WO2013049409A2

WO2013049409A2 - Substrates having nanostructures having biological species immobilized thereon and methods of forming the same and methods of forming nanostructures on surfaces

Info

Publication number: WO2013049409A2
Application number: PCT/US2012/057648
Authority: WO
Inventors: Chad A. Mirkin; Jinan Chai; Lu Shin Wong; Guoliang Liu; Daniel J. EICHELSDOERFER; Keith A. BROWN
Original assignee: Northwestern University
Priority date: 2011-09-27
Filing date: 2012-09-27
Publication date: 2013-04-04
Also published as: WO2013049409A3

Abstract

A method for controlling a number of biological species immobilized on a nanostructure can include selecting a number of biological species to be immobilized on a nanostructure, the biological species having a hydrodynamic diameter, patterning a nanostructure using scanning probe block copolymer lithography, the patterned feature being formed to have a diameter (or line width) to form a nanostructure having a diameter (or line width) corresponding to (a) the hydrodynamic diameter of the biological species and (b) the number of biological species selected to be immobilized on the nanostructure, and immobilizing the biological species on the nanostructure, wherein the selected number of biological species are immobilized on the nanostructure

Description

SUBSTRATES HAVING NANOSTRUCTURES HAVING BIOLOGICAL SPECIES IMMOBILIZED THEREON AND METHODS OF FORMING THE SAME AND METHODS OF FORMING NANOSTRUCTURES ON SURFACES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 61/539,950 filed September 27, 2011, and U.S. Provisional Patent Application No.

61/699,076 filed September 10, 2012 is hereby claimed, and each of the entire disclosure of which is incorporated herein by reference.

STATEMENT OF GOVERNMENTAL INTEREST

[0002] This invention was made with government support under grant number FA9550-08- 01-0124 awarded by the Air Force Office of Scientific Research, grant number N66001-08- 2044 awarded by the Department of Defense, N00244-09-0012 awarded by the Department of Defense, National Security Science and Engineering Faculty Fellowships (NSSEFF), and grant number EEC-0647560 awarded by the National Science Foundation. The government has certain rights in this invention.

BACKGROUND

Field of the Disclosure

[0003] The disclosure is generally directed to a patterning method, and more particularly, to a method of synthesizing and patterning nano structures using block copolymer assisted nanolithography and to the immobilization of biological species on such nanostructures.

Brief Description of Related Technology

[0004] The integration of nanoparticles into devices has enabled applications spanning sensing, catalysis, electronics, photonics, and plasmonics, but synthesizing individual nanoparticles with control over size, composition, and placement on substrates is challenging. With conventional approaches, nanoparticles are synthesized and subsequently positioned on a surface using techniques such as parallel printing, surface dewetting, microdroplet molding, direct writing, and self-assembly). However, it is difficult to use these methods to reliably make and position a single particle on a surface with nanometer scale control.

[0005] Recently, scanning probe block copolymer lithography has emerged as a tool for synthesizing nanoparticles from high mobility precursors, but it is extremely limited from a materials standpoint.

[0006] The challenge of positioning or synthesizing single sub-10 nm nanoparticles in desired locations can be difficult, if not impossible, to achieve using currently available techniques including conventional photolithography. Current lithographic methods produce nanoparticle arrays through either lift-off processes or by prepatterning the surface chemically or geometrically to assist in the assembly of nanoparticles.

[0007] Although techniques such as electron beam (e-beam) lithography offer sub-50 nm resolution, fabricating sub- 10 nm features can be difficult because of proximity effects resulting from electron beam-photoresist interactions. Additionally, the throughput of e- beam lithography is limited by its serial nature. Nanoimprint lithography and micro-contact printing, on the other hand, afford parallel patterning, but do not allow for arbitrary pattern formation. As scanning probe based methods, dip pen nanolithography (DPN) and polymer pen lithography (PPL) are particularly attractive because "inked" nanoscale tips can deliver material directly to a desired location on a substrate of interest with high registration and sub- 50 nm feature resolution. These versatile techniques have been used to generate nanopatterns of alkanethiols, oligonucleotides, proteins, polymers, and inorganic materials on a wide variety of substrates. Previous attempts have been made to pattern nanoparticles directly by DPN, but the strong dependence of this technique on surface interactions, tip inking, and ink transport resulted in inhomogeneous features, whereas nanoparticle assembly via DPN- generated templates are inherently indirect and not ideal for positioning single objects with sub- 10 nm dimensions. Because feature resolution is limited by the AFM tip radius of curvature and the water meniscus formed between tip and substrate, the ultimate resolution of DPN reported to date is 12 nm for an alkanethiol feature formed on crystalline Au (111) substrate, which was achieved by using an ultra sharp tip with a 2 nm radius.

[0008] In contrast with top-down approaches, the self-assembly of block copolymers offers a versatile platform, which affords feature sizes typically in the range of 5 nm to 100 nm, as dictated by the molecular weight of the block copolymers. The well-defined domain structures of the block copolymer system can be used as templates to achieve secondary patterns of functional materials including metals, semiconductors, and dielectrics. However, previous work described the use of block copolymers as thin film templates for the synthesis of nanoparticle arrays in mass, without control over individual particle position or dimensions. These phase separated domains often lack orientation and long-range order, preventing widespread use and adoption in technologically relevant applications. Attempts to improve ordering in block copolymer systems have been explored using external electric fields, shear and flow stresses, thermal gradients, solvent annealing, chemical prepatterning, and graphoepitaxy. Chemical prepatterning and graphoepitaxy provide more control over translational order and feature registration in patterns, but require additional indirect lithographic steps, such as e-beam lithography, which is expensive and low throughput for large area applications. Quasi-long range order of block copolymer microdomains on corrugated crystalline sapphire surfaces was obtained without the use of additional lithographic steps. This technique, however, is limited in the type of substrate that can be patterned and does not allow for positional control of the particles on arbitrary surfaces.

[0009] Protein immobilization on solid substrates with nanoscale control has been utilized in a variety of applications, including chip-based bioassays, proteomics, drug discovery, and cellular biology studies. In cellular biology research, the ability to fabricate protein nanostructures on surfaces has enabled the study of many basic cellular functions including growth, signaling, and differentiation. For combinatorial molecular biology, the

miniaturization of protein nanoarrays allows for smaller and higher density chips and the need for smaller sample volumes. In certain cases, this can translate into diagnostic systems with higher sensitivity and the ability to track disease and biological processes more efficiently. The ability to specifically isolate single biomolecules can also facilitate molecular level studies of such structures. Therefore, being able to nanofabricate bimolecular features at a resolution of 10 nm or less is of significant interest since this length scale approaches the dimensions of single protein molecules and offers an opportunity to address many previously unexplored biological phenomena. Although there are some early examples of using traditional methods such as electron beam lithography to prepare small collections of particles that can support individual protein attachment, the ability to control the placement of biomolecules with this degree of resolution and precision over large areas remains a challenge for current nanolithographic processes. The conventional methods are expensive, have inherently low throughput, and are difficult to implement on the sub- 10 nm scale.

SUMMARY

[0010] In accordance with an embodiment of the disclosure, a substrate can include an array of nanostructures and a biological species immobilized on the nanostructures, wherein about 70% of the nanostructures have a single biological species immobilized thereon.

[0011] In accordance with another embodiment of the disclosure a substrate can include an array of nanostructures and a biological species immobilized on the nanostructures, wherein at least about 70% of the nanostructures have a preselected number of biological species immobilized thereon. [0012] In accordance with an embodiment of the disclosure, a method for controlling a number of biological species immobilized on a nanostructure can include selecting a number of biological species to be immobilized on a nanostructure, the biological species having a hydrodynamic diameter, contacting a substrate with a tip coated with an ink comprising a block copolymer and a nanostructure precursor to form a printed feature comprising the block copolymer and the nanostructure precursor on the substrate, wherein the printed feature is formed to have a diameter (or line width) to form a nanostructure having a diameter (or line width) corresponding to (a) the hydrodynamic diameter of the biological species and (b) the number of biological species selected to be immobilized on the nanostructure, reducing the nanostructure precursor of the printed feature to form the nanostructure having a diameter (or line width) of less than 1 μηι, and immobilizing the biological species on the nanostructure, wherein the selected number of biological species are immobilized on the nanostructure.

