HK1121719B - Nanocontact printing - Google Patents

Description

Nano-contact printing

Background

In recent years, there has been considerable effort aimed at understanding new phenomena on the nanometer scale, and new materials of various nanostructures have been manufactured and characterized. New devices with attractive properties have just begun to be designed. The expectation for a new generation of inexpensive and innovative tools that will change our lives is extremely high. Combining a new set of desirable and undesirable properties with a new family of materials and manufacturing methods will enable devices that we could not even imagine just a decade ago. Coulomb blockade of metal nanoparticles and semiconductor quantum dots, narrow-band fluorescence emission from semiconductor nanoparticles, quantized ballistic conduction of nanowires and nanotubes are just a few new materials/phenomena that affect our way to design optical and electronic devices. For a review of nanodevices and fabrication techniques, see Bashir, superstate and microstuctures (2001), 29 (1): 1 to 16; xia et al, chem.rev. (1999), 99: 1823-1848; and Gonsarves et al, Advanced Materials (2001), 13 (10): 703 and 714, the entire teachings of which are incorporated herein by reference.

The first stage of nanoscience (and mainly nanotechnology) is primarily the development and characterization of new materials and devices based on inorganic semiconductors and metals. One reason for this is that electron beam lithography (one of the earliest tools capable of building nanoscale structures and devices) is a technique for patterning inorganic materials on inorganic substrates. A significant advance in recent years has been the development of new highly versatile nanolithography (nanolithography) based Scanning Probe Microscopy (SPM). Using various types of SPMs, patterns can now be formed on a variety of organic and inorganic substrates by inducing localized chemical modifications or by forming self-assembled monolayers (SAMs). For example, Mirkin and co-workers have developed Atomic Force Microscope (AFM) based techniques (Dip Pen Nanolithography, DPN) in which SAMs can be generated with a resolution of less than 5nm by controlled transfer of molecules from the microscope tip to the substrate (see Lee et al, Science (2002), 295: 1702-1705; Demers et al, Angew.chem.Int.Ed. (2001), 40 (16): 3069-3071; Hong et al, Science (1999), 286: 523-525; Piner et al, Science (1999), 283: 661-. The development of such technologies represents a major breakthrough as devices can now be built not only on inorganic basis, but also on organic and biological material. Organic-based nanomaterials may provide a number of interesting properties that can be effectively tuned at the nanoscale. Due to these new manufacturing techniques and the elucidation of the basic concepts in surface and supramolecular chemistry, new devices are being developed in large quantities.

Many different nanodevices (e.g., nanotransistors, nanosensors, and nanocuiders) are currently being fabricated using organic and inorganic based nanolithography. However, to predict how much the nanotechnology will have an impact, one must estimate the speed of fabrication of complex devices. It has been assumed that device fabrication time (and repeatability) can be a major limiting factor in nanotechnology. In particular, the problem of how to mass-produce has not been solved.

An equivalent with micro-contact printing would be desirable for nanotechnology: stamping techniques designed by Whitesides and co-workers (see U.S. patent nos. 5,512,131, 5,900,160, 6,048,623, 6,180,239, 6,322,979, the entire teachings of which are incorporated herein by reference) have revolutionized the way in which people design microdevices, and have had a tremendous impact by allowing non-chemists to build devices as complex as bio-MEMS. Unfortunately, microcontact printing has some resolution limitations that limit its application in nanotechnology. Chou and collaborators in Princeton have recently addressed this problem. The processes discussed by them in U.S. Pat. nos. 5,772,905 and 6,309,580 and U.S. patent application publications 2002/0167117, 2003/0034329, 2003/0080471 and 2003/0080472, the entire teachings of which are incorporated herein by reference, are based on hard dies (i.e., dies made of inorganic materials) that are stamped onto a soft polymer film that covers a silicon wafer. The printed substrate is typically composed of metal wire or semiconductor material (see Chou et al, Nature (2002), 417: 835-.

One drawback of existing nanolithography techniques for fabricating nanoscale devices is that many parts of the device (features) are fabricated in a series of steps. Thus, these techniques may be used for relatively simple devices, but manufacturing a device with many parts may require an unacceptably large amount of time. One effort to address this problem has been to fabricate multi-tip arrays for SPM (Zhang et al, Nanotechnology (2002), 13: 212, the entire teachings of which are incorporated herein by reference). While such methods may be capable of fabricating tens or hundreds of nano-devices in parallel, it would be desirable to develop nano-stamping techniques that better facilitate mass production by producing many portions of the device in one processing step.

Summary of The Invention

The method of the present invention complements chemically-directed nanolithography techniques that have been developed in recent years and often require complex instrumentation. For example, it has been demonstrated that DNA test arrays can be fabricated using dip pen nanolithography. Once the body of these devices is constructed, the teachings of the present invention can be used to print a large number of inexpensive and extremely sensitive devices for detecting, for example, biohazards, without the need for complex instruments and materials. Since the transfer process is based on self-assembly, all steps except the manufacturing of the body can be performed in parallel over very large areas and on multiple substrates.

In one aspect, the invention is a method of forming a complementary image of a subject. The method includes providing a body comprising a first set of molecules bound to a first substrate to form a pattern. When the first set of molecules comprises nucleic acids, the first set of molecules comprises a plurality of nucleic acids having different sequences. The method further comprises assembling a second set of molecules on the first set of molecules by attraction or formation of a bond. Each molecule of the second set of molecules comprises a reactive functional group and a recognition component attracted to or bound to one or more molecules of the first set of molecules. The method further comprises contacting the reactive functional group of the second set of molecules with a surface of a second substrate to form a bond between the second set of molecules and the second substrate, disrupting the attractive forces or bonds between the first set of molecules and the second set of molecules to form a complementary image of the host, and optionally repeating the assembling, contacting and disrupting steps one or more times.

Each molecule of the second set of molecules may further comprise one or more of the following components: an exposed functional group, a covalent bond or a first spacer linking the reactive functional group to the recognition component or a covalent bond or a second spacer linking the exposed functional group to the recognition component. The second set of molecules may be assembled on the first set of molecules by contacting the subject with a solution comprising the second set of molecules. For example, the body may be held in contact with the second substrate by capillary action of a solution containing the second set of molecules, and the attractive forces or bonds between the first and second sets of molecules may be broken by evaporation of the solution.

The second set of molecules may include two or more different molecules that may have different recognition components, different exposed functional groups, or both different recognition components and different exposed functional groups. The two or more different molecules may form a pattern on the second substrate, the pattern having a profile comprising two or more heights. At least one of the two or more different molecules may include a first spacer, while another of the two or more different molecules may have a second spacer of a different length than the first spacer or may be free of spacers. In another embodiment, the bonds between the first set of molecules and the second set of molecules may be broken by applying heat or by contacting the bonds with a solution having a high ionic strength.

In some embodiments, the component of each molecule of the first set of molecules may be a nucleic acid sequence and the recognition component of the second set of molecules may be a nucleic acid sequence that is at least 80%, at least 90%, at least 95%, or at least 99% complementary to the nucleic acid sequence on the first set of molecules. The bond between the first and second sets of molecules may be broken by contacting the bond with an enzyme. The nucleic acid sequence may comprise DNA, RNA, modified nucleic acid sequences, or combinations thereof. The first set of molecules, the second set of molecules, or both may comprise peptide nucleic acid sequences.

The method can further include forming a pattern of one or more metals, metal oxides, or combinations thereof on a surface of the substrate, and contacting the surface with the first set of molecules. In this embodiment, each molecule of the first set of molecules has a reactive functional group that forms a bond between the metal or metal oxide and the molecules of the first set of molecules to form a body comprising the first set of molecules bonded to the substrate to form the pattern.

In one embodiment, at least a portion of the surface of the second substrate may be free of the second set of molecules. The method may further comprise contacting a surface of the second substrate with a reactant selected to be chemically inert to the second set of molecules and degrade at least a surface layer of the second substrate, thereby degrading the portion of the surface of the second substrate not containing the second set of molecules, and removing the second set of molecules to expose a portion of the surface of the second substrate. For example, the invention can include depositing a material on a portion of the surface of the second substrate that does not contain the second set of molecules, and removing the second set of molecules to expose a portion of the surface of the second substrate. The attractive force between the first and second sets of molecules may be magnetic.

In another aspect, the invention is a method of forming a replica of a body or a portion of a body. The method comprising providing a body comprising a first set of molecules bound to a first substrate to form a pattern, assembling a second set of molecules on the first set of molecules by forming bonds, contacting reactive functional groups of the second set of molecules with a surface of a second substrate to form bonds between the second set of molecules and a second substrate, breaking the bonds between the first set of molecules and recognition components on the second set of molecules to form a complementary image of the body, assembling a third set of molecules on the second set of molecules of the complementary image by forming bonds, contacting reactive functional groups of the third set of molecules with a surface of a third substrate to form bonds between the third set of molecules and the third substrate, breaking the bonds between the second set of molecules and recognition components on the third set of molecules to form a replica of the body or a portion of the body, and optionally repeating the steps of assembling the third set of molecules, contacting the reactive group of the third set of molecules and disrupting the bond between the second set of molecules and the third set of molecules one or more times.

In another aspect, the invention is a composition comprising a first pattern of a first set of molecules bound to a first substrate and a complementary image comprising a pattern of a second set of molecules bound to a second substrate via a reactive functional group on each of the second set of molecules. When the first set of molecules comprises nucleic acid sequences, the first set of molecules comprises a plurality of nucleic acids having different sequences, and each molecule of the second set of molecules has a recognition component that binds to at least a portion of the molecules from the first set of molecules. The first substrate having the first pattern may be a reusable body.

In another aspect, the invention is a kit for printing a molecular pattern on a substrate. The kit comprises a body comprising a pattern of a first set of molecules bound to a substrate and a second set of molecules, each molecule of the second set of molecules comprising a reactive functional group and a recognition component bound to the first set of molecules.

The amount of information stored in molecules such as the DAN chain can be enormous. The method of the invention makes it possible to transfer this information in a massively parallel manner, i.e. in one or only a few steps instead of many. Devices that are now built using multi-step techniques can thus be manufactured in one step. This opportunity will shift the direction of research and device fabrication toward increasing the complexity of the substrates fabricated. As a simple example, if used in a 1mm sample with a series (e.g., 50) of nano-and microfluidic channels (which have 50 different types of DNA strands defining the walls of the channels)²Making the body on a substrate, one can then use the method of the invention to make a body in one printing step, at 1mm²Complementary images of the series of nano-and microfluidic channels were made on a substrate, their respective walls being functionalized in different ways: a real lab-on-a-chip.

A unique feature of the teachings of the present invention is the copying (and thus replication) of the body itself using the parallel process of the present invention. This is a significant advantage over any existing method. Huge production lines usually require many bodies. In combination with the wear of the existing mould, this means that the body needs to be continuously produced. In the method of the invention, once the master is produced, a replica of the master can be produced from it, and the final device can then be printed using these new masters. Repeatability should be improved and only the first body manufacturing equipment that produces parts in a series pattern would have to be used to manufacture the first body.

The method of the present invention is revolutionary not only because it can be used to print organic SAMs, but also because it can be used to convey many types of information (e.g., chemical + shape) and replicate hosts in parallel patterns.

Brief Description of Drawings

The invention will be described with reference to specific embodiments shown in the drawings. The embodiments in the drawings are shown by way of example and are not meant as limitations.

