viscosity layer 28 to uniformly distribute the force from the upper,

second plate 24 onto the lower, first plate 20. The engineered viscosity layer may be selected from oils or other substances having a viscosity chosen so that its resistance to flow is proportionate to the force needed to form the imprints in the selected polymer layer.

[031] A further advantageous improvement is to attract and orient print molecules onto the deposition surface by applying an electric or magnetic field to the print molecules and deposition surface. As incorporated in the pressure imprinting method described above, and illustrated in Figure 3, the oriented molecules 5 in a solution 30 are deposited on a thin polymer layer 32 which has been advantageously placed over a metal layer 34. The polymer layer 32 may be partially polymerized. The molecules 5 are preferentially oriented with respect to an electric field which can be induced between the metal layer and the solution by electrodes 36 placed under the polymer layer 32. Since many macromolecules have an inherent dipole, or are highly polarizable, the molecules will be oriented with respect to the field as they are deposited on the polymer layer 32. The layer of oriented print molecules can be pressed into the polymer layer, as described above. The imprints formed in the polymer are accordingly oriented with respect to the field. Alternatively, the polymer layer and print molecule layer may be deposited using two immiscible fluids, the first being a partially polymerized or monomer layer and the second being a low- viscosity immiscible solvent such as water which holds the print molecules. The print molecules are then aligned using a field as described above.

[032] Figure 3 shows the electrodes in a simple positive/negative alternating arrangement parallel to the surface, however, many electrode arrangements are possible. For example, the electrodes may be arranged at a potential across the layer and solution causing an alignment of molecular dipoles normal to the imprint surface. Other electrode configurations will become apparent to those skilled in the art, so that molecules may be arranged in many predetermined orientations which can conform to a large number of molecular conformations. The molecules can thus be aligned and/or arranged into a variety of patterns. Examples of such arrangements can be dots of varying potential, patterns of dots of varying potential, and patterns of shapes with varying potentials.

[033] An advantage of using field alignment in forming the imprint is that when an oriented molecular imprint is used to adsorb analyte molecules, a similar field can be applied to an analyte solution, which will produce a similar orientation of analyte molecules both in the solution and in the adsorbed molecules. This allows for a great deal of control over how

molecules are oriented onto the surface which can be exploited for subsequent molecular manipulation. Field orientation can also increase the rate of adsorption on the surface because it eliminates two degrees of freedom of rotation of the analyte molecules.

[034] Field orientation may also be used with the process described above that includes a conformal coating to form the imprint. In such case, the print molecules are attracted and oriented to the substrate layer, and the deposited, oriented molecules are coated with a conformal layer as described above.

[035] Of course, a combination of the techniques previously described may be used, as in the following example. A layer of print molecules may deposited and/or placed on a polymer layer and the layer partially polymerized, which will allow impressions to be formed. A conformal layer is formed on the layer of molecules. Pressure is then applied to the conformal layer, which acts to imprint the molecules into the polymer layer. In this way imprints of both functional sides of the molecules may be formed in the polymer layer and in the conformal layer. This is particularly advantageous if a field is used to preferentially orient the molecules. As described below, a second substrate may be attached to the conformal layer so that upon separation of the conformal layer and polymer layer and removal of the print molecules (described below), the imprinted conformal layer is supported on a carrier or functional component.

[036] For all of the examples of the application of fields, the fields may be DC, AC, or AC with a DC bias, where the period of the AC field may be of any type deemed advantageous by those skilled in the art.

[037] Yet another alternative to forming the image by a conformal layer deposition process is a modification of the bulk process in which a photoactive polymer is used. Advantageously, a photoactive polymer can be patterned using standard photolithography techniques. In this way very precise macroscopic patterns of imprinted polymer layers may be formed. According to this embodiment, a component layer is deposited on a substrate and a photoactive polymer layer is subsequently deposited over the component layer. The photoactive polymer is then polymerized with light, and the polymer layer is removed from the substrate. Finally, the bottom edge of the polymer layer is removed until an imprint layer is exposed.

[038] It is also important to recognize that particular molecular conformations are influenced

by conditions in the in- vitro environment; consequently, by controlling the pH, the type and concentrations of other components (including amino acids in the case of biological molecules), the temperature, the molecular flow and various other conditions known to those skilled in the art, the same molecule can be produced in a variety of conformations. This is highly beneficial when combined with any of the techniques previously described since being able to control a molecular topology may be crucial to identification of the molecule when under test in a particular environmentally controlled conformation. In any case it should be clear that any suitable method can be used to form a molecular template layer as deemed suitable by those skilled in the art without fundamentally affecting the intent of this invention.