[0013] In accordance with another embodiment of the disclosure, a method for forming a sub-micron sized nanostructure comprising a single biological species immobilized thereon on a substrate surface can include contacting a substrate with a tip coated with an ink comprising a block copolymer and a nanostructure precursor to form a printed feature comprising the block copolymer and the nanostructure precursor on the substrate, reducing the nanostructure precursor of the printed feature to form a nanostructure having a diameter (or line width) substantially corresponding to a hydrodynamic diameter of the biological species and having a diameter (or line width) of less than 1 μιη, and immobilizing a single biological species on the nanostructure.

[0014] In accordance with yet another embodiment of the disclosure, a method for controlling a number of biological species immobilized on a structure can include selecting a number of biological species to be immobilized on a nanostructure, the biological species having a hydrodynamic diameter, contacting a substrate with a tip coated with a composition comprising a block copolymer and a structure precursor to form a printed feature comprising the block copolymer and the structure precursor on the substrate, wherein the printed feature is formed to have a diameter (or line width) to form a structure having a diameter (or line width) corresponding to (a) the hydrodynamic diameter of the biological species and (b) the number of biological species selected to be immobilized on the nanoparticle, heating the printed feature to a temperature below a decomposition temperature of the block copolymer to aggregate the structure precursor and form a structure precursor aggregated printed feature, heating the structure precursor aggregated printed feature to a temperature above the decomposition temperature of the structure precursor to decompose the polymer, thereby forming the structure, and immobilizing the biological species on the structure, wherein the selected number of the biological species are immobilized on the structure.

[0015] In accordance with an embodiment, a method for forming a sub-micron sized nanostructure comprising a single biological species immobilized thereon on a substrate surface can include contacting a substrate with a tip coated with a composition comprising a block copolymer and a structure precursor to form a printed feature comprising the block copolymer and the structure precursor on the substrate, wherein the printed feature is formed to have a diameter (or line width) to form a nanostructure having a diameter (or line width) substantially equal to the hydrodynamic diameter of the biological species, heating the printed feature to a temperature below a decomposition temperature of the block copolymer to aggregate the structure precursor and form a structure precursor aggregated printed feature, heating the structure precursor aggregated printed feature to a temperature above the decomposition temperature of the structure precursor to decompose the polymer, thereby forming the structure, the structure having a diameter (or line width) substantially

corresponding to a hydrodynamic diameter of the biological species and having a diameter (or line width) of less than 1 μιη, and immobilizing the biological species on the structure, wherein the selected number of the biological species are immobilized on the structure.

[0016] In accordance with an embodiment of the disclosure, a method for forming a structure on a substrate surface that includes contacting a substrate with a tip coated with a composition comprising a block copolymer and a structure precursor to form a printed feature comprising the block copolymer and the structure precursor on the substrate. The method further includes heating the printed feature to a temperature below a decomposition temperature of the block copolymer to aggregate the nanostructure precursor and form a structure precursor aggregated printed feature. Optionally the temperature can be above a glass transition temperature of the block copolymer. The method also includes heating the structure precursor aggregated printed feature to a temperature above the decomposition temperature of the structure precursor to decompose the polymer, thereby forming the structure. In various aspects, the structures are sub-micron sized nanostructures.

[0017] In accordance with an embodiment of the disclosure, a method of forming a structure on a substrate surface, includes heating a substrate comprising a composition comprising a block copolymer and a structure precursor to a temperature below the decomposition temperature of the block copolymer to aggregate the structure precursor to form a structure precursor aggregated composition. The temperature can optionally be above the glass transition temperature of the block copolymer. The method further includes heating the structure precursor aggregated composition to a temperature above the decomposition temperature of the structure precursor to decompose the polymer and form the structure. In various aspects, the structures are sub-micron sized nanostructures.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1A is a schematic drawing illustrating the structure and molecular weight of PEO-&-P2VP;

[0019] Figure IB is a schematic drawing of a method of forming nanostructures in accordance with an embodiment of the disclosure;

[0020] Figure 1C is an atomic force microscopy (AFM) topographical image of a square dot array of PEO-¾-P2VP/AuCl₄ ^~ ink deposited on a Si/SiO_x substrate by dip pen

nanolithography using a method of forming nanostructures in accordance with an

embodiment of the disclosure;

[0021] Figure ID is a graph showing the height profile of one line of PEO-&-P2VP/AuCl₄ ^~ dots from Figure 1C, illustrating the uniformity of the feature size;

[0022] Figure IE is a scanning electron microscopy (SEM) image of sub- 10 nm Au nanoparticles produced by plasma treatment of the square dot array of Figure 1C. The inset is a Fourier transform of the SEM image;

[0023] Figure IF is a high resolution transmission electron microscopy (TEM) image of a crystalline Au nanoparticle formed by a method in accordance with the disclosure, illustrating that the nanoparticle has a diameter of 8 nm and the crystal has an interplanar spacing of 0.24 nm. The inset is a typical electron diffraction pattern of the Au (111) nanoparticle;

[0024] Figure 2A is a TEM image of PEO-£>-P2VP/AuCl₄ ^~ micelles prepared by dropping the solution on a carbon-coated copper grid;

[0025] Figure 2B is a TEM image of Au nanoparticles formed within the polymer matrix after DPN patterning using a method in accordance with an embodiment of the disclosure;

[0026] Figure 3 is an X-ray photoelectron spectroscopy spectra of Au nanoparticles formed by a method in accordance with an embodiment of the disclosure using a PEO-b- P2VP/HAuCl₄ ink; [0027] Figure 4A is an SEM image of a large array of single Au nanoparticles formed by a method in accordance with an embodiment of the disclosure;

[0028] Figure 4B is a graph illustrating a registry analysis of the array of 400 particle features over different areas, with the distribution error being defined as the ratio of the distance of the particles away from the center of the block copolymer feature to the feature diameter;

[0029] Figure 5A is an AFM topographical image of a 5 x 5 dot pattern of a PEO-b- P2VP/AuCi₄ ^~ ink with different sizes deposited on a Si/SiO_x substrate generated by a method in accordance with an embodiment of the disclosure in which the tip-substrate contact time was intentionally increased. The tip-substrate contact time from bottom to top of the image is 0.01, 0.09, 0.25, 0.49, and 0.81 seconds;

[0030] Figure 5B is a graph showing the height profile of one line of PEO-^-P2VP/AuCl₄ ^~ dots of Figure 5A, demonstrating the time-dependent polymer transport volume;

[0031] Figure 5C is an SEM image of Au particles (bright dots) with different sizes formed within the block copolymer matrix (dark circles) after brief plasma exposure of the PEO-b- P2VP/AuCl₄ ^" dots of Figure 5A;

[0032] Figure 5D is a scanning TEM image of the pattern of Figure 5A, confirming the formation of single Au nanoparticles (black dot) within the block copolymer matrix (grey surrounding dot);

[0033] Figure 5E is a graph illustrating the size distribution of the PEO- ?-P2VP/AuCi₄ ^~ dots of Figure 5A and the size distribution of the corresponding Au nanoparticles formed by reduction of the PEO-&-P2VP/AuCl₄ ^" dots of Figure 5A;

[0034] Figure 6 is a scanning TEM image of a 5 x 5 dot array of PEO-£>-P2VP/AuCl₄ ^~ dots with different sizes formed on a S1₃N4 substrate generated by a method in accordance with an embodiment of the disclosure in which the tip-substrate contact time was intentionally increased. The tip-substrate contact time from bottom to top of Figure 6 is 1, 4, 9, 16, and 25 seconds. Single Au nanoparticles (bright white spot) formed within the block copolymer matrix (gray surrounding) except in the circled features where two nanoparticles were found;

[0035] Figure 7A is a dark field optical microscopy image of the Northwestern University Wildcat logo pattern made of individual PEO- ?-P2VP/AuCl₄ ^" dots features formed by a method in accordance with an embodiment of the disclosure; [0036] Figure 7B is an SEM image of a magnified portion of Figure 7A showing the formation of a Au nanoparticle arrays embedded in the block copolymer matrix upon plasma exposure. The inset is a magnified SEM image of a single gold nanoparticle after polymer removal;