Fig. 1A-D are illustrations of one embodiment of the method of the present invention for generating complementary images.

FIG. 2 is a schematic representation of a first set of molecules bound to a second set of molecules.

Fig. 3A and 3B are AFM images of a host having a monolayer of nucleic acid molecules bound to a surface of a substrate.

Fig. 3C is an AFM image of a complementary image of the body shown in fig. 3A.

Fig. 3D is an AFM image of a complementary image of the body shown in fig. 3B.

Figure 4A is an AFM image with a host of nucleic acids bound to a substrate in a grid pattern.

Fig. 4B is an AFM image of a complementary image of the body shown in fig. 4A.

Definition of

As used herein, a "host" is a substrate having a first set of molecules bound to the surface of the substrate in a random or non-random pattern. In one embodiment, the first set of molecules is bound to the body in a non-random pattern. The first set of molecules may comprise one or more different molecules. The information encoded in the pattern may be derived from the location of each molecule on the surface of the substrate and/or the chemical nature of the molecule (e.g. molecules from a first set of molecules having a particular nucleic acid sequence will specifically bind to nucleic acid molecules having a complementary sequence).

As used herein, "complementary image of a body" refers to an image on a substrate that is a mirror image of spatial and/or chemical information encoded in the body or portion thereof when the pattern on the body is asymmetric, or that is a duplicate of spatial and/or chemical information encoded in the body or portion thereof when the image on the body is symmetric. In one embodiment, the complementary image is formed by binding a second set of molecules to a second substrate. For example, if the first set of molecules bound to the body are nucleic acid molecules that form a non-centrosymmetric pattern, the complementary image of the body will be a mirror image of the body formed on a second substrate with a second set of molecules that are nucleic acids having a sequence complementary to at least a portion of the nucleic acid sequences from the first set of molecules. In some embodiments, the chemical information imparted onto the complementary image is not identical to the information on the subject, but is sufficient information to allow at least a portion of the information from the subject to be replicated. For example, when the first and second sets of molecules are nucleic acid molecules, at least three or more consecutive bases in the molecules from the first set of molecules may be complementary to three or more consecutive bases from the second set of molecules. For example, at least 80%, at least 90%, at least 95%, or at least 99% of the nucleic acid sequences on the first and second sets of molecules may be complementary. By selecting for the second set of molecules that bind to only a fraction of the molecules of the first set of molecules bound to the body, a complementary image can be formed from a fraction of the pattern on the body. The height profile of the complementary image may have two or more levels when the second set of molecules binds to only a portion of the molecules of the first set of molecules. Further, the complementary image may encode only a mirror image of the spatial information encoded in the subject, or may encode both the chemical and spatial information encoded in the subject. For example, if the first set of molecules bound to the body are nucleic acid molecules forming an asymmetric pattern, the complementary image of the body will be a mirror image of the body formed on a second substrate with a second set of molecules, the second set of molecules being nucleic acids having a sequence complementary to at least a portion of the nucleic acid sequences from the first set of molecules. In this example, both spatial and chemical information is transferred from the subject to the complementary image. Moreover, only a portion of the chemical information may be transferred to the complementary image. For example, when the first set of molecules on the subject are nucleic acid molecules, then the second set of molecules forming the complementary image may be nucleic acid sequences that are complementary to only a portion of the nucleic acid sequences on the subject (e.g., not the entire sequence).

As used herein, a "replica of a subject" is a copy of spatial and/or chemical information encoded in a pattern of the subject. The replica may be a copy of only a portion of the pattern of the subject, or may be a copy of the entire pattern of the subject. Further, a replica of a subject may replicate only the spatial information of the subject, or may replicate both the spatial and chemical information encoded in the subject. Furthermore, a replica of the master may replicate only a portion of the chemical information.

"chemical information encoded in a molecule" refers to the ability of the molecule to specifically bind (usually in a particular conformation) to another molecule or to a particular type of molecule. For example, a particular nucleic acid sequence may specifically bind to a complementary sequence, or protein a may specifically bind to an immunoglobulin.

As used herein, the term "pattern" refers to the spatial location of each molecule in a set of molecules bound to a substrate and the chemical structure of each molecule in the set of molecules.

As used herein, the term "silane" refers to a functional group having the following structural formula:

r in the above formula₂Each occurrence is independently selected from-H, alkyl, aryl, alkenyl, alkynyl, and arylalkyl.

As used herein, the term "chlorosilane" refers to a functional group having the following structural formula:

r in the above formula₆Each occurrence is independently selected from-Cl OR-OR₂Provided that at least one R is₆is-Cl. Preferably, each R₆Are all-Cl.

As used herein, the term "spacer" refers to a divalent group that links two components of a molecule. Exemplary spacers include alkylene, heteroalkylene, heterocycloalkylene, alkenylene, alkynylene, arylene, heteroarylene, aralkylene, and heteroarylalkylene, wherein the alkylene, heteroalkylene, heterocycloalkylene, alkenylene, alkynylene, arylene, heteroarylene, aralkylene, or heteroaralkylene can be substituted or unsubstituted.

As used herein, the term "alkyl" refers to a fully saturated straight or branched chain C₁-C₂₀Hydrocarbons or cyclic C₃-C₂₀A hydrocarbon. Alkyl groups may be substituted or unsubstituted.

The term "alkylene" refers to an alkyl group having at least two points of attachment to at least two moieties (e.g., methylene, ethylene, isopropylene, etc.). The alkylene group may be substituted or unsubstituted.

"alkenyl" is a straight or branched chain C having one or more double bonds₂-C₂₀Hydrocarbons or cyclic C₃-C₂₀A hydrocarbon. Alkenyl groups may be substituted or unsubstituted.

"alkenylene" refers to an alkenyl group having at least two points of attachment to at least two moieties. Alkenylene groups may be substituted or unsubstituted.

"alkynyl" is a straight or branched chain C having one or more triple bonds₂-C₂₀Hydrocarbons or cyclic C₃-C₂₀A hydrocarbon. Alkynyl groups may be substituted or unsubstituted.

"alkynylene" refers to an alkynyl group having at least two points of attachment to at least two moieties. Alkynylene groups may be substituted or unsubstituted.

"Heteroalkylidene" means a compound having the formula-X- { (alkylene) -X }_qA group of (A) wherein X is-O-, -NR₁-or-S-; and q is an integer of 1 to 10. R₁Is hydrogen, alkyl, aryl, arylalkyl, alkenyl, alkynyl, heteroaryl, heteroarylalkyl or heterocycloalkyl. Heteroalkylene groups may be substituted or unsubstituted.

The term "aryl" when used herein alone or as part of another moiety (e.g., arylalkyl, etc.) refers to a carbocyclic aromatic group such as phenyl. Aryl also includes fused polycyclic aromatic ring systems in which a carbocyclic aromatic ring is fused to another carbocyclic aromatic ring (e.g., 1-naphthyl, 2-naphthyl, 1-anthracenyl, 2-anthracenyl, etc.), or in which a carbocyclic aromatic ring is fused to one or more carbocyclic non-aromatic rings (e.g., tetralin, indane, etc.). The point of attachment of the arylene group fused to the carbocyclic, non-aromatic ring may be on the aromatic or non-aromatic ring. The aryl group may be substituted or unsubstituted.

"arylene" refers to an aryl group having at least two points of attachment to at least two moieties (e.g., phenylene, etc.). The arylene group may be substituted or unsubstituted.

"arylalkyl" refers to an aryl group attached to another moiety through an alkylene linker. Arylalkyl groups may be substituted or unsubstituted. When the arylalkyl group is substituted, the substituent may be on the aromatic ring or alkylene portion of the arylalkyl group.

As used herein, "arylalkyl" refers to an arylalkyl group having at least two points of attachment to at least two moieties. The second point of attachment can be on an aromatic ring or alkylene. The arylenealkyl group may be substituted or unsubstituted. When the arylenealkyl group is substituted, the substituent may be on the aromatic ring or alkylene portion of the arylenealkyl group.

As used herein, the term "heteroaryl" refers to an aromatic heterocycle containing 1, 2, 3, or 4 heteroatoms selected from nitrogen, sulfur, or oxygen. Heteroaryl groups may be fused to one or two rings such as cycloalkyl, heterocycloalkyl, aryl or heteroaryl. The point of attachment of the heteroaryl group to the molecule may be on the heteroaryl, cycloalkyl, heterocycloalkyl, or aryl ring, and the heteroaryl group may be attached through a carbon or heteroatom. Examples of heteroaryl groups include imidazolyl, furyl, pyrrolyl, thienyl, oxazolyl, thiazolyl, isoxazolyl, thiadiazolyl, oxadiazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, quinolinyl, isoquinolinyl, indazolyl, benzoxazolyl, benzofuranyl, benzothiazolyl, indolizinyl, imidazopyridinyl, pyrazolyl, triazolyl, isothiazolyl, oxazolyl, tetrazolyl, benzimidazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzooxadiazolyl, indolyl, tetrahydroindolyl, azaindolyl, imidazopyridinyl, quinazolinyl, purinyl, pyrrolo [2, 3] pyrimidinyl, pyrazolo [3, 4] pyrimidinyl, or benzo (b) thienyl, each of which is optionally substituted. Heteroaryl groups may be substituted or unsubstituted.

"heteroarylene" refers to a heteroaryl group having at least two points of attachment to at least two moieties. Heteroarylenes may be substituted or unsubstituted.

"heteroarylalkyl" refers to a heteroaryl group that is linked to another moiety through an alkylene linker. Heteroarylalkyl groups may be substituted or unsubstituted. When heteroarylalkylene is substituted, the substituent may be on the aromatic ring or alkylene portion of the heteroarylalkyl. Heteroarylalkyl groups may be substituted or unsubstituted.

"heteroarylenealkyl" refers to a heteroarylalkyl group having at least two points of attachment to at least two moieties. Heteroarylenealkyl groups may be substituted or unsubstituted.

"heterocycloalkyl" refers to a non-aromatic ring containing one or more (e.g., one to four) oxygens, nitrogens, or sulfur (e.g., morpholine, piperidine, piperazine, pyrrolidine, and thiomorpholine). Heterocycloalkyl groups may be substituted or unsubstituted.

"heterocycloalkylene" refers to a heterocycloalkyl group having at least two points of attachment to at least two moieties. Heterocycloalkylene groups may be substituted or unsubstituted.

Suitable substituents for alkyl, alkylene, alkenyl, alkenylene, alkynyl, alkynylene, heteroalkyl, heteroalkylene, heterocycloalkyl, heterocycloalkylene, aryl, arylene, arylalkyl, arylenealkyl, heteroaryl, heteroarylene, heteroarylalkyl, and heteroarylalkylene include any substituent that is stable under the reaction conditions used in the methods of the present invention. Examples of substituents include aryl (e.g., phenyl), arylalkyl (e.g., benzyl), nitro, cyano, halo (e.g., fluoro, chloro, and bromo), alkyl (e.g., methyl, ethyl, isopropyl, cyclohexyl, and the like), haloalkyl (e.g., trifluoromethyl), alkoxy (e.g., methoxy, ethoxy, and the like), hydroxy, -NR₃R₄、-NR₃C(O)R₅、-C(O)NR₃R₄、-C(O)R₃、-C(O)OR₃、-OC(O)R₅Wherein R is₃And R₄Each occurrence is independently-H, alkyl, aryl, or arylalkyl; and R is₅Each occurrence is independently alkyl, aryl, or arylalkyl.