[039] After forming the complementary, negative image of the print molecule, the next step is the removal of the print molecules from the host matrix, whether a conformal coating layer, impressed polymer layer, or bulk polymer layer. This may be accomplished, for example, by rinsing or dissolution in a choice of solvents and/or baths deemed suitable to one skilled in the art. Such solvents or baths may include, but are not limited to: pH controlled aqueous rinses, organic solvents, emulsions, and jet rinses comprised of such fluids. The final cleaning step, however chosen, should produce a pristine surface with the negative images of the print molecule formed in the surface capable of adsorbing analyte or print molecules.

[040] The next step in the method according to the invention is to form a master template containing positive images of print molecules from the imprinted layer containing the complementary, negative images. The master template becomes an imprint tool from which other imprints (negative images) are formed. To accomplish this, a variety of techniques may be used.

[041] According to one embodiment of the method, the formation of the master template includes the step of conformally coating the imprinted layer with a metal. According to one embodiment, atomic layer deposition (ALD) may be used to form the conformal metal layer; however, other deposition means may be used if capable of reproducing the critical features of the imprint, reproducing the required fidelity for idiotypic interaction with analyte molecules, and providing adequate materials strength for the subsequent steps which are disclosed below. The ALD layer may be applied in a layer sufficiently thick to form a conformal layer with its own backing layer as a unitary body. Alternatively, ALD may be used to form a seed layer and electro or electroless plating may subsequently be applied over the ALD seed layer to form a

thick solid backing layer. According to yet another alternative, a layer of ALD may form a thin sacrificial layer and a thicker backing layer may be formed over the sacrificial layer. For the purposes of this description, the layer formed on the imprinted layer, whether of a single material formed in one or more applications, or of layers of different material, is referred to here as the conformal layer.

[042] Alternatively, one skilled in the art may choose another suitable means to form the conformal layer. Examples of other suitable layers may be found in the literature of chemical vapor deposition (CVD), plasma enhanced chemical vapor deposition (PECVD) ₅ ALD, liquid phase deposition (LPD), and polymerization reactions. Such layers may be comprised of any suitable material where in this case suitable means that the formed layer may be separated from the template layer without, at the very least, damage to the formed layer and preferably no damage to the template layer. Suitable materials for such a layer may be metals, Ti, Ta, Al, Cu, Sn, Au, Ag, Cr, Ni, etc., silicate glasses with varying degrees of additives, ceramics such as TaN, Si ₃ N ₄ , AI ₂ O3, etc., or polymers such as BCB, Parylene, PDMS, and similar materials. Such conformal layers may be formed in a single layer, or a thin conformal layer may be formed with a thicker backing layer overlaid to form a composite conformal layer.

[043] According to another embodiment, bulk metallic glass may be used to form the conformal layer for the master template, according to a process such as described in "Superplastic Forming of Bulk Metallic Glass," Schroers et al., in Journal of Materials, May 2005, pps 35-39. The bulk metallic glass conformal layer may also be formed thick to provide a backing layer, or a layer of different material may be deposited for a backing layer.

[044] After the conformal layer is formed, it may optionally be planarized. Planarization may be accomplished by polishing, by reflowing a suitable low-temperature material, such as wax, over the conformal layer, or by a suitable choice of a planarizing adhesive, or by cold flow planarization using In or In-based solders, or in fact any suitable technique known to those skilled in the art.

[045] A carrier substrate is next adhered directly to the conformal layer to facilitate the next step, which is separating the imprint layer containing negative images and the conformal layer containing the positive images. Figure 4 illustrates a negative image layer 40 formed according to any suitable process carried on the carrier substrate 16. A thin sacrificial layer 42 formed by

ALD is deposited directly on the negative image layer 40 and a subsequent thicker solid backing layer 44 which may be Parylene, vapor deposited metal, electroplated metal, electroless metal, or any other suitable material is deposited on the sacrificial layer 42. A second carrier layer 46 is applied to the planarized, composite conformal contact layer 42 and backing 44 layer.