[0037] Figure 8A is an SEM image of a 3 x 3 array of Au nanoparticles having sub-5 nm diameters formed in by a method in accordance with an embodiment of the disclosure;

[0038] Figure 8B is scanning TEM images of the individual Au nanoparticles of Figure 8 A, showing the size of the nanoparticles;

[0039] Figure 8C is a histogram showing the size distribution of the sub-5 nm Au nanoparticles of Figure 8A;

[0040] Figure 9A is a dark field optical microscopy image of a large scale pattern of PEO-?-P2VP/AuCi₄ ^~ dots formed by polymer pen lithography (15,000 pen array) on a Si/SiO_x substrate using a method in accordance with an embodiment of the disclosure. The inset shows a 20 x 20 dot array with 2 μιη spacing for each pattern formed by an individual pen of the pen array;

[0041] Figure 9B is an SEM image of Au particles (bright dot) formed within the patterned array of Figure 9A after the block copolymer matrix was removed by oxygen plasma. The inset shows a single Au nanoparticle has a diameter of 9.5 nm; and

[0042] Figure 10 is an SEM image of sub-5 nm Pt nanoparticles formed in a PEO-&-P2VP block copolymer matrix by dip pen nanolithography using a method in accordance with an embodiment of the disclosure.

[0043] Figure 11 a is a schematic illustration of a method for forming a nanoparticle in accordance with embodiments of the disclosure;

[0044] Figure 1 lb is a temperature profile of first and second thermal treatments of a method of forming a nanoparticle in accordance with embodiments of the disclosure;

[0045] Figure 12a is a scanning electron microscopy (SEM) image of large-area patterned nanoreactors loaded with gold precursors on a hydrophobic silicon substrate;

[0046] Figure 12b is an atomic force microscopy image of a patterned array of nanoreactors, the diameters of which are 400 nm; [0047] Figure 12c are ex-situ SEM images illustrating diffusion and segregation of gold precursors inside the polymer matrix during a method of forming nanoparticles in accordance with an embodiment of the disclosure;

[0048] Figure 12d is an SEM image of an array of synthesized gold nanoparticles on a hydrophobic silicon substrate and a magnified view of a single gold nanoparticle, the dashed circle denotes the original size of the nanoreactor;

[0049] Figure 12e is an SEM image illustrating that multiple nanoparticles are formed when the first thermal treatment step is eliminated, the dashed circle denotes the original size of the nanoreactor;

[0050] Figure 13a is a schematic illustration of the pathways for formation of a nanoparticle using methods in accordance with embodiments of the disclosure, Mⁿ⁺ and M° denote metal ions and fully reduced metal, respectively. Δι and Δ₂ correspond to the first and second thermal treatments at 7i_ow and T_hi_gh, respectively;

[0051] Figure 13b are XPS spectra collected for exemplary precursors for each pathway before thermal treatment (top), after the first thermal treatment (middle), and after the second thermal treatment (bottom). All spectra are shifted for clarity and the dashed lines denote the initial and final peak positions;

[0052] Figures 14a and 14b are high-angle annular dark-field (HAADF) STEM (z- contrast) images of Pt nanoparticle synthesis in accordance with an embodiment of the disclosure. After the first thermal treatment (Figure 14a) the precursor,

aggregated within the polymer nonreactor. After the second thermal treatment (Figure 14b), the precursor decomposed and formed a single nanoparticle. The polymer nanoreactors were also decomposed. The dashed circles outline the boundary of the polymer nanoreactors;

[0053] Figure 15 is HRTEM images illustrating the cyrstallinity of nanoparticles form in accordance an embodiment of the disclosure;

[0054] Figures 16a and 16b are TEM images of a patterned array of PEO-£>-P2VP nanoreactors on hydrophobic silicon nitride window after the first thermal treatment at 150°C (Figure 16a) and after the second thermal treatment at 500°C (Figure 16b). Ag nanoparticles were observed after the first annealing step. The dotted circles denote the position of the patterned printed features (nanoreactors);

[0055] Figure 17 is an EDX spectra of synthesized metal nanoparticles formed in accordance with a method in accordance with the disclosure. Si signal is from the silicon nitride membrane. Al and Cu signals are from the TEM sample holder. Since a Cu signal is always present in the background, an EDX spectrum of Cu-containing nanoparticles is not shown;

[0056] Figure 18 is an XPS spectra of nanoparticles composed of Ag, Pd, C0₂O₃, NiO, and CuO after formation using a method in accordance with an embodiment of the disclosure;

[0057] Figure 19 is a graph of a thermogravimetric analysis of PEO-&-P2VP illustrating that the thermal decomposition peak of PEO-&-P2VP is at 409°C. The temperature ramping rate was 10°C/min

[0058] Figure 20 is HRTEM images of gold nanoparticles formed by a method in accordance with an embodiment of the disclosure with the size of the nanoparticle being controlled by the concentration of the nanostructure precursor in the block-copolymer nanostructure precursor ink;

[0059] Figure 21 is TEM images of patterned arrays of nanoreactors of PEO-¾-P2VP on a silicon nitride window after the first thermal treatment illustrating the effect of protonation of PEO-&-P2VP on the loading of the precursors;

[0060] Figure 22a is a photograph of HAuCl₄ in PEO-¾-P2VP aqueous solution (Au^m:2VP = 4.1) after 1 day and 14 days illustrating the reduction of Auⁱⁿ to Au° and formation of Au nanoparticles in the solution after 14 days;

[0061] Figure 22b is an SEM image of representative Au nanoparticles formed in solution after 14 days; and

[0062] Figure 23 is representative STEM images of arrays of nanoparticles for precursors having varying reduction potentials. Dotted circles highlight the position of nanoparticles. For clarity, zoomed-in images of nanoparticles are shown in the inset,. The scale bars apply to all images and inset images. The difference size of the nanoparticles are determined by the ink concentration and amount of polymer delivered to the synthesis sites.

[0063] Figure 24a is an AFM topographical image of a dot array of PEO- ?-P2VP/AuCl₄ ^~ ink on a S1₃N₄ TEM membrane patterned by scanning probe block copolymer lithography;

[0064] Figure 24b is an STEM image of a single AU nanoparticle array produced after polymer removal by plasma treatment; [0065] Figure 24c is an STEM image of controlled formation of Au nanoparticles (white dots) across a range of sizes within the block copolymer matrix (grey background) after brief plasma exposure;

[0066] Figure 24d is a graph of the size distribution of Au nanoparticles synthesized as a function of tip-substrate dwell time;

[0067] Figure 24e is an SEM image illustrating an arbitrary pattern made of individual PEO-£-P2VP/AuCl₄ ^~ dot features printed in the form of "NU";

[0068] Figure 24f is a magnified SEM image of the Au nanoparticles after polymer removal;

[0069] Figure 25 a is a schematic diagram of streptavidin-CdSe/ZnS quantum dot conjugates immobilized onto scanning probe block copolymer lithography patterned Au nanoparticles functionalized with biotin thiol conjugates;

[0070] Figure 25b is an STEM image of a square array pattern of AU nanoparticles with associated CdSe/ZnS quantum dots. The magnified image of each particle defined by the box at each corner shows that all of the Au nanoparticles are associated with the grey rods of the quantum dot labels;

[0071] Figure 26 is an STEM image of a square array pattern of about 10 nm Au nanoparticles with associated smaller 1.4 nm Au nanoparticles. The magnified images defined by the box at each corner show that each of the large Au nanoparticles patterned on the substrate has three smaller Au nanoparticles (indicated by the arrows) on average, indicating the immobilization of three streptavidin molecules at each site;

[0072] Figure 27a is an STEM image showing the association of 1.4 nm Au nanoparticle labels with the patterned Au nanoparticles. The number of Au particle labels and in turn the number of immobilized streptavidin molecules is dependent on the size of the patterned Au nanoparticles;

[0073] Figure 27b is a histogram of the number of the associated small Au nanoparticle labels as a function of the size of the patterned Au nanoparticle;