Alkyl, alkylene, heterocycloalkylene, and any saturated moiety of alkenyl, alkenylene, alkynyl, alkynylene may also be substituted with ═ O and ═ S.

When the heteroalkylene, heterocycloalkyl, heterocycloalkylene, heteroaryl, or heteroarylene group contains a nitrogen atom, it may be substituted or unsubstituted. When a nitrogen atom in an aromatic ring of a heteroaryl or heteroarylene group has a substituent, the nitrogen may be a quaternary nitrogen.

As used herein, the term "nucleic acid" or "oligonucleotide" refers to a polymer of nucleotides. Typically, a nucleic acid comprises at least three nucleotides. The polymer may comprise natural nucleosides (i.e., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine) or modified nucleosides. Examples of the modified nucleotide include base-modified nucleosides (e.g., cytarabine, inosine, isoguanosine, nebularine, pseudouridine, 2, 6-diaminopurine, 2-aminopurine, 2-thiothymidine, 3-deaza-5-azacytidine, 2' -deoxyuridine, 3-nitropyrrole, 4-methylindole, 4-thiouridine, 4-thiothymidine, 2-aminoadenosine, 2-thiothymidine, 2-thiouridine, 5-bromocytidine, 5-iodouridine, inosine, 6-azauridine, 6-chloropurine, 7-deazaadenosine, 7-deazaguanosine, 8-azaadenosine, 8-azidoadenosine, benzimidazole, M1-methyladenosine, pyrrolopyrimidine, 2-amino-6-chloropurine, 3-methyladenosine, 5-propynyl cytidine, 5-propynyl uridine, 5-bromouridine, 5-fluorouridine, 5-methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O (6) -methylguanine, and 2-thiocytidine), chemically or biologically modified bases (e.g., methylated bases), modified sugars (e.g., 2 ' -fluororibose, 2 ' -aminoribose, 2 ' -azidoribose, 2 ' -O-methylribose, L-enantiomeric nucleoside arabinose, and hexoses), modified phosphate groups (e.g., phosphorothioate and 5 ' -N-phosphoramidite linkages), and combinations thereof. Natural and modified nucleotide monomers for chemical synthesis of nucleic acids are commercially available.

As used herein, the term "Peptide Nucleic Acid (PNA)" refers to a polymer having a peptide backbone in which natural or unnatural nucleobases are attached to each amino acid residue. Peptide nucleic acids are described in Hanvey et al, Science (1992), 258: 1481-1485, the entire teachings of which are incorporated herein by reference. A PNA is capable of specifically binding to a nucleic acid or another PNA having a sequence of at least three consecutive bases (e.g. six consecutive bases) complementary to the sequence of said PNA. In one embodiment, the PNA is at least 80%, at least 90%, at least 95% or at least 99% complementary to the nucleic acid or second PNA.

As used herein, the term "attractive force" refers to a force that pulls two or more molecules together. Examples of attractive forces include attraction of molecules with a net positive charge to molecules with a net negative charge, dipole-dipole attraction, and magnetic attraction.

Unless indicated as covalent bonds, the term "bond" includes covalent and non-covalent bonds, such as hydrogen bonds, ionic bonds, electrostatic interactions, magnetic interactions, covalent bonds, and van der waals bonds.

As used herein, the term "recognition component" is a molecular component that is capable of specifically binding to another molecule.

As used herein, "specifically binds" refers to a molecule or complex that, when its recognition component binds to one or more other molecules or complexes, is specific enough to distinguish the molecule or complex from other components or contaminants of the sample. The molecules comprising the recognition component and their targets are conventional and not described in detail herein. Techniques for making and using such systems are well known in the art and are exemplified by Tijssen, P., "Laboratory Techniques in Biochemistry and Molecular Biology practices and procedures of enzyme Immunoas

As used herein, "aptamer" refers to a non-naturally occurring nucleic acid that selectively binds to a target. Aptamer-forming nucleic acids can be composed of naturally occurring nucleosides, modified nucleosides, naturally occurring nucleosides with hydrocarbon linkers (e.g., alkylene) or polyether linkers (e.g., PEG linkers) interposed between one or more nucleosides, modified nucleosides with hydrocarbon or PEG linkers interposed between one or more nucleosides, or combinations thereof.In one embodiment, the nucleotide or modified nucleotide of the nucleic acid ligand can be replaced with a hydrocarbon linker or a polyether linker, provided that the substitution does not substantially reduce the binding affinity and selectivity of the nucleic acid ligand (e.g., the dissociation constant of the aptamer to the target should be no greater than about 1 x 10^-6M). The target molecule of an aptamer is a three-dimensional chemical structure that binds to the aptamer. However, the aptamers are not simple linear complementary sequences of nucleic acid targets, but may include regions that bind by complementary Watson-Crick base pairing interrupted by other structures such as hairpin loops.

Detailed Description

The method of the present invention relates to molecular pattern stamping and/or devices based on reversible self-assembly of molecules, especially organic molecules. The method is suitable for stamping almost any nanostructured inorganic and/or organic device and can be used to transfer a large amount of information from one substrate to another. The working principle of this technique is completely different from any existing nano-structuring technique.

In one embodiment of the invention, the assembly of the second set of molecules is induced by reversible supramolecular chemistry (e.g., hydrogen bonding, ionic bonding, covalent bonding, electrostatic interactions, van der waals interactions, magnetic interactions, or combinations thereof) using a host comprising a substrate having the first set of molecules bound in a pattern to at least one surface. The second set of molecules is attached to the surface of the substrate by using a substantially irreversible surface chemistry, and the reversible bonds between the first set of molecules and the second set of molecules are subsequently broken. As used herein, the term "substantially irreversible" means that the second set of molecules is attached to the surface of the substrate by a bond that is stable under conditions that will disrupt the bond between the first and second sets of molecules. Supramolecular bonds may serve as a mechanism for shape transfer; this avoids the need for mechanical contact between the body and the substrate being stamped and therefore constitutes a major advance over nano-imprinting developed by Chou and co-workers. The method is tailored to reliably deliver organic patterns. The use of organic molecules allows for a large number of variations and enables the simultaneous delivery of multiple surface features.

Referring to FIG. 1, in one embodiment, a method of forming a complementary image of a host includes providing a host 10 comprising a first set of molecules 12 bound to a first substrate 14 to form a pattern. The first set of molecules 12 may include a spacer 11 and a recognition component 13. The second set of molecules 16 is assembled on the first set of molecules by forming bonds. The second set of molecules comprises a reactive functional group 18 and a recognition component 20, the recognition component 20 being bound to the recognition component 13 of the first set of molecules 12 (see fig. 2, which provides an enlarged view of the second set of molecules bound to the first set of molecules). The reactive functional groups 18 of the second set of molecules 16 are then brought into contact with the surface of a second substrate 22. The reactive functional group reacts with a surface of the second substrate to form a bond between the second set of molecules and the second substrate. In one embodiment, the remaining exposed surface of the second substrate may be further contacted with another set of molecules 24, each of the other set of molecules 24 having a reactive functional group, such as an alkane having a thiol substituent, e.g., mercaptohexanol, which is capable of binding to the surface to cover the exposed surface of the second substrate. The bonds between the first set of molecules and the second set of molecules are then broken and the second set of molecules bound to the second substrate form a complementary image 26 of the body 10. Once the body and the complementary image have been separated by breaking the bonds between the first and second sets of molecules, the body can be reused one or more times to form additional complementary images. In one embodiment, the lateral dimension of at least one portion of the complementary image is less than 200nm or less, such as 100nm or less, 50nm or less, or 20nm or less.

In one embodiment, the second set of molecules may further comprise one or more of the following components: exposed functional groups 28; a covalent bond or first spacer 30 linking the reactive functional group to the recognition component; and a covalent bond or a second spacer linking the exposed functional group to the recognition component.

The second set of molecules may include two or more different molecules. For example, two or more molecules of the second set of molecules may have different recognition components, such as different nucleic acid sequences, or two or more molecules of the second set of molecules may have both different recognition components and different exposed functional groups. In some embodiments, one or more molecules from the first set of molecules determine where each molecule from the second set of molecules binds.

In one embodiment, two or more different molecules of the second set of molecules form a pattern on the second substrate, the pattern having a profile comprising two or more heights. For example, the molecules of the second set of molecules may include two or more spacers 30 of different lengths. The difference in length of the spacers may be such that the molecular image transferred to the second substrate has a different height.

In one embodiment, the second set of molecules is assembled on the first set of molecules by contacting the subject with a solution comprising the second set of molecules. In one method of transferring a pattern on a body to a second substrate, the body is held in contact with the second substrate by capillary action of a solution containing the second set of molecules. Mechanical force (e.g., 10) may also be applied^-3Pa to 1GPa) to hold the two substrates together. For example, the force may be on the order of about 10^-3Pa, 1KPa, 1MPa, or 1 GPa. The solution containing the second set of molecules is then slowly evaporated, bringing the host and the second substrate closer together and facilitating the binding of the second set of molecules to the second substrate.

The bonds between the first set of molecules and the second set of molecules may be formed by hydrogen bonds, ionic bonds, covalent bonds, electrostatic interactions, van der waals interactions, magnetic interactions, pi-bond interactions, or combinations thereof. In one embodiment, the bond between the first set of molecules and the second set of molecules is broken by the application of heat. Alternatively or additionally, the bonds between the first set of molecules and the second set of molecules are broken by contacting the bonds with a solution or polar solvent having a high ionic strength. In yet another embodiment, the bonds between the first set of molecules and the second set of molecules are broken by contacting the bonds with a solution having a high ionic strength and applying heat. Alternatively, the bonds between the first set of molecules and the second set of molecules are disrupted by contacting them with a solution containing an enzyme that disrupts the bonds. Typically, the bond between the first set of molecules and the second set of molecules can be broken without breaking the bond between the majority of the second set of molecules and the second substrate.

The reactive functional groups on the second set of molecules may be groups capable of binding to the surface of the second substrate. For example, when the reactive functional group on the second set of molecules is a thiol group or a protected thiol group, the surface of the second substrate may be gold, silver, copper, cadmium, zinc, palladium, platinum, mercury, lead, iron, chromium, manganese, tungsten, or a mixture or alloy of any of these metals. As used herein, the term "reactive functional group" is a group that is capable of reacting to form a bond with the surface of the substrate. Methods for protecting and deprotecting thiol Groups can be found in Greene and Wuts, "Protective Groups in organic Synthesis", John Wiley & Sons (1991), the entire teachings of which are incorporated herein by reference. The protected thiol groups may be deprotected and then reacted with the substrate surface. In another example, the reactive functional groups on the second set of molecules are silanes or chlorosilanes and the surface of the second substrate is doped or undoped silicon, glass, fused silica, or any substrate having an oxidized surface, such as silicon dioxide, aluminum oxide, calcium phosphate ceramic, and hydroxylated polymers. The non-hydroxylated surface may be plasma etched to produce oxidized groups capable of reacting with silane. In another example, the reactive functional group on the second set of molecules is a carboxylic acid and the surface of the second substrate is an oxide, such as silica, alumina, quartz or glass, or an oxidized polymer surface. In another example, the reactive functional group on the second set of molecules is a nitrile or isonitrile and the surface of the second substrate is platinum, palladium, or any alloy thereof. In another example, the active functional group on the second set of molecules is a hydroxamic acid and the surface of the second substrate is copper or aluminum. Phosphonic acids may also be used to attach the second set of molecules to the aluminum substrate.