[046] Separation may be accomplished in any way known to those skilled in the art, such as by utilizing a mismatch in thermal coefficients to create stress between the surfaces of the molecular imprint layer and conformal layer containing the positive image to cause them to separate. An alternative is the selective etching of a predeposited sacrificial layer, hi this case a first deposited thin metallic sacrificial layer is etched away and the material of this layer is chosen so that it possesses etch selectivity with respect to at least the formed backing layer. It should be recognized that any suitable sacrificial layer, other than a metal, may be chosen without fundamentally affecting the intent of the subsequently disclosed methods, where suitable in this case implies all of the required suitable properties of conformality and preservation of image fidelity as well as appropriate etch selectivity.

[047] One consequence of the use of a sacrificial layer is that it reduces the direct size of the produced positive image although preserving the aspect ratio. This can have the subsequent effect of making the imprinted image too small to idiotypically interact with the original print molecule. Of course, this may be corrected by the subsequent deposition of another conformal layer over the positive mold to restore the surface to the correct proportions. In fact this process may be used to adjust the aspect ratio of the positive to offset the subsequent use of sacrificial layers in any downstream process step and may be used as well to control selectivity.

[048] The separated template 50, illustrated in Figure 5, contains a surface 52 bearing an image identical to the layer of print molecules. This surface may now be substituted for the print molecules to form molecular imprints directly or to form intermediates for forming additional positive templates, as illustrated by the following exemplary embodiments.

[049] The formed template may be used to stamp molecular imprints having functional surfaces identical to the original imprint layer into other material layers. This may be done serially for as long as the template retains adequate fidelity. Figure 6 illustrates a positive image template 50 stamping molecular imprints into a polymer layer 60. An advantageous

method of stamping is to use an engineered viscosity layer as described in connection with Figure 2 between the template 50 and the force applying instrument to ensure the equal application of pressure across the imprinted polymer layer.

[050] Alternatively, the positive image templates may be used to form imprints by acting as forming substrates, rather than as stamps. By this embodiment, the template is used in the same manner as the layer of print molecules in the examples described above. For example, the positive image template may be conformally coated with a sacrificial layer. The conformal layer deposition is then followed by the deposition of a relatively thick layer of the desired functional material. An example of this process is an ALD deposition of Siθ ₂ of 1-10 nm on the template surface, followed by the ALD deposition of athin Ni layer 0.1 — 100 nm, and finally by a thick layer of electro or electroless deposited Ni 100 nm — 1 mm. After layer formation is complete the conformal SiO ₂ layer may be etched away and the Ni faced negative mold is used as indicated above, where it is understood that aspect ratio correction may be required as previously disclosed.

[051 ] The use of the technique of conformally coating the positive template as disclosed above allows one to choose from a wide number of material systems provided that they are adequately conformal and capable of a suitable separation step. In fact, the negative images thus formed may be metallic, ceramic, metallic glass, or glass, or even of polymeric composition. The wide variety of materials which may now be chosen adds considerably to the functionality of the imprint and to the overall functionality of devices formed from such imprints. It should be clear that there are any number of combinations and iterations of positive and negative layers, materials, layer types, layer functions, etc., of the above examples which may also be used with any number of materials and layer deposition choices not specified that may be used without substantially altering the intent of this teaching.

[052] According to another aspect of the invention, the positive image may be used to form one or more new negative images that may in turn be used to form one or more additional positive images. In this way the original template made directly from the virus, cell or molecule is not used repeatedly, preserving its fidelity, while subsequent copies are used to form imprints. To make additional templates, the first template is stamped into a suitable material to form a new negative or imprint. The new negative is then used make a new template in any of the methods described above.

[053] According to another example, a template is used to form a multiplicity of negative images, which may conveniently be formed on a sheet. This sheet of negatives can then be used to form a number of positive templates, which may advantageously also be formed on a sheet. The sheet of templates can be used in a batch process to stamp new imprints having functional surfaces, thus speeding up the production of imprints.

[054] It should be recognized the invention provides that functional imprinted surfaces may be formed with combinations of molecular imprints. For example, one set of molecules might be an enzyme for catalyzing a reaction while next to it might be one of the reagents. In this way a complex functional surface may be prepared for localized chemical reactivity. In a medical diagnostic application, for example, sets of molecules may represent different pathogens causing similar symptoms in a patient, such as mononucleosis, streptococcus, and adenovirus that a practitioner may wish to test for. Accordingly, a template may also be formed with combinations of molecule images to facilitate forming such functional surfaces.