[0074] Figure 28 is a graph of UV-Vis absorbance at 652 nm over time for the TMB assay, measured for various surface substrates including Au nanoparticle immobilized arrays of avidin-HRP; [0075] Figure 29a is a schematic diagram of antibody-antigen binding on the patterned Au nanoparticles bearing NTA-functionalized self-assembled monolayers;

[0076] Figure 29b is an STEM image of a square array pattern of patterned Au

nanoparticles formed by scanning probe block copolymer lithography. The magnified images on each corner show that each of the large Au nanoparticles capped with NTA groups is in close proximity to a single small Au nanoparticle label (indicated by the arrows) from the IgG and a quantum dot label (indicated by the arrows) from the F_ab;

[0077] Figure 30 is a STEM image (left panel) and an energy-dispersive X-ray

spectroscopy (EDX) elemental mapping of a patterned Au nanoparticle having a CdSe/ZnS quantum dot bound thereto to mark the interaction of biotin and immobilized streptavidin. The middle panel denotes the patterned Au nanoparticle and the right panel denotes the quantum dot label in EDX mapping; and

[0078] Figure 31 is an STEM image (left panel) and an EDX elemental mapping of a patterned Au nanoparticle having an antigoat IgG molecule labeled with 1.4 nm Au nanoparticle bound thereto, which was in turn bound to a goat-derived F_ab antibody (labeled by CdSe/ZnS quantum dot). The middle panel denotes the patterned Au nanoparticle, while the right panel denotes the quantum dot label in EDX mapping. Due to its small size, the small 1.4 nm Au nanoparticle label conjugated to the antigoat IgG could not be observed in EDX mapping.

DETAILED DESCRIPTION

[0079] The methods disclosed herein can allow for patterning of sub-10 nm size single nanostructures, for example, nanoparticles, while enabling one to control the growth and position of individual nanostructures in situ. The methods can also allow for patterning of larger structures, for example, up to 100 nm sized structures. The process is advantageously based on an understanding of the pathways for polymer-mediated diffusion and reduction and can allow for the generation of single nanoparticles of a variety of materials, including, for example, metals, metal oxides, or metal alloys, independent of precursor mobility.

Nanoparticles exhibit size-dependent photonic, electronic, and chemical properties that could lead to a new generation of catalysts and nanodevices, including single electron transistors, photonics, and biomedical sensors. [0080] In order to realize many of these targeted applications, the methods of the disclosure can advantageously provide for the synthesis of monodisperse particles while controlling individual particle position on technologically relevant surfaces.

[0081] In various embodiments, methods of the disclosure advantageously provide a patterning method that can allow for the control of the number of biological species (e.g., a number of molecules of a biological species) immobilized at specific locations on a surface. Such control can be down to the single-molecule level. Embodiments of methods of the disclosure advantageously recognize that a biological species can be immobilized on nanostructures formed using scanning probe block copolymer lithography. Further, methods of the disclosure recognize that the size of the nanostructure can be used and tailored to control the number of biological species that attach to a patterned nanostructure. The methods of the disclosure can, therefore, allow for arbitrary patterns of nanostructures having a selected number biological species immobilize thereon to be generated.

[0082] In one embodiment, a method in accordance with the disclosure includes selecting a number of biological species to be immobilized on a patterned nanostructure. The method further include contacting a substrate with a tip coated with an ink containing a block copolymer and a nanostructure precursor to form a printed feature comprising the block copolymer and the nanostructure precursor on the substrate. The printed feature is formed to have a diameter (or line width) to form a nanostructure having a diameter (or line width) that corresponds to (a) the hydrodynamic diameter and/or the diameter of gyration of the biological species, and (b) the number of biological species selected to be immobilized on the nanostructure. As used herein, the "hydrodynamic diameter" is twice the hydrodynamic radius. As used herein, the "diameter of gyration" is twice the radius of gyration. The nanostructure is then formed by reducing the nanostructure precursor of the printed feature. Once the nanostructure is formed, the biological species is immobilized on the nanostructure. For example, the nanostructure can be immersed in a solution containing the biological species.

[0083] By selecting the diameter (or line width) of the nanostructure when the printed feature is formed to correspond to (a) the hydrodynamic diameter and/or the diameter of gyration and (b) the number of biological species to be immobilized on the nanostructure, the selected number of biological species can be immobilized on the nanostructure. It has been advantageously determined that by controlling the size of the nanostructure, the number of biological species immobilized on the nanostructure can be controlled and tailored. [0084] In an embodiment, the method includes printing the substrate with a printed feature having a diameter (or line width) such that the resulting nanostructure is formed to have a diameter (or line width) that is substantially the same as the hydrodynamic diameter and/or the diameter of gyration of the biological species. In such an embodiment, a single biological species, for example, a protein, can be immobilized on the nanostructure.

[0085] Patterning the nano structures using scanning probe block copolymer lithography can advantageously allow for fine control over the particle size and thereby, based on the recognition of the relationship between size of the nanostructure and the biological species demonstrated herein, control over the number of biological species immobilized on the nanostructure.

[0086] For example, when a single biological species is to be immobilized on the nanostructure, the diameter (or line width) of the nanostructure can be equal to or approximately equal to the hydrodynamic diameter and/or equal to the diameter of gyration (also referred to herein as the "size of the biological species") of the biological species. For example, the nanostructure can have a diameter (or line width) that is within a tolerance (plus or minus) of about 0% to about 30%, about 0% to about 5%, about 0% to about 3%, about 1% to about 25 %, about 2% to about 20% about 10% to about 30%, about 0% to about 10%, and about 0% to about 15%, about 0% to about 20% of the hydrodynamic diameter and/or the diameter of gyration of the biological species in order to have a single biological species immobilized on the nanostructure. Other suitable tolerance values include, for example, plus or minus, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30% of the hydrodynamic diameter and/or the diameter of gyration of the biological species. If it is desirable to have multiple biological species immobilized on the nanostructure, the nanostructure can have a diameter (or line width) that is within a tolerance (plus or minus) about 0% to about 30%, about 0% to about 5%, about 0% to about 3%, about 1% to about 25 %, about 2% to about 20% about 10% to about 30%, about 0% to about 10%, and about 0% to about 15%, about 0% to about 20% of the hydrodynamic diameter and/or the diameter of gyration of the biological species times the number of species to be immobilized on the nanostructure. Other suitable tolerance values include, for example, plus or minus, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30% of the hydrodynamic diameter and/or the diameter of gyration of the biological species times the number of biological species to be immobilized on the nanostructure [0087] In various embodiments, scanning probe block copolymer lithography can be used to generate sub- 10 nm nanostmctures, on which proteins can be immobilized. Scanning Probe Block Copolymer Lithography can allow for patterning of sub- 10 nm size single nanostmctures, for example, nanoparticles, while enabling one to control the growth and position of individual nanostmctures in situ.

Substrates having Nanostructures with Biological Species Immobilized thereon

[0088] Embodiments of the disclosure advantageously provide a substrate having an array of nanostmcture patterned on the substrate. Substantially all of the nanostmctures can have a preselected number of biological species immobilized on an individual nanostmcture. For example, the preselected number of biological species can be a single biological species immobilized on an individual nanostmcture. For example, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or 100% of the

nanostmctures patterned on the substrate can have the preselected number of biological species immobilized thereon. For example, the preselected number of biological species can be a single biological species immobilized on a single nanostmcture. In one exemplary embodiment, a substrate includes an array of nanoparticles and 80% of the nanoparticles include only a single protein immobilized thereon.

[0089] As discussed above, the nanostmctures can have a diameter (or line width) that is substantially equal to (i.e., corresponds to) the hydrodynamic diameter of the biological species times the preselected number of biological species to be immobilized thereon. For example, where a single biological species is to be immobilized on an individual nanostmcture, the nanostmctures can have a diameter or line width that is substantially equal to the hydrodynamic diameter of the biological species. As noted above, as used herein, the "hydrodynamic diameter" is twice the hydrodynamic radius. The radius of gyration can also be a suitable parameter for use in determining a suitable diameter of a nanostmcture based on the number of biological species to be immobilized thereon. For example, where a single biological species is to be immobilized on an individual nanostmcture, the nanostmctures can have a diameter that or line width that is substantially the same as the diameter of gyration. As noted above, as used herein, the "diameter of gyration" is twice the radius of gyration.