In one embodiment, at least some of the molecules of the first set of molecules comprise a recognition component that binds to one or more molecules of the second set of molecules. For example, each molecule of the first set of molecules may comprise a nucleic acid sequence recognition component. In one embodiment, each molecule of the first set of molecules comprises a nucleic acid sequence, such as a DAN, an RNA, a modified nucleic acid sequence, or a combination thereof, and the recognition component of the second set of molecules is a nucleic acid sequence. In one embodiment, the nucleic acid recognition component of each molecule of the second set of molecules may be complementary to at least a portion of the nucleic acid sequence of at least one molecule from the molecules of the first set of molecules. For example, three or more consecutive nucleobases (e.g., six or more nucleobases) in molecules from the second set of molecules are complementary to three or more consecutive nucleobases (e.g., six or more nucleobases) in molecules from the first set of molecules. In another example, at least 80%, at least 90%, at least 95%, or at least 99% of the nucleotides on the first set of molecules are complementary to those molecules from the second set of molecules to which they bind. When the second set of molecules is assembled onto the first set of molecules, the second set of molecules will hybridize to molecules from the first set of molecules having a sequence complementary to the nucleic acid recognition component of the second set of molecules, or a portion thereof. In this embodiment, the first set of molecules bound to the subject is contacted with a solution of the second set of molecules under conditions that promote hybridization. Conditions to promote hybridization are known to those skilled in the art. A general description of hybridization conditions is discussed in Ausebel, F.M. et al, Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience, 1989, the teachings of which are incorporated herein by reference. Factors such as sequence length, base composition, percent mismatch between hybridizing sequences, temperature, and ionic strength affect the stability of nucleic acid hybrids.

In one embodiment, the first set of molecules comprises two or more different molecules having different nucleic acid sequence recognition components. In this embodiment, the second set of molecules comprises molecules having a nucleic acid sequence, or portion thereof, complementary to at least one molecule of the first set of molecules. In one embodiment, the hydrogen bonds between the hybrid molecules from the first set of molecules and the second set of molecules are disrupted by contacting the hydrogen bonds with an enzyme. For example, enzymes from the helicase family can be used to break bonds between hybridized nucleic acid molecules. Various helicases have been reported to dehybridize double-stranded oligonucleotides. For example, E.coli Rep, E.coli DnaB, E.coli UvrD (also known as helicase II), E.coli RecBCD, E.coli RecQ, bacteriophage T7 DNA helicase, the human RECQL series; WRN (RECQ2), BLM (RECQL3), RECQL4, RECQL5, s.pombe rqh1, caenorhabditis elegans (c.elegance) T04A11.6 (typically, the helicase name is derived from the organism of origin of the enzyme). Helicases can be divided into two types: 1) a helicase that moves in a 3' direction along a nucleic acid strand; and 2) a helicase that moves in a 5' direction along a nucleic acid strand. In general, the particular helicase type used to disrupt the hydrogen bonds between the hybridizing nucleic acids is selected by taking into account the structural hindrance of the particular hybridizing nucleic acid. Cofactors such as single-stranded DNA binding proteins (SSBs) that stabilize single-stranded DNA may be added.

Another method of disrupting the bond between two hybridized nucleic acids is to use restriction endonucleases that recognize specific base sequences and cleave both strands at specific positions of the nucleic acid sequence. Examples of restriction endonucleases include BamHI, EcoRI and BstXI. Other methods of dehybridization of nucleic acids using enzymes can be found in Lubert Stryer, Biochemistry, 4th Edition; benjamin Lewin, Gene VII; kristen Moore Picha and Smita S.Patel, "Bacteriophage T7 DNAheliase bonds dTTP, Forms Hexamers, and bonds DNA in the Absence of Mg2+," J.biol.chem. (1998), Vol.273, Issue 42, 27315-; sheng Cui, Raffaella Klima, Alex Ocem, Daniel Arosio, Arturo Falaschi and AlessanddroVintigni, "Characterization of the DNA-unwinding Activity of Human RECQ1, a pharmaceutical specific engineered by Human reproduction Protein A," J.biol.chem. (2003), Vol.278, Issue 3, 1424-; umezu, k, and Nakayama, h. (1993), j.mol. Biol,. 230: 1145-; nakayama, k., Irino, n., and Nakayama, h., mol.gen.genet. (1985), 200: 266-271; kusano, k., Berres, m.e., and Engels, w.r., Genetics (1999), 15: 1027-1039; ozsoy, a.z., Sekelsky, j.j. and Matson, s.w., Nucleic Acids Res (2001), 29: 2986-299, the entire teachings of which are incorporated herein by reference.

In another embodiment, the component of each molecule of the first set of molecules is a Peptide Nucleic Acid (PNA) and the recognition component of the second set of molecules is a PNA sequence. Alternatively, the component of each molecule from the first set of molecules is a Peptide Nucleic Acid (PNA) and the recognition component of the second set of molecules is a nucleic acid sequence, or vice versa. PNA molecules hybridize to other PNA molecules and nucleic acid sequences in a manner similar to the hybridization of nucleic acids to other nucleic acids. In one embodiment, at least one or more molecules from the second set of molecules must have at least three consecutive bases (e.g., six consecutive bases) that are complementary to at least three consecutive bases (e.g., six consecutive bases) in the molecules from the first set of molecules. In another example, at least 80%, at least 90%, at least 95%, or at least 99% of the nucleotides on the first set of molecules are complementary to those from the second set of molecules to which they bind.

Alternatively, the bonds between the first set of molecules and the second set of molecules are broken by applying heat, by contacting the bonds with a solution having a high ionic strength or polarity, by applying a magnetic or electric field, or any combination of the above.

When a set of molecules binds to the substrateAt the surface, the molecules may overlap or stack on top of each other, such that a portion of the molecules will be exposed at the surface of the substrate. The exposed functional groups may be hydrophobic, hydrophilic, or amphiphilic functional groups. In addition, the exposed functional group may be a functional group that selectively binds various biological or other chemical substances such as proteins, antibodies, antigens, sugars, and other carbohydrates, and the like. The exposed functional groups may include members of any specific or non-specific binding pair, such as any member of the following non-limiting list: antibodies/antigens, antibodies/haptens, enzymes/substrates, enzymes/inhibitors, enzymes/cofactors, binding proteins/substrates, carrier proteins/substrates, lectins/carbohydrates, receptors/hormones, receptors/effectors, complementary strands of nucleic acids, repressors/inducers, and the like. Other examples of the exposed functional group include, but are not limited to, -OH, -CONH-, -CONHCO-, -NH₂、-NH-、-COOH、-COOR、-CSNH-、-NO₂ ^-、-SO₂、-SH、-RCOR-、-RCSR-、-RSR、-ROR-、-PO₄ ^-3、-OSO₃ ^-2、-SO₃ ^-、-COO^-、-SOO^-、-RSOR-、-CONR₂、-(OCH₂CH₂)_nOH (wherein n is 1-20, preferably 1-8), -CH₃、-PO₃H^-2-imidazole, -N (CH)₃)₂、-N(R)₂、-PO₃H₂、-CN、-(CF₂)_nCF₃(wherein n is 1-20, preferably 1-8) and alkenes, wherein R is hydrogen, a hydrocarbon, a halogenated hydrocarbon, a protein, an enzyme, a carbohydrate, a lectin, a hormone, a receptor, an antigen, an antibody or a hapten.

The exposed functional groups may include protecting groups that can be removed to further modify the complementary image or replica of the host. For example, a photo-removable protecting group may be used. Various positive photoactive groups are known in the art, for example nitroaromatics such as ortho nitrobenzyl derivatives or benzylsulfonyl groups. Photo-removable protecting groups are described, for example, in U.S. patent No. 5,143,854, the entire teachings of which are incorporated herein by reference, and Patchornik, JACS, 92: 6333 (1970) and Amir et al, JOC, 39: 192(1974), both of which are incorporated herein by reference.

In one embodiment, the complementary image may be further modified by binding the exposed functional group of at least one of the molecules of the second set of molecules to a metal or metal ion. For example, the exposed functional group may include an amine, an amide, a nitrosyl, a cyano, a carbonyl, a thiol, a thiocarbonyl, a selenocarbonyl, an alkenyl, an aryl, an arylalkyl, a heteroaryl, a heteroarylalkyl, or a cyclopentadienyl group at a terminal end thereof, or include a linear or cyclic organic group having one or more double bonds or a conjugated pi system. These groups may be coordinated with atoms or ions of metals such as iron, cobalt, nickel, gold, silver, zinc, potassium, phosphorus, selenium, sodium, platinum, palladium, titanium, vanadium, molybdenum, magnesium, rhenium, ruthenium and osmium. When the appropriate chelating group is too large or otherwise unsuitable for placement on the second set of molecules during deposition, the second set of molecules may be modified to attach the appropriate chelating group to at least a portion of the molecules. For example, porphyrin or corrin rings may be attached to at least a portion of the second set of molecules using the same coupling chemistry described below for attaching the recognition component of the second set of molecules to the spacer. Alternatively or additionally, the second set of molecules may comprise peptide sequences or a stretch of enzyme or other protein which function to bind metal atoms or ions. In some embodiments, the metal atom or ion may be coordinated to two, three or more functional groups of the second set of molecules.

The first and second spacers of the first and second sets of molecules may be independently selected from the group consisting of alkylene, heteroalkylene, heterocycloalkylene, alkenylene, alkynylene, arylene, heteroarylene, arylenealkyl, and heteroarylenealkyl. The alkylene, heteroalkylene, heterocycloalkylene, alkenylene, alkynylene, arylene, heteroarylene, arylenealkyl, and heteroarylenealkyl spacers may be substituted or unsubstituted. In one embodiment, either of the first or second spacers or both the first and second spacers are substituted with one or more halogens and/or hydroxyl groups.

In another embodiment, the deposited substrate is fabricated with spacers that are fixed to the substrate by silanes or other reactive functional groups. The end of the spacer includes a reactive group such as an epoxy or carboxylate. In this embodiment, the recognition component 20 of the second set of molecules comprises a reactive group which reacts with a reactive group on the spacer to create a covalent bond between the spacer and the recognition component. For example, the recognition component can be an amine-terminated molecule, such as amine-terminated DNA. The carboxyl-terminated molecules also react with the epoxy groups to form anhydrides. Other chemistries that can be used to couple the second set of molecules to the set of spacers on the substrate include anhydride-hydroxyl, carbodiimide coupling, reaction of carboxylate salts with amines, hydroxyl groups and other groups, and other coupling reactions known to those skilled in the art. The reaction conditions may be selected to maintain the stability of the recognition component. For example, while some identified components are unstable to heat or particular solvents, they are stable to exposure to such conditions for short periods of time (e.g., hours). In some embodiments, the second set of molecules and the reactive group on the end of the spacer do not react with themselves to prevent the deposited spacer or molecules from attaching to each other rather than to the substrate.