[055] The invention further discloses a method of formation of a controlled number of imprint sites which are to be located near the structure where the local modification of the macromolecule is to be performed. In general it will be difficult to exactly control the number of molecules which might be present when an imprint is formed. The print molecules, or idiotypic precursors used to form imprints are small and it is likely only limited control of the numbers will be possible. However, methods are known which could be used to determine the location of unwanted sites and thus allow for the deactivation of the unwanted sites. A method for identification and deactivation according to the invention includes using a focused ion beam to image the metallic surface of the embossing template tool and to determine the location of both wanted and unwanted features. If the initial positioning scan is performed with low energy ions then it is possible to return to the undesired features and while increasing the power of the ions remove the unwanted features through focused ion beam etching. In this way any number of imprints in virtually any location can be realized. Of course, this is just one example of how unwanted features might be removed. In general, the method is to image and locate the features and then to remove the undesired features either by etching or by selectively filling them. This advantage can be extended when one then considers that an almost limitless number of patterns of a single or combinations of single imprints can be formed with almost any imaginable two dimensional form.

[056] Further, although the use of layers comprised of single molecules has been described, it should be recognized that the techniques above are readily applicable to molecule layers formed using crystallized proteins as well as from LB films, or any other layer comprised of molecules with which one might wish to form an imprint. The only difference in the use of such layers is in the application of the layers to the initial substrate for the formation of the original negative image, but many such techniques are well known to those skilled in the art and are herein included by reference. The advantage of being able to replicate crystals is that the replicated imprints of such crystals may act as synthetic seed crystals. A further advantage of using a crystallized surface is that more molecules will be added per unit of surface area on a crystalline surface than on a flat surface. The crystal form may be imprinted as a crystal, but such an advantage may also be realized if a crystal is placed on a surface and melted. By containing the melt on the surface, another type of high density imprint may be realized. These high density imprints provide substantial advantages for uses in sensors and chemical separation systems because of the increased number of molecules which may be adsorbed in a predetermined locality. Increased absorption density can improve respectively the signal to noise ratio (sensitivity) of a sensor response and the efficiency of the separation system. The advantage of LB films is that the LB arrangement provides a very dense packing of molecules and as such can improve the interaction density of the surface, which again has advantages in sensors and separations. Another advantage of the use of LB films is to ensure the orientation of the molecules used to form the LB layer.

[057] It should also be recognized that although these examples have dealt only with biological molecules, the techniques disclosed herein may be extended to include any molecular type whether organic, inorganic, of hybrids thereof. The method may be used with bacteria, viruses, proteins, cells, gases, liquids, powders or any molecule whose image may be formed using techniques similar to those disclosed herein are included. Further master templates may be formed from cultured antigens themselves bypassing the imprinting steps disclosed above. They may also be imprints of chemical surfaces which are sensitive to the molecules being analyzed and not just direct idiotypes.

[058] The ability to form layers of selected materials with selected functionality is a major improvement to the art and this improvement is further enhanced by the realization that such functionality may be localized. One method for localizing functionality is, as mentioned, using

individual templates to imprint selected locations on a surface. Another method forms a template itself with localized functionality. A sheet with positive images is formed, and selected portions are masked using standard photolithographic techniques. The mask can then be used to form a protective layer around which localized regions of conformal material with specifically chosen chemical functionality are formed. Then the mask layer may be stripped off and another conformal layer deposited until the structure is finished as detailed above.

[059] In a similar way, localized functionality may be realized through the use of localized materials deposition capability. For example it is known that certain types of self-assembled monolayers (SAMs) adhere preferentially to Au, an example being octadecanethiol. Further, it has been demonstrated that a wide variety of molecules can be chemically attached to functional groups at the heads of SAMs molecules. A localized chemical reactor may be formed by depositing Au between imprint features on a surface. Then, the entire structure is dipped into an appropriate series of baths, the first bath immobilizing the source molecule in the imprint features, followed by a rinse and a second bath to form the SAM. In this way, regions of localized chemical functionality are formed. The resultant localized chemical reactor may, for example, be used for gene modification or localized DNA cleaving.