[0090] In various embodiments, the nanostmctures can be formed to have different sizes such different nanostmctures patterned on the substrate can have a different number of biological species selected to be immobilized thereon and/or different types of biological species can be immobilized on different nanostmctures. For example, the nanostmcture can have a diameter (or line width) that is within a tolerance (plus or minus) of about 0% to about 30%, about 0% to about 5%, about 0% to about 3%, about 1% to about 25 %, about 2% to about 20% about 10% to about 30%, about 0% to about 10%, and about 0% to about 15%, about 0% to about 20% of the hydrodynamic diameter and/or diameter of gyration of the biological species in order to have a single biological species immobilized on the

nanostructure. Other suitable tolerance values include, for example, plus or minus, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30% of the hydrodynamic diameter and/or the diameter of gyration of the biological species. If it is desirable to have multiple biological species immobilized on the nanostructure, the nanostructure can have a diameter (or line width) that is within a tolerance (plus or minus) of about 0% to about 30%, about 0% to about 5%, about 0% to about 3%, about 1% to about 25 %, about 2% to about 20% about 10% to about 30%, about 0% to about 10%, and about 0% to about 15%, about 0% to about 20% of the hydrodynamic diameter or the diameter of gyration of the biological species times the number of species to be immobilized on the nanostructure. Other suitable tolerance values include, for example, plus or minus, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, and 30% of the hydrodynamic diameter and/or the diameter of gyration of the biological species times the number of biological species to be immobilized on the nanostructure.

[0091] The substrate can include any suitable number of nanostructures.

Advantageously, the substrates can include a large number of nanostructures with substantially all of the nanostructures having only a single biological species or a preselected number of biological species immobilized on each nanostructure. For example, the substrate can have at least about 10,000 nanostructures, at least about 20,000 nanostructures, at least about 30,000 nanostructures, at least about 40,000 nanostructures, at least about 50,000 nanostructures, at least about 60,000 nanostructures, at least about 70,000 nanostructures, at least about 80,000 nanostructures, at least about 90,000 nanostructures, at least about 100,000 nanostructures, at least about 200,000 nanostructure, at least about 300,000 nanostructures, at least about 400,000 nanostructures, at least about 500,000 nanostructures, at least about 600,000 nanostructures, at least about 700,000 nanostructures, at least about 800,000 nanostructures, at least about 900,000 nanostructures, or at least about 1,000,000

nanostructures. The nanostructures can be patterned on the substrate in any arbitrary pattern. Scanning Probe Block Copolymer Lithography with Precursor Reduction

[0092] U.S. Patent Application Publication No. 2011/0165341, the disclosure of which is incorporated herein by reference in its entirety, discloses one exemplary method of scanning probe block copolymer lithography. In accordance with embodiments of the disclosure, the scanning probe block copolymer lithography method can utilize dip-pen nanolithography or polymer pen lithography printing methods to transfer phase-separating block copolymer- nanostructure precursor inks to a substrate. After patterning, nanostructure formation can be induced by reduction of the nanostructure precursor in the printed features and removal of the block copolymer matrix. The printed features and accordingly the formation of the nanostructures can be arranged in any arbitrary pattern using the method of the disclosure. Any nanostructure having any shape can be formed by the method of the disclosure. The nanostructures can be, for example, nanoparticles or nanowires.

[0093] Advantageously, methods in accordance with embodiments of the disclosure can allow for in situ synthesis of nanostructures having a size 10 or more times smaller than the originally printed features. For example, the printed features, which include the block- copolymer matrix and the nanostructure precursor, can have a diameter or line width of about 20 nm to about 1000 nm, about 40 nm to about 800 nm, about 60 nm to about 600 nm, about 80 nm to about 400 nm, or about 100 nm to about 200 nm. Other suitable printed feature diameters or line widths include about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, 600, 620, 640, 660, 680, 700, 720, 740, 760, 780, 800, 820, 840, 860, 880, 900, 920, 940, 960, 980, and 1000 nm. The resulting nanostructures can have a diameter or line width of about 1 nm to about 100 nm, about 1 nm to about 25 nm, about 2 nm to about 20 nm, about 4 nm to about 15 nm, about 6 nm to about 10 nm, about 50 nm to about 80 nm, or about 40 nm to about 60 nm. Other suitable nanostructure diameters or line widths include, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, and 100 nm. The size of the printed feature and the resulting nanostructure can be tailored to control the number of biological species that immobilize on the nanostructure.

[0094] Referring to Figure IB, a method of forming nanostructures can include loading a tip with the ink that includes a block copolymer matrix and a nanostructure precursor. Figure IB illustrates the use of a dip-pen nanolithography (DPN) tip for patterning. However, other tip-based lithography methods, such as polymer pen lithography (PPL) and gel pen lithography, can be used. The coated tip is then brought into contact with a substrate to deposit the ink on the substrate in the form of printed features. The printed features include the block copolymer matrix and the nanostructure precursor contained in the block copolymer matrix. The nanostructure precursor in the printed features can then be reduced to form the nanostructures and block copolymer matrix can be removed. Referring to Figures 7A and 7B, embodiments of the method of the disclosure can allow for arbitrary pattern control of single nanostructures, for example, nanoparticles, by patterning with tip-based patterning methods such as DPN and PPL.

[0095] The block copolymer material should be selected so as to be capable of transferring from a scanning probe tip to a substrate in a controllable way and sequestering the nanostructure precursor. Suitable block copolymer materials include, for example, polyethylene oxide)-&-poly(2-vinylpyridine) (PEO-6-P2VP), PEO-6-P4VP, and PEO-b- PAA. Figure 1A illustrates the PEO-&-P2VP block copolymer. When using a PEO-6-P2VP block copolymer, the P2VP is responsible for concentrating the nanostructure precursor, while the PEO acts as a delivery block to facilitate ink transport. The block copolymer separates into nanoscale micelles, which not only localizes the nanostructure precursor, but also cause the amount of nanostructure precursor in each feature to be substantially lower than if the feature was made from pure metal ion ink.

[0096] The molar ratio of the nanostructure concentrating or precursor-coordinating block to the nanostructure precursor can be about 1:0.1 to about 64: 1, about 1:0.1 to about 10:1, about 1:0.5 to about 8:1, about 1:1: to about 10:1, about 2:1 to about 8:1, about 4:1 to about 6:1, about 10:1 to about 64:1, about 15:1 to about 60:1, or about 30:1 to about 40:1. Other suitable molar ratios include about 1:0.1, 1:0.2, 1:0.25, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 22:1, 24:1, 26:1, 28:1, 30:1, 32:1, 34:1, 36:1, 38:1, 40:1, 42:1, 44:1, 46:1, 48:1, 50:1, 52:1, 54:1, 56:1, 58:1, 60:1, 62:1, and 64:1.

[0097] The nanostructure precursor can be, for example, any precursor material suitable for forming a metal nanostructure, a semiconductor nanostructure, or a dielectric

nanostructure. For example, the nanostructure precursor can be a metal salt, such as, HAuCl₄, H₂PtCl₆, Na₂PdCl₄, Co(N0₃)₂, CdCl₂, ZnCl₂, FeCl₃, AgN0₃, Cu(N0₃)₂, Ni(N0₃) and other inorganic compounds. Figure 8 A illustrates a pattern of Au nanoparticles formed by a method in accordance with an embodiment of the disclosure using the metal salt HAuCl₄ and the block copolymer PEO-&-P2VP. Figure 10 illustrates a pattern of Pt nanoparticles formed by a method in accordance with an embodiment of the disclosure using the metal salt Na₂PtCl₄ and the block copolymer PEO-&-P2VP, with the molar ratio of P2VP to Pt being 1 to 0.25.