The subject may be prepared by any method known to those skilled in the art (see Xia et al, chem. Rev. (1999), 99: 1823-1848, the entire teachings of which are incorporated herein by reference). For example, the method of forming the body may be a nanopatterning (nanopatterning) method. In one embodiment, the body is prepared by forming a pattern of one or more metals, metal oxides, or combinations thereof on a surface of a substrate using electron beam lithography. The surface of the substrate is then contacted with a first set of molecules. In this embodiment, each of the first set of molecules has a reactive functional group that forms a bond between the metal or metal oxide and a molecule in the first set of molecules, thereby binding the first set of molecules to a substrate, forming a host with the first set of molecules bound to the substrate to form a pattern. The reactive groups and substrate material used to form the host may be the same or different from the reactive groups and substrate material used to pattern the second set of molecules.

Alternatively, dip pen nanolithography may be used to prepare the body. Methods for preparing molecularly patterned substrates using dip pen nanolithography are described in Schwartz, Langmuir (2002), 18: 4041-: 661 663, both of which are incorporated herein by reference in their entirety.

Alternatively, the body may be prepared using alternative lithography (replacement lithography), nano-shading (nanostamping), or nano-grafting (nanostamping). These methods are described in Sun et al, JACS (2002), 124 (11): 2414-; amro et al, Langmuir (2000), 16: 3006-3009; liu et al, Nano Letters (2002), 2 (8): 863-867; and Liu et al, acc, chem, res, (2000), 33: 457- > 466; the entire teachings of these documents are incorporated herein by reference.

Another embodiment is a photolithography (lithographic method) wherein at least a portion of the surface of the second substrate is free of the second set of molecules. In this embodiment, the exposed surface of the second substrate is contacted with a reactant selected to be chemically inert to the second set of molecules as a stain resist (resist) and to degrade at least a surface layer of the second substrate, thereby degrading the portion of the surface of the second substrate not containing the second set of molecules. For example, the reactant is a reactive ion etching compound. The second set of molecules is then removed to expose a portion of the surface of the second substrate.

In another embodiment, at least a portion of the second substrate surface is free of the second set of molecules, and a material is deposited onto the portion of the second substrate surface that is free of the second set of molecules. Examples of deposited materials include semiconductors, dielectrics, metals, metal oxides, metal nitrides, metal carbides, and combinations thereof. The second set of molecules is then removed to expose a portion of the surface of the second substrate.

In one aspect of the invention, a method of forming a complementary image of a subject includes assembling a second set of molecules on the first set of molecules by attractive forces. Examples of attractive forces include attraction of molecules with a net positive charge to molecules with a net negative charge, dipole-dipole attraction, and magnetic attraction. In one embodiment, the attractive force is a magnetic force. In one example, when the attractive force is a magnetic force, one or more molecules from the first set of molecules and from the second set of molecules comprise an iron or iron oxide component. In this embodiment, the attractive force between the first set of molecules and the second set of molecules can be destroyed by applying a magnetic field.

In another aspect of the invention, the method includes forming a replica of the body or portions thereof. The host used in this embodiment of the method of the invention comprises a first set of molecules bound to a first substrate to form a pattern. Assembling a second set of molecules on the first set of molecules by forming bonds. The second set of molecules includes a reactive functional group and a recognition component that binds to the first set of molecules. The reactive functional groups of the second set of molecules are then contacted with the surface of a second substrate. The reactive functional group reacts with a surface of the second substrate to form a bond between the second set of molecules and the second substrate. The bond between the first set of molecules and the second set of molecules is then broken, and the second set of molecules bound to the second substrate forms a complementary image of the host. A third set of molecules is then assembled on the second set of molecules of the complementary image by forming bonds. Each molecule of the third set of molecules comprises a reactive functional group and a recognition component bound to the second set of molecules. The reactive functional groups of the third set of molecules are then contacted with the surface of a third substrate. The surface of the third substrate reacts with the reactive functional groups of the third set of molecules to form bonds between the third set of molecules and the third substrate. The bond between the second set of molecules and the third set of molecules is then broken, and the third set of molecules bound to the third substrate forms a replica of the pattern or portion thereof of the body. Once the complementary image and the replica have been separated, the complementary image may be reused one or more times to form additional replicas. In one embodiment, at least one portion of the replica has a lateral dimension of 200nm or less, such as 100nm or less, 50nm or less or 20nm or less.

The method of forming the replica is the same as that used to form the complementary image, except that the complementary image of the master is used as a template (or "master") to transfer the pattern to the third substrate. Thus, the embodiments and examples disclosed above with respect to the second set of molecules and the second substrate also apply to the third set of molecules and the third substrate, respectively. Furthermore, the examples of conditions for assembling a second set of molecules on the first set of molecules and for disrupting the bonds between the first and second sets of molecules can equally apply to the conditions for assembling the third set of molecules on the second set of molecules and for disrupting the bonds between the third and second sets of molecules.

In another embodiment, the invention relates to a molecular printer for producing a complementary image of a host, wherein the host has a first set of molecules bound to a first substrate. The molecular printer comprises means for delivering a solution of a second set of molecules to the surface of the body and means for contacting the second set of molecules with a second substrate. In this embodiment, the second set of molecules includes a reactive functional group; and a recognition component bound to the first set of molecules.

The device may comprise one or more reservoirs containing the second set of molecules and one or more containers or components for immobilizing the body in a position to deliver the solution containing the second set of molecules. Furthermore, the apparatus may comprise computer controlled means for transferring the solution of the second set of molecules from the reservoir to the surface of the subject for use. The apparatus may further comprise a clip to secure the body to the second substrate. The temperature of the solution of the second set of molecules and the container containing the body may also be controlled. The device may further comprise a reservoir containing a solution for breaking the bond between the first and second molecules, such as a solution with a high ionic strength or a solution containing an enzyme that will break the bond, and a means for delivering the solution. Furthermore, after the second substrate has bound to the second set of molecules, a heating element may be used to heat the solution in contact with the bound first and second sets of molecules to break the bonds. The computer controlled means for delivering the solution and controlling the temperature may be any of a variety of commonly used laboratory automation instruments such as those described by Harrison et al, Biotechniques, 14: 88-97 (1993); fujita et al, Biotechniques, 9: 584-591 (1990); wada et al, rev.sci.instrum, 54: 1569-1572(1983), the teachings of all of which are incorporated herein by reference. Suitable laboratory robots are also commercially available, for example, Applied Biosystems model 800 Catalyst (Foster City, Calif.). In one embodiment, the apparatus further comprises means for separating the second substrate and the host after the bond between the first set of molecules and the second set of molecules has been broken.

These and other aspects of the present invention will be further understood upon consideration of the following examples, which are intended to illustrate certain specific embodiments of the present invention, and are not intended to limit its scope, as defined by the claims.

Examples

Example 1:preparation of complementary images of DNA monolayers

A.Preparation of DNA solution

Before use, 75% H is used₂SO₄And 25% H₂O₂The solution of (2) washes all glassware. All water used is ultrapure water (18MΩ/cm)。

The first DNA 5 '-/5-thiol MC6-D/ACG CAA CTT CGG GCT CTT-3' was purchased from Integrated DNA Technologies, Inc. (IDT), Coraville, IA. All DNA strands were used in the state accepted from the manufacturer. The first DNA was dissolved in water at a concentration of 1. mu.g/mL, divided into smaller aliquots of 50. mu.L, and stored at-20 ℃. When a portion of this solution was used, it was reduced by placing an aliquot in 40mM buffer solution (0.17M sodium phosphate, pH 8) containing Dithiothreitol (DTT) for 16 hr. Size exclusion chromatography (NAP 10 column from Pharmacia Biotech) was used to separate the oligonucleotides and the by-products of the DTT reaction according to the manufacturer's instructions. The column was equilibrated and the oligonucleotide eluted using 10mM sodium phosphate buffer (pH 6.8). The concentration of the resulting DNA solution was calculated from the absorbance of the solution at 260 nm. In the case of the first DNA (i.e., the DNA was used to form a host), 1M potassium phosphate buffer solution (pH 3.8) was added to the DNA solution to increase the ionic strength of the solution. The final concentration of DNA was 4-5. mu.M.

In the case of the second DNA solution (i.e., complementary image formation using DNA), 1M NaCl in TE buffer (10mM Tris buffer pH 7.2 and 1mM EDTA) was added to increase the ionic strength of the solution. The second DNA used was purchased from Integrated DNA Technologies, Inc (IDT), claville, IA, and has the following structure: 5 '-/5 mercaptan MC6-D/AAG AGC CCG AAG TTG CGT-3'.

B.Preparation of hosts with DNA monolayers

Clean and atomically flat gold on mica was used as the substrate. The substrate was left in the first DNA solution prepared above for 5 days to allow DAN to bind to the surface of the substrate. The substrate was rinsed twice with 1M potassium phosphate buffer and five times with water. The substrate was exposed to 1mM aqueous spacer thiol 6-mercapto-1-hexanol for 2hr to minimize non-specific adsorption of single-stranded DNA, and then rinsed five times with water.

C.Preparation of complementary images

Soaking the subject prepared in step B in the second DNA solution for 2 hours to allow the complementary DNA to hybridize with the DNA bound to the subject. The substrate was rinsed twice with 1M NaCl in TE buffer and five times with water.

Clean gold on the second mica substrate was brought into contact with the bulk such that the two gold surfaces faced each other with a small amount of water between them. A small mechanical force is applied to push the two substrates together. As the water between the two substrates evaporates, the spacing between the surfaces decreases due to the increase in capillary attraction. Thus, the thiol group of the second DNA approaches the second substrate and binds to it. After about 5hr, the substrate was soaked in TE buffer for 20min with 1M NaCl (70 ℃). The substrates (i.e., bulk and complementary images) were autosegregating, rinsed twice with 1m nacl in TE buffer, and rinsed five times with water, then air dried. Both the subject (see fig. 3A and 3B) and the complementary images (see fig. 3C and 3D) were imaged using the AFM tapping mode.

D.Results

The DNA completely covers the first substrate surface. Due to the strong interaction between the monolayer and the tip, thorough coverage makes AFM imaging difficult. The layer transferred to the second substrate is also completely covered.

Example 2:pattern transfer of gold grids

The AFM calibration gold grid was soaked for 5 days in a 4 μ M solution of the first DNA molecule described in example 1 to create a patterned body. The subject was exposed to 1mM 6-mercapto-1-hexanol in water for 2hr to minimize non-specific adsorption of single-stranded DNA, and then rinsed 5 times with water and air-dried. The subject was then exposed to a 6 μ M solution of the second DNA described in example 1 for 2 hours, whereby hybridization occurred. The gold substrate on the second mica was placed on the body so that the two gold surfaces faced each other with a small amount of water between them. A small mechanical force is applied to push the two substrates together. After about 5hr, the substrate was soaked in 1M NaCl (70 ℃) in TE buffer for 20 min. The two substrates (i.e., bulk and complement images) were autosegregating, rinsed twice with 1M NaCl in TE buffer and five times with water, and then air dried. Both the subject and the complementary image were imaged using an AFM tapping mode (see fig. 4A and 4B, respectively).