[060] The added ability to define localized materials and localized functionality greatly leverages the advance in the art of this invention, as exemplified in the following device structures which may now be realized. For example, it is now possible to fabricate an imprint template with separated regions of imprinted molecular functionality. These separated regions may be fabricated from a metallic layer, separated by a dielectric and covered with a dielectric. The advantage gained from such an arrangement is that a potential may be applied between the two regions or even between the two regions and a solution which may contain samples of print molecule which are in turn to be detected. This is useful since many molecules contain strong dipoles as well as a strong but pH dependent regional charge. This not only helps in bringing the molecule near to the substrate, but the application of a potential is also advantageous since it can allow molecules to align prior to arrival at the imprinted feature, thus enhancing the interaction between the imprint and the print molecule's idiotypic region. The electrical field may be DC, AC or AC with a DC bias, and the period of the AC field may be of any type deemed advantageous by those skilled in the art.

[061] Similarly, the imprinted layers may also be adhered directly to sensors such as a quartz

mass balance (QMB) or a surface acoustic wave (SAW) device or any other type of desired and appropriately chosen mass sensitive sensor. A SAWR or other mass sensitive sensor responds to a change in mass by shifting its resonant frequency. By placing a molecular imprint on the surface of a SAWR, the change in mass becomes a function of the molecules adsorbed on the sensor. Using an appropriate interrogator (frequency measurement system) with the ability to detect frequency shifts, a sensor with the ability to adsorb an analyte molecule (i.e., print molecules whose imprints have been placed over the resonator structure) can be used to detect the presence of the analyte molecule in, for example, a solution in which the sensor is placed. Such a system using imprints formed directly from print molecules has been proposed by Dickert et al. for an oil quality sensor as described in US Patent No. 6,223,589, which is incorporated herein by reference. The present invention, on the other hand, allows for the production of multiple, identical sensors in a facilitated manner. Further, the ability to provide localized functionality allows the device designer to better optimize device performance. For example, the mass adsorption functionality (i.e., the imprints) can be located at the most sensitive region of the device. Further, since the active region of the SAWR may be completely covered by the layer comprising the imprints, the response is also highly selective to the analyte in question. The imprint layer serves to shield the active region of the device, which minimizes the effects of the fluid and other components of the solution.

[062] hi this exemplary embodiment, illustrated in Figure 7, a SAWR 70 is prepared with an electrode 72 as an outer ring, with two additional electrodes 74, 76 arranged in parallel inside the ring. A dielectric material layer is applied to the area between the outer electrode 72 and the parallel electrodes 74, 76. A polymer layer 78 is deposited over the electrodes. Imprints of a selected print molecules 80 are then stamped into the polymer layer 78 using a template tool as described herein. An advantage of preparing a sensor according to the invention is that the materials chosen to form the chemically selective layer may be chosen for optimum performance of the SAWR device. The electrodes will help analyte molecules align for better adsorption onto the surface.

[063] Another improvement is in controlling the pressure used to form the imprints on the sensor surface using the response from the sensing elements to the imprint pressure. To accurately reproduce a template image in an imprint, the applied pressure must neither be too light, which may not cause the features to be imprinted, or too high, which might damage the

structure or critical features of the structure should be avoided. As explained above, adsorbed mass can cause a frequency shift in a SAWR device. Similarly, applied pressure on the SAWR device will also cause a frequency shift. A sensor being imprinted on can therefore be monitored for frequency change during the imprinting process to determine when and if sufficient force has been applied for imprint fidelity. The measurement of the frequency can be used as feedback into the pressure apparatus via the imprinting tool. In this way it is possible to prevent the application of excessive force and simultaneously determine that adequate pressure has been applied so that an impression of required fidelity has been formed.

[064] A further improvement to this device can be realized if a heater is added to the SAWR structure. Such a heater could quite simply be a metallic resistor deposited on the surface near the imprinted area or an element disposed beneath the polymer layer. This heater can be used to selectively raise the temperature of the sensor and thus cause the analyte to desorb. Heating can be controlled via a wireless link to a transmitter, making it possible to use the sensor indefinitely without maintenance or intervention. This improvement provides the ability to analyze the rate of repopulation of the imprint sites, which can determine the concentration of analyte molecules in the solution. Another advantageous aspect is that the monitoring and control system may remotely control the detection process in real-time using feedback from the sensor, which can measure concentration and reaction states.