[0098] In one embodiment, the nanostructure precursor is HAuCl₄ and the block copolymer is PEO-&-P2VP. The protonated pyridine units have a strong affinity to AuCl₄ ^" moieties because of electrostatic interactions, while the PEO block enables good transport properties in DPN experiments. Referring to Figure IB, when the block copolymer and the nanostructure precursor are mixed in an aqueous solution, micelles with a water insoluble P2VP core surrounded by a PEO corona form, confining the AuCLf to the P2VP core.

[0099] The block copolymer-nanostructure precursor ink can be printed on any suitable substrate, including, for example, Si/SiOx substrates, S1₃N₄ membranes, glassy carbon, and Au substrates.

[00100] After patterning, the nanostructures are formed by reduction of the nanostructure precursor in the printed features. The reducing agent can be any suitable agent for transforming the nanostructure precursor to a nanostructure. Subsequent reduction of the patterned block copolymer-nanostructure precursor micelles results in formation of nanostructures within the aggregated micelles. For example, oxygen or argon plasma can be used as the reducing agent and to remove the block copolymer. Reduction of the

nanostructure precursor material by oxygen plasma can be facilitated by hydrocarbon oxidation. Other suitable reducing agents include, for example, gases such as H₂. The reducing agent can also be used to remove the block copolymer after formation of the nanostructures. The block copolymer can also be removed after formation of the

nanostructures using a heat treatment. For example, the block copolymer can be removed by heat treating the substrate to a temperature higher than the decomposition temperature of the polymer.

[0100] The size of the nanostructures synthesized by a method in accordance with embodiments of the disclosure can be controlled, for example, by controlling the chain length of the copolymer block, the loading concentration of the nanostructure precursor, and the type of reducing agent. For example, increasing the loading concentration of the

nanostructure precursor results in nanostructures having an increased size. Additionally, without intending to be bound by theory, it is believed that increasing the molecular weight of the copolymer block results in a larger micelle cores, and hence, larger nanostructures. The nanostructure precursor determines the local concentration of ions within the polymer micelle. The lower the concentration, the smaller the synthesized nanostructures. For example, referring to Figure 8B, sub-5 nm nanoparticles can be formed by using a salt- copolymer mixture having a molar ratio of nanoparticle concentrating block to nanoparticle precursor of about 4 to 1.

[0101] The dwell time (also referred to herein as the tip-substrate contact time) during patterning of the block copolymer-nanostructure precursor inks can be about 0.01 seconds to about 30 seconds, about 0.01 second to about 10 seconds, about 0.05 seconds to about 8 seconds, about 0.1 seconds to about 6 seconds, about 0.5 seconds to about 4 seconds, about 1 second to about 2 seconds, about 10 seconds to about 30 seconds, about 8 seconds to about 26 seconds, about 6 seconds to about 24 seconds, about 15 seconds to about 20 seconds, or about 10 seconds to about 15 seconds. Other suitable dwell times includes, for example, about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30 seconds.

[0102] The size of the nanostructures synthesized by a method in accordance with embodiments of the disclosure can also be controlled by varying the dwell time when patterning by DPN or polymer pen lithography methods. The feature size dependence on tip- substrate contact time (dwell time) exhibited when using DPN or polymer pen lithography methods can be used to control both the size of the printed feature (having the block copolymer and the nanostructure precursor) and the size of the resulting nanostructure.

Referring to Figure 5E, for example, nanostructures synthesized using a method in accordance with embodiments of the disclosure and patterned by DPN can have a diameter that is linearly dependent on the square root of the tip-substrate contact time (dwell time).

[0103] Without intending to be bound by theory, it is believed that the number of nanostructures, for example, nanoparticles formed within a block copolymer printed feature can be controlled by controlling the size of the block copolymer-nanostructure precursor printed feature. For example, referring to Figure 6, multiple nanoparticles can be formed within a block copolymer matrix, when the block copolymer patterned feature has a diameter of 450 nm or greater.

Scanning Probe Block Copolymer Lithography with Precursor Aggregation

[0104] An embodiment of the disclosure includes patterning methods that can allow for patterning of sub- 10 nm size single nanostructures, for example, nanoparticles, while enabling one to control the growth and position of individual nanostructures in situ. These methods can also allow for patterning of larger structures, for example, up to 100 nm sized structures. The process is advantageously based on an understanding of the pathways for polymer-mediated and can allow for the generation of single nanoparticles of a variety of materials, including, for example, metals, metal oxides, or metal alloys, independent of precursor mobility. Nanoparticles exhibit size-dependent photonic, electronic, and chemical properties that could lead to a new generation of catalysts and nanodevices, including single electron transistors, photonics, and biomedical sensors. Further, as discussed above, the precise control over feature size that can advantageously be achieved with these methods can allow for control and tailorability of the number of biological species that can be attached to a nanostructure.

[0105] In order to realize many of these targeted applications, the methods of the disclosure can advantageously provide for the synthesis of monodisperse particles while controlling individual particle position on technologically relevant surfaces. The method of the disclosure allows for a materials general approach to synthesizing individual

nanoparticles as well as nanostructures with control over size, composition, and surface placement, thereby allowing for the synthesis of a diverse class of nanoparticles and structures, including, for example, Au, Ag, Pt, Pd, Fe₂0₃, C0₂O₃, NiO, CuO, and alloys of Au and Ag. The methods of the disclosure can advantageously provide simple and materials general method for synthesizing nanostructures with tailored size, composition, and placement. The nanostructures can be synthesized on site and can be rapidly integrated into functional devices, with, in some embodiments, no need for post-synthetic processing or assembly. The ability to synthesize homogenous or combinatorial arrays of specified nanoparticles on surfaces can enable fundamental studies and technological applications in fields such as catalysis, nanomagnetism, microelectronics, and plasmonics. The

understanding of polymer-mediated nanoparticle synthesis can also enable the utilization of block copolymers as a matrix to synthesize three dimensional nanoparticle lattices, both in thin films and in the bulk.

[0106] In accordance with embodiments of the disclosure, the method can utilize dip-pen nanolithography or polymer pen lithography printing methods to transfer block copolymer- nanostructure precursor inks to a substrate. "Block copolymer-nanostructure precursor inks" and block copolymer structure precursor inks" are used herein interchangeable and refer to an ink or coating composition for patterning or coating a substrate that includes a block copolymer and a precursor. In alternative embodiments, an ink containing the block copolymer and structure precursor can be applied to a substrate using any know non-tip based method, such as micro-contact printing, dip coating, spin coating, vapor coating, spray coating, and brushing. Figure 11 A is a schematic illustration of a method in accordance with the disclosure, exemplifying application of the block copolymer- structure precursor ink using dip-pen nanolithography.

[0107] As illustrated in Figure 11, after application of the block copolymer- structure precursor ink to a substrate (whether by tip-based or non-tip based application methods), structure formation can be induced by thermal annealing. In one embodiment, a first thermal treatment Δ1 is performed in which the applied ink can be annealed at temperature Ti_ow that is above the decomposition temperature T^P _d of the polymer. Optionally, the temperature Ti_ow can be between the glass transition temperature T_g of the polymer and the decomposition temperature T^Pa of the polymer (T_g < Ti_ow < T^P _d). The first thermal treatment initiates phase separation and aggregation of the nanoparticle precursor materials within the printed feature or coating. In various embodiments, as detailed below, structure precursor ion reduction can occur during the first thermal treatment. Subsequently, a second thermal treatment Δ2 can be performed at a temperature T_hi_gh at a temperature above the decomposition temperature of the structure precursor T^S _d. Optionally the temperature T_hi_gh can be between the decomposition temperature of the structure precursor T^S _d and the melting point of the structure precursor T^m (T^S _d < T_hig_h < T^m) to facilitate one or more of nanostructure precursor ion reduction, particle formation, and polymer decomposition. Figure 1 IB is a schematic illustration of the heating profiles of the first and second thermal treatments.

[0108] The methods of the disclosure advantageously utilize polymer-mediated diffusion of the structure precursor within the block copolymers. The block copolymer can acts as a transport vehicle for precursor deposition, a diffusion media for structure precursor aggregation, a reducing agent for precursor reduction, and/or a spatially confined nanoreactor for particle synthetic reactions. In an embodiment, the block copolymer sequentially acts as a transport vehicle for precursor deposition, a diffusion media for structure precursor aggregation, a reducing agent for precursor reduction, and a spatially confined nanoreactor for particle synthetic reactions.