Example 3:production of DNA chip

E.g., Demer et al, angelw.chem.int.ed. (2001), 40: 3071 preparation of bodies using dip-pen nanolithography as described in 3073, the entire teachings of which are incorporated herein by reference. To prepare the host, the gold surface on the mica substrate was contacted with a 1mM solution of 1-Octadecanethiol (ODT) in ethanol for 5min to cover the exposed gold surface with ODT molecules. The substrate was then immersed in a 1mM solution of 1, 16-mercaptohexadecanoic acid (MHA) and the ODT molecules bound to the surface were displaced using the tip of an atomic force microscope by contacting the surface with a force of about 0.5nN to create a 100nm circle. The MHA in solution binds to the exposed gold surface of the dot. The carboxylic acid groups of MHA were activated with a 10mg/mL solution of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC) in 0.1M morpholine/ethanesulfonic acid at pH 4.5, followed by rinsing with a 0.1M sodium borate/boric acid buffer solution at pH 9.5. A25. mu.M solution of DNA modified with a 1-hexylamine group in borate buffer was placed on the surface of the substrate. The amine groups of the DNA bind to the activated MHA molecules, forming DNA circles of 100nm diameter. The procedure of forming MHA dots and binding DNA molecules to the MHA dots was repeated multiple times with different amine modified DNA molecules to form a body with a DNA array portion of about 100 nm.

Printing an array of complementary images of the DNA sequences using the body onto a second substrate, wherein each DNA sequence is complementary to a DNA molecule on the body and is located on the second substrate in a position that is a mirror image of its complementary sequence on the body. Complementary image arrays are prepared by modifying a set of DNA molecules, including all DNA molecules complementary to DNA molecules on a subject, with a hexyl thiol linker. The thiol-modified DNA molecule was placed in a phosphate buffer containing 1M NaCl at pH 6.8. The host was soaked in the solution containing the thiol-modified DNA molecule for 2hr, then the host was removed from the solution, rinsed once with 1M NaCl in TE buffer, and rinsed five times with water.

Clean gold on the second mica substrate was brought into contact with the bulk such that the two gold surfaces faced each other with a small amount of water between them. A small mechanical force is applied to push the two substrates together. As the water between the two substrates evaporates, the spacing between the surfaces decreases due to the increase in capillary attraction. Thus, the thiol group of the thiol-modified DNA molecule approaches the second substrate and binds to it. After about 5hr, the substrate was soaked in 1M NaCl (70 ℃) in TE buffer for 20 min. The substrates were autosegregating, rinsed twice with 1M NaCl in TE buffer and five times with water and then air dried. One or more additional complementary images can be prepared using the subject.

Example 4:preparation of complementary images of DNA arrays

The DNA chip was purchased and used as a first body. The DNA chip has a 12X 12 square array in which each square is 300nm X300 nm and has a different DNA sequence attached to the substrate for a total of 144 different DNA sequences. The 300nm by 300nm squares are spaced 100nm apart along the x-axis and y-axis of the substrate surface.

An array of 12 x 12 complementary images of DNA sequences, each complementary to a DNA molecule on the host, are printed using the host on a second substrate, in positions on the second substrate that are mirror images of their complementary sequences on the host. A set of DNA molecules, including all DNA molecules complementary to DNA molecules on the subject (i.e., 144 different complementary DNA sequences), is modified with a hexyl thiol linker. The thiol-modified DNA molecule was placed in a phosphate buffer containing 1M NaCl at pH 6.8. The host was soaked in the solution containing the thiol-modified DNA molecule for 2hr, then the host was removed from the solution, rinsed once with 1M NaCl in TE buffer, and rinsed five times with water.

Clean gold on the mica substrate was brought into contact with the bulk such that the gold surface of the new substrate faced a 12 × 12 array of DNA molecules. There is a small amount of water between the two surfaces. A small mechanical force is applied to push the two substrates together. As the water between the two substrates evaporates, the spacing between the surfaces decreases due to the increase in capillary attraction. Thus, the thiol group of the thiol-modified DNA molecule approaches the second substrate and binds to it. After about 5hr, the substrate was soaked in 1M NaCl (70 ℃) in TE buffer for 20 min. The substrates were autosegregating, rinsed twice with 1m nacl in TE buffer, and five times with water, then air dried. The complementary image has a 12 x 12 array of DNA molecules that are complementary to the DNA molecules on the host. Following the same procedure, one or more additional complementary image arrays can be prepared using the subject.

Furthermore, following this procedure, the first body can be replicated one or more times, except that the complementary image is used instead of the body, and a third set of 144 DNA molecules, having the same sequence as the DNA molecules on the first body and modified with a hexyl thiol linker, is assembled on the complementary image. A third gold substrate on the mica was then contacted with the complementary image as described above with respect to the first host and second substrate. A third set of DNA bound to a third substrate and separated from the complementary image is a replica of the first master.

Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification or practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

Claims (139)

1. A method of forming a complementary image of a subject, comprising:

a) providing a host comprising a first set of molecules bound to a first substrate to form a pattern, wherein when the first set of molecules comprises nucleic acids, the first set of molecules comprises a plurality of nucleic acids having different sequences;

b) assembling a second set of molecules on the first set of molecules by attraction or formation of bonds, wherein each molecule of the second set of molecules comprises:

i) a reactive functional group; and

ii) a recognition component attracted to or bound to one or more molecules of the first set of molecules;

c) contacting the reactive functional group of the second set of molecules with a surface of a second substrate, thereby forming a bond between the second set of molecules and the second substrate;

d) disrupting the attractive forces or bonds between the first set of molecules and the second set of molecules, thereby forming a complementary image of the subject; and

e) optionally repeating b) to d) one or more times.

2. The method of claim 1, wherein each molecule of the second set of molecules further comprises one or more of the following components:

a) exposed functional groups;

b) a covalent bond or a first spacer linking the reactive functional group to the recognition component; and

c) a covalent bond or a second spacer that links the exposed functional group to the recognition component.

3. The method of claim 2, wherein the second set of molecules is assembled on the first set of molecules by contacting the subject with a solution comprising the second set of molecules.

4. The method of claim 3, wherein the body is held in contact with the second substrate by capillary action of a solution containing the second set of molecules.

5. The method of claim 4, wherein disrupting the attractive forces or bonds comprises evaporating the solution.

6. The method of claim 3, wherein the second set of molecules comprises two or more different molecules.

7. The method of claim 6, wherein two or more different molecules of the second set of molecules have different recognition components.

8. The method of claim 6, wherein two or more different molecules of the second set of molecules have both different recognition components and different exposed functional groups.

9. The method of claim 6, wherein two or more different molecules of the second set of molecules form a pattern on the second substrate, the pattern having a profile that includes two or more heights.

10. The method of claim 9, wherein at least one of the two or more different molecules comprises a first spacer and another of the two or more different molecules comprises no spacer or a second spacer of a different length than the first spacer.

11. The method of claim 2, wherein the at least one portion of the complementary image has a lateral dimension of 200nm or less.

12. The method of claim 11, wherein the at least one portion of the complementary image has a lateral dimension of 100nm or less.

13. The method of claim 12, wherein the at least one portion of the complementary image has a lateral dimension of 50nm or less.

14. The method of claim 13, wherein the at least one portion of the complementary image has a lateral dimension of 20nm or less.

15. The method of claim 2, wherein the bonds formed between the first set of molecules and the second set of molecules are hydrogen bonds, ionic bonds, pi-bond interactions, covalent bonds, van der waals bonds, or a combination thereof.

16. The method of claim 2, wherein the attractive force between the first set of molecules and the second set of molecules is a magnetic interaction, an electrostatic interaction, or a combination thereof.

17. The method of claim 15, wherein the bond between the first set of molecules and the second set of molecules is broken by the application of heat.

18. The method of claim 15, wherein the bonds between the first set of molecules and the second set of molecules are broken by contacting the bonds with a solution having a high ionic strength.

19. The method of claim 2, wherein the reactive functional group on the second set of molecules is a thiol, silane, chlorosilane, carboxylic acid, nitrile, isonitrile, hydroxamic acid, or phosphonic acid.

20. The method of claim 19, wherein the thiol group is a protected thiol group.

21. The method of claim 2, wherein the surface of the second substrate is doped or undoped silicon, glass, fused silica, calcium phosphate ceramic, hydroxylated polymer, oxidized polymer surface, oxide, platinum, palladium, aluminum, gold, silver, copper, cadmium, zinc, mercury, lead, iron, chromium, manganese, tungsten, an alloy comprising at least one of platinum, palladium, aluminum, gold, silver, copper, cadmium, zinc, mercury, lead, iron, chromium, manganese, and tungsten, or a mixture comprising at least one of platinum, palladium, aluminum, gold, silver, copper, cadmium, zinc, mercury, lead, iron, chromium, manganese, and tungsten.

22. The method of claim 21, wherein the oxide is silica or alumina.

23. The method of claim 2, wherein the component of each molecule in the first set of molecules is a nucleic acid sequence and the recognition component of the second set of molecules is a nucleic acid sequence that is at least 80% complementary to the nucleic acid sequence on the first set of molecules.

24. The method of claim 23, wherein the recognition component of the second set of molecules is a nucleic acid sequence that is at least 90% complementary to the nucleic acid sequence on the first set of molecules.

25. The method of claim 24, wherein the recognition component of the second set of molecules is a nucleic acid sequence that is at least 95% complementary to the nucleic acid sequence on the first set of molecules.

26. The method of claim 25, wherein the recognition component of the second set of molecules is a nucleic acid sequence that is at least 99% complementary to the nucleic acid sequence on the first set of molecules.

27. The method of any one of claims 23-26, wherein the bond between the first set of molecules and the second set of molecules is disrupted by contacting the bond with an enzyme.

28. The method of any one of claims 23-26, wherein the nucleic acid sequences of the first and second sets of molecules are selected from the group consisting of DNA, RNA, modified nucleic acid sequences, and combinations thereof.

29. The method of claim 2, wherein the component of each molecule of the first set of molecules is a Peptide Nucleic Acid (PNA) sequence and the recognition component of the second set of molecules is a PNA sequence.

30. Claim 2Wherein the exposed functional group of each molecule of the second set of molecules is absent or independently selected from-OH, -CONH-, -CONHCO-, -NH₂、-NH-、-COOH、-COOR、-CSNH-、-NO₂ ^-、-SO₂、-SH、-RCOR-、-RCSR-、-RSR、-ROR-、-PO₄ ^-3、-OSO₃ ^-2、-SO₃ ^-、-COO^-、-SOO^-、-RSOR-、-CONR₂Wherein n is 1-20- (OCH)₂CH₂)_nOH、-CH₃、-PO₃H^-2-imidazole, -N (CH)₃)₂、-NR₂、-PO₃H₂- (CF) of, -CN, where n is 1-20₂)_nCF₃Porphyrins, corrin rings, peptide sequences and alkenes,

wherein R is hydrogen, a hydrocarbon, a halogenated hydrocarbon, a protein, an enzyme, a carbohydrate, a lectin, a hormone, a receptor, an antigen, an antibody or a hapten.

31. The method of claim 30, wherein at each occurrence, n-1-8.

32. The method of claim 30, further comprising binding the exposed functional group of at least one molecule of the second set of molecules to a metal or metal ion.

33. The method of claim 2, wherein the second set of molecules has a first spacer, a second spacer, or first and second spacers, and the spacers are independently selected from the group consisting of alkylene, heteroalkylene, heterocycloalkylene, alkenylene, alkynylene, arylene, heteroarylene, arylenealkyl, and heteroarylenealkyl, wherein the alkylene, heteroalkylene, heterocycloalkylene, alkenylene, alkynylene, arylene, heteroarylene, arylenealkyl, or heteroarylenealkyl may be substituted or unsubstituted.