[065] Another advantage may be realized if ultrasonic energy is used to help the aligning molecules arrange themselves in their lowest energy form with respect to the electrical potential and the molecules' own dipole moments. An ultrasonic transducer can be attached to the surface of the substrate in an advantageous manner so that the desired molecules are moved into their lowest surface energy potentials and molecules which are not desired and/or not in the correct position are vibrated off the electrodes. It is clear that ultrasonic energy can be used in this way to enhance the rate of molecular uptake onto the electrodes and to improve the yield of correctly oriented molecules.

[066] Devices that are sensitive to mass in this way and which are subjected to temperature changes will often need to have calibration curves to account for changes in sensor response with respect to temperature. The invention provides a manner to accomplish this calibration, using differential measurement, a concept which is widely used in SAW sensor technology. According to an exemplary embodiment of the differential technique, two SAWRs are each

coated with similar layers over similar regions of the SAWR. The layers differ only in that one resonator has a functional coating and the other does not. Both SAWs are placed into the identical environment, with the non-imprinted device providing a baseline because the difference in response between the two SAWs is that one is sensitive to the analyte and the other is not. Alternatively, devices may be placed on a single substrate with localized functionality to eliminate the need for calibration; however, in the most sensitive applications calibration may be required to account for deviation between the two applied layers, hi this case calibration coefficients can be derived and stored for use with the sensor. The storage of the coefficients can be proximate to the devices by utilizing writeable RFEDs or they can be stored at the interrogator and programmed into the interrogator before use.

[067] One advantageous method for such a calibration is to use a solution identical to that which will be measured but devoid of the analyte. Then known and varying amounts of the analyte may be introduced into the solution and calibration versus temperature and concentration derived, hi this way the response of the sensors to temperature and environment can be completely accounted for and very precise measurement of the analyte becomes possible.

[068] It should be realized that although this example has dealt with SAWR devices it is possible to use, without affecting the Intent of this teaching, a SAW delay line or a BAW based sensor, a QMB, an FBAR, or in fact, any mass sensitive device. AU of these sensors have been previously studied, are well known to those skilled in the art, and will benefit from the advantages of this teaching.

[069] Another benefit of the approaches described herein is that the localization of the imprint features allows the layers to be placed over transducers which can in turn produce a local effect. For example if the localized choice of materials were to be designed to focus acoustic energy in a specific location then this energy could be applied quite locally to desorb an adsorbed surface species.

[070] Another type of functionality that is now available has to do with the formation of imprint structures on surfaces which allow for localized heating. This ability combined with the ability to localize the materials provides a means whereby there may a degree of thermal isolation. For example, a rather complex protein molecule may be locally bound chemically or

held electrostatically after being localized in the imprint. The molecule may then be thermally unwrapped for subsequent reaction in a local chemical reactor. In addition, by varying heat and/or electrostatic field strength, an adsorbed molecule can be desorbed locally into a fluid flow for the purposes of reaction elsewhere on the device structure or at some point downstream. This allows for control of the concentration of reactants during the course of a reaction. Further, since the molecular orientation is controlled by the imprint feature this also allows for a great deal of control during molecular modification and when combined with the localized reactor described above there is now means for very precise molecular modification reactions and subsequently the controlled release of modified species in ways heretofore unavailable. It is clear that many types of reactions both synthetic and analytic can utilize the capability for localized heating and localized chemical functionality and that is now available as a result of this teaching. Another advantageous example of localized heating is that now temperature programmed desorption can be measured directly on the area of local heating. In this way it is easy to determine the concentration of the analyte under study. In this case the desorption rate is functionally dependent upon the depth of the potential well into which the molecule is located when it adsorbs into the imprint versus the reduction in this potential barrier due to thermal energy and the reduction in the potential due to conformational changes.

[071] Finally, it should be recognized that the methods used for formation of the imprints disclosed herein lend themselves readily to the formation of molecular imprints with device functionality as a part of integrated circuit manufacture, thus providing a means whereby all manner of devices, both monolithic and multichip, may be realized, for example it would be possible to fabricate a single chip QMB, SAWR, or any other suitable device complete with the frequency source and the frequency measurement circuitry with a highly adsorbate selective surface on the sensor. In particular, it should be recognized that atomic layer deposition techniques described here for imprinting molecular images may be used for imprinting other surfaces of similar scale.

[072] Of course it is possible to imagine a very large number of permutations of the various aspects of the disclosed invention, however it should be clear that these permutations as will occur to those skilled in the art are within the scope of the invention.