[0109] The block copolymer matrix can then be removed. The printed features and accordingly the formation of the structures can be arranged in any arbitrary pattern using the method of the disclosure. Any structure having any shape can be formed by the method of the disclosure. The nanostructures can be, for example, nanoparticles or nanowires. [0110] Advantageously, methods in accordance with embodiments of the disclosure can allow for synthesis of nanostructures having a size 10 or more times smaller than the originally printed features. For example, the printed features, which include the block- copolymer matrix and the nanostructure precursor, can have a diameter or line width of about 20 nm to about 1000 nm, about 40 nm to about 800 nm, about 60 nm to about 600 nm, about 80 nm to about 400 nm, or about 100 nm to about 200 nm. Other suitable printed feature diameters or line widths include about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, 600, 620, 640, 660, 680, 700, 720, 740, 760, 780, 800, 820, 840, 860, 880, 900, 920, 940, 960, 980, and 1000 nm. The resulting nanostructures (also referred to herein as "structures") can have a diameter or line width of about 1 nm to about 100 nm, about 1 nm to about 25 nm, about 2 nm to about 20 nm, about 4 nm to about 15 nm, about 6 nm to about 10 nm, about 50 nm to about 80 nm, or about 40 nm to about 60 nm. Other suitable nanostructure diameters or line widths include, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, and 100 nm.

[0111] Referring to Figure 11 A, a method of forming nanostructures can include loading a tip with the ink that includes a block copolymer matrix and a nanostructure precursor. Figure 11 A illustrates the use of a dip-pen nanolithography (DPN) tip for patterning. However, other tip-based lithography methods, such as polymer pen lithography (PPL) and gel pen lithography, can be used. The coated tip is then brought into contact with a substrate to deposit the ink on the substrate in the form of printed features. Embodiments of the method of the disclosure can allow for arbitrary pattern control of single nanostructures, for example, nanoparticles, by patterning with tip-based patterning methods such as DPN and PPL.

[0112] Alternatively, non-tip based coating and patterning methods can be used. Non-tip based methods can include any known application methods including, but not limited to, micro-contact printing, dip coating, spin coating, vapor coating, spray coating, brushing, and combinations thereof.

[0113] As used herein "printed features," generally refers to features patterned by both tip- based and non-tip based patterning methods as well as coatings applied to a substrate. The printed features include the block copolymer matrix, which is also referred to herein as a nanoreactor, and the structure precursor contained in the block copolymer matrix. [0114] The block copolymer material should be selected so as to be capable of

sequestering the structure precursor. In various embodiments in which tip-based patterning methods are used, the block copolymer should also be selected so as to be capable of transferring from a scanning probe tip to a substrate in a controllable way. Suitable block copolymer materials include, for example, poly(ethylene oxide)-¾-poly(2-vinylpyridine) (PEO-&-P2VP), PE0-6-P4VP, and PEO-&-PAA. Figure 11 A illustrates the PEO-&-P2VP block copolymer. When using a PEO-&-P2VP block copolymer, the P2VP is responsible for concentrating the nanostructure precursor, while the PEO acts as a delivery block to facilitate ink transport. The block copolymer can separate into micelles, for example, nanoscale micelles, upon patterning or coating, which can facilitate localizing the structure precursor.

[0115] The molar ratio of the nanostructure concentrating or precursor-coordinating block to the structure precursor can be about 1:0.1 to about 300: 1, about 1:0.1 to about 10:1, about 1:0.5 to about 8:1, about 1:1: to about 10:1, about 2:1 to about 8:1, about 4:1 to about 6:1, about 10:1 to about 64:1, about 15:1 to about 60:1, about 30:1 to about 40:1, about 2:1 to about 256:1, about 10:1 to about 200:1, about 20:1 to about 150:1, about 30:1 to about 100:1, about 40: 1 to about 50: 1, about 100: 1 to about 256: 1, about 80: 1 to about 200: 1, about 60: 1 to about 100:1, about 2:1 to about 4:1, about 2:1 to about 25:1, about 6:1 to about 20:1, about 10:1 to about 40:1, or about 25:1 to about 75:1. Other suitable molar ratios include about 1:0.1, 1:0.2, 1:0.25, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1,8:1,9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1,20:1,22:1,24:1,26:1, 28:1, 30:1, 32:1, 34:1, 36:1, 38:1, 40:1, 42:1, 44:1, 46:1, 48:1, 50:1, 52:1, 54:1, 56:1, 58:1, 60:1, 62:1, 64:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 100:1, 120:1, 140:1, 160:1, 180:1, 190:1, 200:1, 210:1, 220:1, 230:1, 240:1, 250:1, and 256:1.

[0116] The structure precursor can be, for example, any precursor material suitable for forming a metal nanostructure, a semiconductor nanostructure, or a dielectric nanostructure, as well as larger feature sized metal, semiconductor, and dielectric structures. For example, the structure precursor can be a metal salt, such as, of HAuCl₄, AgN03, H₂PtCl6, Na₂PdCl₄, Fe(N0₃)₃, Co(N0₃)₂, Ni(N0₃)₂, Cu(N0₃)₂, Na₂PtCl₄, CdCl₂, ZnCl₂, FeCl₃, NiCl₂, and combinations thereof. In one embodiment, metal alloy structures can be formed by blending metal precursors in the ink. For example, metal alloy nanoparticles can be formed by blending metal precursors in the ink. [0117] In one embodiment, when the block copolymer and the structure precursor are mixed in an aqueous solution, micelles with a water insoluble P2VP core surrounded by a PEO corona form, confining the structure precursor, for example, AuCLf, to the P2VP core.

[0118] The block copolymer-structure precursor ink can be printed on or applied to any suitable substrate, including, for example, Si/SiOx substrates, S1₃N4 membranes, glassy carbon, and Au substrates.

[0119] After patterning, a first thermal treatment Δ1 is performed to effect structure precursor ion aggregation. Phase separation during the first thermal treatment Δ1 can concentrate the precursor ions in a single or concentrated region, which for example can enable formation of single structures in each printed feature. In an embodiment, this concentration enables formation of a single nanoparticle. The first thermal treatment is carried out at a temperature Ti_ow that is below the decomposition temperature T^Pa of the polymer. Optionally the temperature Ti_ow can be above the glass transition temperature T_g of the polymer. For example, depending on the block copolymer used, the temperature Ti_ow of first thermal treatment can performed at a temperature Ti_ow in a range of about 70°C to about 400°C, about 78°C to about 400°C, about 80°C to about 350°C, about 100°C to about 300°C about 120°C to about 250°C, about 140°C to about 225°C, about 150°C to about 200°C, about 70°C to about 78°C, about 76°C to about 80°C, or about 78°C to about 200°C. Other suitable temperatures include for example, about 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, or 400°C . A temperature that is above the glass transition temperature of the block copolymer can be a temperature that is above the glass transition temperature of each polymer of the block copolymer. For example, when a PEO-&-P2VP block copolymer is used, the thermal treatment can be performed at a temperature Ti_ow of about 150°C. The glass transition temperature of PEO is about -76°C, the glass transition temperature of P2VP is about 78°C, and the decomposition temperature of PEO-¾-P2VP is about 400°C. Other suitable temperatures can be used depending on the decomposition temperature of the polymer T^P _d and/or optionally the glass transition temperature of the polymer T_g. The thermal treatment can be performed, for example, in a tube furnace under a flow of Ar gas. In one embodiment, the substrate containing the printed feature can be placed in a furnace and the temperature can be ramped up to Ti_ow from ambient temperature in about one hour. The ramping rate for reaching the temperature Ti_ow of the first thermal treatment can be, for example, about l°C/min to about 10°C/min, about 2°C/min to about 8°C/min, about 4°C/min to about 6°C/min, or about 3°C/min to about 7°C/min. Other suitable ramping rates include about 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10°C/min. The first thermal treatment Alcan be carried out at the temperature Ti_ow for about 2 hours to about 24 hours, about 4 hours to about 24 hours, about 6 hours to about 22 hours, about 8 hours to about 20 hours, about 10 hours to about 18 hours, about 14 hours to about 16 hours and about 2 hours to about 6 hours. Other suitable times include about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, or 24. The first thermal treatment Δ1 can be carried out for any suitable time to allow for full phase separation between the precursor and the polymer.