34. The method of claim 33, wherein the substituents of said alkylene, heteroalkylene, heterocycloalkylene, alkenylene, alkynylene, arylene, heteroarylene, arylenealkyl, and heteroarylenealkyl are selected from the group consisting of halogen and hydroxy.

35. The method of claim 2, further comprising:

a) forming a pattern of one or more metals, metal oxides, or combinations thereof on a surface of the first substrate;

b) contacting the surface with the first set of molecules, wherein each molecule of the first set of molecules has a reactive functional group that forms a bond between the metal or metal oxide and a molecule of the first set of molecules, thereby forming a body comprising the first set of molecules bound to the substrate to form a pattern.

36. The method of claim 2, wherein providing the body comprises forming the body using dip pen nanolithography.

37. The method of claim 2, wherein at least a portion of the second substrate surface is free of the second set of molecules.

38. The method of claim 37, further comprising:

a) contacting a surface of the second substrate with a reactant selected to be chemically inert to the second set of molecules and degrade at least a surface layer of the second substrate, thereby degrading the portion of the surface of the second substrate not containing the second set of molecules; and

b) removing the second set of molecules to expose a portion of the surface of the second substrate.

39. The method of claim 38, wherein the reactant is a reactive ion etching compound.

40. The method of claim 37, further comprising:

a) depositing a material on the portion of the surface of the second substrate that does not contain the second set of molecules; and

b) removing the second set of molecules to expose a portion of the surface of the second substrate.

41. The method of claim 40, wherein the deposited material is selected from the group consisting of semiconductors, dielectrics, metals, metal oxides, metal nitrides, metal carbides, and combinations thereof.

42. The method of claim 2, wherein the second set of molecules is assembled on the first set of molecules by magnetic attraction.

43. The method of claim 42, wherein the attractive force between the first set of molecules and the second set of molecules is disrupted by applying a magnetic field.

44. The method of claim 42, wherein the recognition component of one or more molecules of the second set of molecules is iron or iron oxide particles.

45. A method of forming a replica of a body or part thereof, comprising:

a) providing a host comprising a first set of molecules bound to a first substrate to form a pattern;

b) assembling a second set of molecules on the first set of molecules by forming bonds, wherein each molecule in the second set of molecules comprises:

i) a reactive functional group; and

ii) a recognition component bound to said first set of molecules;

c) contacting the reactive functional group of the second set of molecules with a surface of a second substrate, thereby forming a bond between the second set of molecules and the second substrate;

d) disrupting the bonds between the first set of molecules and the second set of molecules, thereby forming a complementary image of the subject;

e) assembling a third set of molecules on the second set of molecules of the complementary image by magnetic interaction, electrostatic interaction, or a combination thereof, or forming a bond, wherein each molecule of the third set of molecules comprises:

i) a reactive functional group; and

ii) a recognition component bound to said second set of molecules;

f) contacting the reactive functional group of the third set of molecules with a surface of a third substrate, thereby forming a bond between the third set of molecules and the third substrate;

g) disrupting the bonds between the second set of molecules and the third set of molecules, thereby forming a replica of the body or portion thereof; and

h) optionally repeating e) to g) one or more times.

46. The method of claim 45, wherein each molecule of the second set of molecules optionally further comprises a spacer linking the active functional group of the second set of molecules to the recognition component bound to the first set of molecules, and each molecule of the third set of molecules further comprises one or more of the following components:

a) exposed functional groups;

b) (ii) a covalent bond or a first spacer linking the reactive functional group of the third set of molecules to the recognition component bound to the second set of molecules; and

c) attaching the exposed functional group to the covalent bond or a second spacer bound to the recognition component on the second set of molecules.

47. The method of claim 46, wherein the third set of molecules is assembled on the second set of molecules by contacting the complementary images with a solution comprising the third set of molecules.

48. The method of claim 47 wherein said complementary image is maintained in contact with said third substrate by capillary action of a solution containing said third set of molecules.

49. The method of claim 48, wherein the solution is evaporated to break bonds between the second set of molecules and the third set of molecules.

50. The method of claim 46, wherein the third set of molecules comprises two or more different molecules.

51. The method of claim 50, wherein two or more different molecules of the third set of molecules have different recognition components.

52. The method of claim 50, wherein two or more different molecules of the third set of molecules have both different recognition components and different exposed functional groups.

53. The method of claim 50, wherein two or more different molecules of the third set of molecules form a pattern on the third substrate, the pattern having a profile comprising two or more heights.

54. The method of claim 46, wherein at least one portion of the replica of the body or portion thereof has a lateral dimension of 200nm or less.

55. The method of claim 54, wherein at least one portion of the replica of the body or portion thereof has a lateral dimension of 100nm or less.

56. The method of claim 55, wherein at least one portion of the replica of the body or portion thereof has a lateral dimension of 50nm or less.

57. The method of claim 56, wherein at least one portion of the replica of the body or portion thereof has a lateral dimension of 20nm or less.

58. The method of claim 46, wherein the bonds formed between the second set of molecules and the third set of molecules are hydrogen bonds, ionic bonds, covalent bonds, pi-bond interactions, van der Waals bonds, or combinations thereof.

59. The method of claim 58, wherein the bond between the second set of molecules and the third set of molecules is broken by the application of heat.

60. The method of claim 58, wherein the bonds between the second set of molecules and the third set of molecules are broken by contacting the bonds with a solution having a high ionic strength.

61. The method of claim 46, wherein the component of each molecule of the second set of molecules is a nucleic acid sequence and the recognition component of the third set of molecules is a nucleic acid sequence that is at least 80% complementary to the recognition component of the second set of molecules.

62. The method of claim 61, wherein the recognition component of said third set of molecules is a nucleic acid sequence that is at least 90% complementary to the recognition component of said second set of molecules.

63. The method of claim 62, wherein the recognition component of said third set of molecules is a nucleic acid sequence that is at least 95% complementary to the recognition component of said second set of molecules.

64. The method of claim 63, wherein the recognition component of said third set of molecules is a nucleic acid sequence that is at least 99% complementary to the recognition component of said second set of molecules.

65. The method of any one of claims 61-64, wherein the second set of molecules comprises two or more molecules having different nucleic acid sequences.

66. The method of any one of claims 61-64, wherein two or more different molecules of the third set of molecules have different nucleic acid sequences.

67. The method of any one of claims 61-64, wherein the bond between the second set of molecules and the third set of molecules is disrupted by contacting the bond with an enzyme.

68. The method of any one of claims 61-64, wherein the component of one or more molecules of the first set of molecules is a nucleic acid sequence.

69. The method of claim 68, wherein the nucleic acid sequences of said first, second and third sets of molecules are selected from the group consisting of DNA, RNA, modified nucleic acid sequences, and combinations thereof.

70. The method of claim 46, wherein the component of each molecule of the first set of molecules is a Peptide Nucleic Acid (PNA) sequence and the recognition component of the second set of molecules is a PNA sequence.

71. The method of claim 46, wherein the exposed functional group of each molecule of the third set of molecules is absent or independently selected from-OH, -CONH-, -CONHCO-, -NH₂、-NH-、-COOH、-COOR、-CSNH-、-NO₂ ^-、-SO₂、-SH、-RCOR-、-RCSR-、-RSR、-ROR-、-PO₄ ^-3、-OSO₃ ^-2、-SO₃ ^-、-COO^-、-SOO^-、-RSOR-、-CONR₂Wherein n is 1-20-(OCH₂CH₂)_nOH、-CH₃、-PO₃H^-2-imidazole, -N (CH)₃)₂、-NR₂、-PO₃H₂- (CF) of, -CN, where n is 1-20₂)_nCF₃Porphyrins, corrin rings and olefins,

wherein R is hydrogen, a hydrocarbon, a halogenated hydrocarbon, a protein, an enzyme, a carbohydrate, a lectin, a hormone, a receptor, an antigen, an antibody or a hapten.

72. The method of claim 71, wherein at each occurrence, n-1-8.

73. The method of claim 71, further comprising the step of binding the exposed functional group of at least one molecule of the third set of molecules to a metal or metal ion.

74. The method of claim 46, wherein the molecules of the second set of molecules have a spacer and the molecules of the third set of molecules have a first and second spacer, and the spacers are independently selected from the group consisting of alkylene, heteroalkylene, heterocycloalkylene, alkenylene, alkynylene, arylene, heteroarylene, aralkylene, and heteroarylalkylene, wherein the alkylene, heteroalkylene, heterocycloalkylene, alkenylene, alkynylene, arylene, heteroarylene, aralkylene, or heteroaralkylene can be substituted or unsubstituted.

75. The method of claim 74, wherein the substituents of said alkylene, heteroalkylene, heterocycloalkylene, alkenylene, alkynylene, arylene, heteroarylene, arylenealkyl, and heteroarylenealkyl are selected from the group consisting of halogen and hydroxy.

76. The method of claim 46, further comprising:

a) forming a pattern of one or more metals, metal oxides, or combinations thereof on a surface of the first substrate using electron beam lithography;

b) contacting the surface with the first set of molecules, wherein each molecule of the first set of molecules has a reactive functional group that forms a bond between the metal or metal oxide and a molecule of the first set of molecules, thereby forming a body comprising the first set of molecules bound to the substrate to form a pattern.

77. The method of claim 46, wherein at least a portion of the third substrate surface is free of the third set of molecules.

78. The method of claim 77, further comprising:

a) contacting a surface of the third substrate with a reactant selected to be chemically inert to the third set of molecules and degrade at least a surface layer of the third substrate, thereby degrading the portion of the surface of the third substrate not containing the third set of molecules; and

b) removing the third set of molecules to expose a portion of the surface of the third substrate.

79. The method of claim 78, wherein the reactant is a reactive ion etching compound.

80. The method of claim 77, further comprising:

a) depositing a material on the portion of the surface of the third substrate that does not contain the third set of molecules; and

b) removing the third set of molecules to expose a portion of the surface of the third substrate.

81. The method of claim 80, wherein the deposited material is selected from the group consisting of semiconductors, dielectrics, metals, metal oxides, metal nitrides, metal carbides, and combinations thereof.

82. A composition comprising:

a) a first pattern of a first set of molecules bound to a first substrate, wherein when the first set of molecules comprises nucleic acid sequences, the first set of molecules comprises a plurality of nucleic acids having different sequences; and

b) a complementary image comprising a pattern of a second set of molecules bound to a second substrate via an active functional group on each molecule in the second set of molecules, wherein each molecule in the second set of molecules has a recognition component bound to at least a portion of the molecules from the first set of molecules.

83. The composition of claim 82, wherein each molecule of the second set of molecules further comprises one or more of the following components:

a) exposed functional groups;

b) a covalent bond or a first spacer linking the reactive functional group to the recognition component; and

c) a covalent bond or a second spacer that links the exposed functional group to the recognition component.

84. The composition of claim 83, wherein the second set of molecules comprises two or more different molecules.

85. The composition of claim 84, wherein two or more different molecules of the second set of molecules have different recognition components.

86. The composition of claim 84, wherein two or more different molecules of the second set of molecules have both different recognition components and different exposed functional groups.

87. The composition of claim 86, wherein two or more different molecules of the second set of molecules form a pattern on the second substrate, the pattern having a profile comprising two or more heights.

88. The composition of claim 87, wherein at least one of said two or more different molecules comprises a first spacer and another of said two or more different molecules does not comprise a spacer or comprises a second spacer of a different length than said first spacer.

89. The composition of claim 83, wherein at least one portion of the complementary image has a lateral dimension of 200nm or less.

90. The composition of claim 89, wherein at least one portion of said complementary image has a lateral dimension of 100nm or less.

91. The composition of claim 90, wherein at least one portion of the complementary image has a lateral dimension of 50nm or less.

92. The composition of claim 91, wherein at least one portion of the complementary image has a lateral dimension of 20nm or less.

93. The composition according to claim 83, wherein there is a magnetic interaction, an electrostatic interaction, or a combination thereof, or a bond is formed between the first set of molecules and the second set of molecules, and the bond is a hydrogen bond, an ionic bond, a covalent bond, a pi-bond interaction, a van der waals bond, or a combination thereof.

94. The composition of claim 83, wherein the reactive functional group on the second set of molecules is a thiol, silane, chlorosilane, carboxylic acid, nitrile, isonitrile, hydroxamic acid, or phosphonic acid.

95. The composition of claim 94, wherein said thiol group is a protected thiol group.

96. The composition of claim 83, wherein the surface of the second substrate is doped or undoped silicon, glass, fused silica, calcium phosphate ceramic, hydroxylated polymer, oxidized polymer surface, oxide, platinum, palladium, aluminum, gold, silver, copper, cadmium, zinc, mercury, lead, iron, chromium, manganese, tungsten, an alloy comprising at least one of platinum, palladium, aluminum, gold, silver, copper, cadmium, zinc, mercury, lead, iron, chromium, manganese, and tungsten, or a mixture comprising at least one of platinum, palladium, aluminum, gold, silver, copper, cadmium, zinc, mercury, lead, iron, chromium, manganese, and tungsten.

97. The composition of claim 96, wherein the oxide is silica or alumina.

98. The composition of claim 83, wherein the component of each molecule of said first set of molecules is a nucleic acid sequence and the recognition component of said second set of molecules is a nucleic acid sequence that is at least 80% complementary to the nucleic acid sequence of said first set of molecules.

99. The composition of claim 98, wherein the recognition component of said second set of molecules is a nucleic acid sequence that is at least 90% complementary to the nucleic acid sequence of said first set of molecules.

100. The composition of claim 99, wherein the recognition component of said second set of molecules is a nucleic acid sequence that is at least 95% complementary to the nucleic acid sequence of said first set of molecules.

101. The composition of claim 100, wherein said second set of molecules' recognition component is a nucleic acid sequence that is at least 99% complementary to the nucleic acid sequence of said first set of molecules.

102. The composition of any one of claims 98-101, wherein the nucleic acid sequences of the first and second sets of molecules are selected from the group consisting of DNA, RNA, modified nucleic acid sequences, and combinations thereof.

103. The composition of any one of claims 98-101, wherein the component of each molecule of said first set of molecules is a Peptide Nucleic Acid (PNA) sequence and the recognition component of said second set of molecules is a PNA sequence.

104. The composition of claim 93, wherein the exposed functional group of each molecule of the second set of molecules is absent or independently selected from-OH, -CONH-, -CONHCO-, -NH₂、-NH-、-COOH、-COOR、-CSNH-、-NO₂ ^-、-SO₂、-SH、-RCOR-、-RCSR-、-RSR、-ROR-、-PO₄ ^-3、-OSO₃ ^-2、-SO₃ ^-、-COO^-、-SOO^-、-RSOR-、-CONR₂Wherein n is 1-20- (OCH)₂CH₂)_nOH、-CH₃、-PO₃H^-2-imidazole, -N (CH)₃)₂、-NR₂、-PO₃H₂- (CF) of, -CN, where n is 1-20₂)_nCF₃Porphyrins, corrin rings and olefins,

wherein R is hydrogen, a hydrocarbon, a halogenated hydrocarbon, a protein, an enzyme, a carbohydrate, a lectin, a hormone, a receptor, an antigen, an antibody or a hapten.

105. The composition of claim 104, wherein at each occurrence, n-1-8.

106. The composition of claim 104, further comprising a metal or metal ion bound to an exposed functional group of at least one molecule from the second set of molecules.

107. The composition of claim 83, wherein each molecule in said second set of molecules has a first spacer, a second spacer, or first and second spacers, and said spacers are independently selected from the group consisting of alkylene, heteroalkylene, heterocycloalkylene, alkenylene, alkynylene, arylene, heteroarylene, aralkylene, and heteroarylalkylene, wherein said alkylene, heteroalkylene, heterocycloalkylene, alkenylene, alkynylene, arylene, heteroarylene, aralkylene, or heteroaralkylene can be substituted or unsubstituted.

108. The composition of claim 107 wherein the substituents for said alkylene, heteroalkylene, alkenylene, alkynylene, arylene, heteroarylene, heterocycloalkylene, arylenealkyl, and heteroarylenealkyl are selected from the group consisting of halogen and hydroxy.

109. The composition of claim 83, wherein at least a portion of the second substrate surface is free of the second set of molecules.

110. The composition of claim 82, wherein the first substrate having the first pattern is a reusable host.

111. A kit for printing a molecular pattern on a substrate, comprising:

a) a body comprising a pattern of a first set of molecules bound to a substrate; and

b) a second set of molecules, wherein each molecule of the second set of molecules comprises:

i) a reactive functional group; and

ii) a recognition component bound to the first set of molecules.

112. The kit of claim 111, wherein each molecule of the second set of molecules further comprises one or more of the following components:

a) exposed functional groups;

b) a covalent bond or a first spacer linking the reactive functional group to the recognition component; and

c) a covalent bond or a second spacer that links the exposed functional group to the recognition component.

113. The kit of claim 112, wherein the second set of molecules comprises two or more different molecules.

114. The kit of claim 113, wherein two or more different molecules of the second set of molecules have different recognition components.

115. The kit of claim 113 wherein two or more different molecules of the second set of molecules have both different recognition components and different exposed functional groups.

116. The kit of claim 115, wherein at least one of the two or more different molecules comprises a first spacer and another of the two or more different molecules does not comprise a spacer or comprises a second spacer of a different length than the first spacer.

117. The kit of claim 112, wherein at least a portion of the body has a lateral dimension of less than 200 nm.

118. The kit of claim 112, wherein the recognition component of each molecule of the second set of molecules is capable of binding to at least one molecule of the first set of molecules via hydrogen bonds, ionic bonds, covalent bonds, van der waals bonds, magnetic interactions, pi-bond interactions, electrostatic interactions, or any combination thereof.

119. The kit of claim 118, further comprising a solution having a high ionic strength capable of breaking the bond between the first set of molecules and the second set of molecules.

120. The kit of claim 112, further comprising a second substrate bound to the reactive functional groups of the second set of molecules.

121. The kit of claim 112, wherein the reactive functional group on the second set of molecules is a thiol, silane, chlorosilane, carboxylic acid, nitrile, isonitrile, hydroxamic acid, or phosphonic acid.

122. The kit of claim 121, wherein the thiol group is a protected thiol group.

123. The kit of claim 112, wherein the surface of the second substrate is doped or undoped silicon, glass, fused silica, calcium phosphate ceramic, hydroxylated polymer, oxidized polymer surface, oxide, platinum, palladium, aluminum, gold, silver, copper, cadmium, zinc, mercury, lead, iron, chromium, manganese, tungsten, an alloy comprising at least one of platinum, palladium, aluminum, gold, silver, copper, cadmium, zinc, mercury, lead, iron, chromium, manganese, and tungsten, or a mixture comprising at least one of platinum, palladium, aluminum, gold, silver, copper, cadmium, zinc, mercury, lead, iron, chromium, manganese, and tungsten.

124. The kit of claim 123, wherein the oxide is silica or alumina.

125. The kit of claim 120, wherein the component of each molecule of the first set of molecules is a nucleic acid sequence and the recognition component of the second set of molecules is a nucleic acid sequence that is at least 80% complementary to at least one nucleic acid sequence from the first set of molecules.

126. The kit of claim 125, wherein the recognition component of the second set of molecules is a nucleic acid sequence that is at least 90% complementary to at least one nucleic acid sequence from the first set of molecules.

127. The kit of claim 126, wherein the recognition component of the second set of molecules is a nucleic acid sequence that is at least 95% complementary to at least one nucleic acid sequence from the first set of molecules.

128. The kit of claim 127, wherein the recognition component of the second set of molecules is a nucleic acid sequence that is at least 99% complementary to at least one nucleic acid sequence from the first set of molecules.

129. The kit of any one of claims 125-128 wherein the second set of molecules comprises two or more different molecules.

130. The kit of claim 129, wherein the first set of molecules comprises two or more molecules having different nucleic acid sequences.

131. The kit of claim 129, wherein two or more different molecules of the second set of molecules have different nucleic acid sequences.

132. The kit of claim 118, further comprising a solution having an enzyme capable of disrupting the bond between the first set of molecules and the second set of molecules.

133. The kit of any one of claims 125-128, wherein the nucleic acid sequences of the first and second sets of molecules are selected from the group consisting of DNA, RNA, modified nucleic acid sequences, and combinations thereof.

134. The kit of any one of claims 125-128, wherein the component of each molecule of the first set of molecules is a Peptide Nucleic Acid (PNA) sequence and the recognition component of the second set of molecules is a PNA sequence.

135. The kit of claim 112, wherein the exposed functional group of each molecule of the second set of molecules is absent or independently selected from-OH, -CONH-, -CONHCO-, -NH₂、-NH-、-COOH、-COOR、-CSNH-、-NO₂ ^-、-SO₂、-SH、-RCOR-、-RCSR-、-RSR、-ROR-、-PO₄ ^-3、-OSO₃ ^-2、-SO₃ ^-、-COO^-、-SOO^-、-RSOR-、-CONR₂Wherein n is 1-20- (OCH)₂CH₂)_nOH、-CH₃、-PO₃H^-2-imidazole, -N (CH)₃)₂、-NR₂、-PO₃H₂- (CF) of, -CN, where n is 1-20₂)_nCF₃Porphyrins, corrin rings and olefins,

wherein R is hydrogen, a hydrocarbon, a halogenated hydrocarbon, a protein, an enzyme, a carbohydrate, a lectin, a hormone, a receptor, an antigen, an antibody or a hapten.

136. The kit of claim 135, wherein at each occurrence, n-1-8.

137. The kit of claim 135, further comprising a metal or metal ion capable of binding to an exposed functional group of at least one molecule of the second set of molecules.

138. The kit of claim 112, wherein each molecule in the second set of molecules has a first spacer, a second spacer, or first and second spacers, and the spacers are independently selected from alkylene, heteroalkylene, heterocycloalkylene, alkenylene, alkynylene, arylene, heteroarylene, arylenealkyl, and heteroarylenealkyl, wherein the alkylene, heteroalkylene, heterocycloalkylene, alkenylene, alkynylene, arylene, heteroarylene, arylenealkyl, or heteroarylenealkyl may be substituted or unsubstituted.

139. The kit of claim 138, wherein the substituents of said alkylene, heteroalkylene, heterocycloalkylene, alkenylene, alkynylene, arylene, heteroarylene, arylenealkyl, and heteroarylenealkyl are selected from the group consisting of halogen and hydroxy.