[0120] The printed features can then be cooled to ambient temperature prior to performing the second thermal treatment. For example, the temperature of the furnace can be cooled to ambient temperature in one hour.

[0121] Once the first thermal treatment for effecting nanostructure precursor ion aggregation is complete, a second thermal treatment Δ2 at a temperature T_hi_gh can be performed. The second thermal treatment can allow for reduction of the precursor and/or decomposition of the polymer. The temperature T_hi_gh is above the thermal decomposition T^S _d of the nanostructure precursor material and preferably below the melting point of the precursor T^m. For example, depending on the nanostructure precursor, the temperature T i_gh can be in a range of about 400°C to about 800°C, about 450°C to about 750°C, about 500°C to about 700°C, about 550°C to about 650°C. For example, the temperature can be about 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, and 800°C. Other suitable temperatures can be used depending on the decomposition and melting temperatures of the precursor used. The second thermal treatment Δ2 can be performed in a furnace, for example, a tube furnace under Ar gas. The second thermal treatment Δ2 can be performed, for example, by ramping the temperature of the furnace from ambient to the temperature T_hi_gh of the second thermal treatment Δ2. For example, the temperature can be ramped to the second thermal treatment temperature T_hi_gh in one hour. The ramping rate for reaching the temperature T_hi_gh of the second thermal treatment can be, for example, about l°C/min to about 10°C/min, about 2°C/min to about 8°C/min, about 4°C/min to about 6°C/min, or about 3°C/min to about 7°C/min. Other suitable ramping rates include about 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10°C/min. The second thermal treatment Δ2 can be performed for about 2 hours to about 10 hours, about 4 hours to about 8 hours, about 6 hours to about 10 hours, about 2 hours to about 4 hours, or about 3 hours to about 7 hours. Other suitable times include about 2, 3, 4, 5, 6, 7, 8, 9, and 10 hours. The second thermally treated substrate can then be cooled for example by ramping the furnace from the temperature T_hi_gh to ambient temperature.

[0122] Referring to Figure 13, it has advantageously been determined that the structure formation process, for example nanoparticle formation, can proceed in at least three different pathways. The structure formation process was investigated by ex-situ scanning electron microscopy (SEM) with respect to formation of nanoparticles. Figure 12a illustrates a pattern of printed features with polymer nanoreactors loaded with gold precursors. Figure 12b illustrates an AFM image of a patterned array of printed features having diameters of about 400 nm. Referring to Figure 12c, this allows for the monitoring of the polymer nanoreactors at various time points during annealing. Figure 12c was generated using an Au precursor in a PEO- P2VP polymer matrix. As illustrated in Figure 12c, as the Au precursor phase separates inside the polymer matrix and forms an aggregate; the even contrast is attribute to a homogeneous metal ion distribution. As illustrated in panel 2 of Figure 12c, during particle formation, there is a transition to a more heterogeneous appearance with one bright area being attributable to a localized concentration of metal ions. Because PEO is a weak reducing agent, further annealing at Ti_ow was performed to reduce the Au precursor and form an Au seed.

[0123] Figure 22 illustrates that weakly reducing nature of PEO. Figure 22a is a photograph of HAuCl₄ in PEO-&-P2VP aqueous solution (Au^m: 2 VP = 4: 1) after 1 day and 14 days. After 1 day, the Au^m was not yet reduced and was yellow in color. After 14 days, the solution changed to dark red, indicating the reduction of Au^m to Au° and the formation of Au nanoparticles in solution. The ratio Au:2VP was selected to highlight the color change in the exemplification of Figure 22a. Figure 22b is an SEM image of representative Au nanoparticles formed in the solution of Figure 22a after 14 days. The nanoparticles have various shapes and sizes. In inks containing high reduction potential precursor materials, like Au and Ag, it can be advantageous to use such inks within three days of preparation to avoid reduction of the precursor in the ink solution.

[0124] After annealing at Ti_ow for a sufficient time the Au precursor can be fully reduced and a single nanoparticle can be formed inside each polymer nanoreactor. Figure 12d illustrates an array of synthesized gold nanoparticles on a hydrophobic silicon substrate and a magnified view of a single gold nanoparticle, formed by methods in accordance with the disclosure. The dashed circle in the inset of Figure 12d illustrates the original size of the printed feature prior to thermal treatment and removal of the polymer nanoreactor.

[0125] For a nanoparticle that is formed by reduction of the precursor material, the precursor is either reduced by the polymer or through its thermal decomposition depending on the reduction potential of the precursor. For example, depending on the reduction potential of the precursor, the precursor can either be reduced by the polymer when annealed during the first thermal treatment at temperature Ti_ow (pathway 1) or during the second thermal treatment during T_hi_gh (pathway 2). Figure 12c illustrates an example of pathway 1. In other embodiments, the nanoparticle can have the same oxidation state as the precursor after the first and second thermal treatments (pathway 3). Standard reduction potentials of various precursor materials are provided in Table 1, below.

Table 1: Standard Reduction Potential of Precursor Materials

Half Reaction E°(Volts)

AuCl₄ ^"(aq) + 3e^" → Au(s) + 4Cl^"(aq) E° = 1.00

Ag⁺ + e^" →Ag(s) E° = 0.80

Fe³⁺ + e^" → Fe²⁺ E° = 0.77

[PtCl₄ (aq) + 2e^" _{→ Pt(s) + 4cr(aq)} E° = 0.73

[PtCl₆]² (aq) + 2e^" _→ [PtCl₄]²-(aq) + 2Cl (aq) E° = 0.68

[PdCl₄]^2~(aq) + 2e^" → Pd(s) + 4Cl^"(aq) E° = 0.59

Cu²⁺ + 2e^" → Cu(s) E° = 0.34

2H⁺ + 2e^~ → H₂(g) E° = 0.00

Ni²⁺ + 2e^~ → Ni(s) E° = -0.25

Co²⁺ + 2e^" → Co(s) E° = -0.28

Fe²⁺ + 2e^~ → Fe(s) E° = -0.44

[0126] As shown in Figure 12e, in embodiments in which the precursor reduces during the second thermal treatment, the elimination of the first thermal treatment can result in multiple nanoparticles being formed in a single printed feature, as precursor aggregation does not occur prior to particle formation.

[0127] Figure 13a provides a schematic illustration of the three pathways along with the x- ray photoelectron spectroscopy (XPS) images demonstrating formation of the nanoparticle along a given pathway. Table 2 below provides a listing of various decomposition pathways for precursor materials. Table 2: Decomposition Pathways

[0128] Figure 13b (left panel) provides XPS data for representative precursors for each pathway. For example, the XPS data in Figure 13b illustrates the formation of Au particles via pathway 1. The Au 4f_{7 2} peak for the HAuCl₄ salt precursor ink examined in Figure 3b is at 84.9 eV, which is within the expected range for Au¹. The partial reduction illustrated in Figure 13b prior to heat treatment may be attributed to either the reduction by PEO or by photoreduction during the measurement. After the first thermal treatment Δ1 at temperature Tiow, the Au 4f₇/2 peak shifts to 83.8 eV, indicating that the Au precursor has been reduced further by PEO. This peak lies slightly lower in energy than expected for bulk gold (84.0 eV), which may be attributed to the presence of electron-donating surface ligands from the PEO. This effect and shift in energy has been noted for gold nanoparticles suspended in electron-donating surface ligands. After performing the first thermal treatment Δ2 and thermal decomposition at Thigh, the positions of the Au 4f peaks shift slightly higher in energy to match those of bulk gold.

[0129] Metals with slightly lower reduction potentials, such as Pt and Pd, follow reduction Pathway 2 (Figure 13b, middle panel). In the case of Pt, for both the precursor containing ink (prior to the first thermal treatment) and after the first thermal treatment at r_low, the Pt 4f_7/2 peak lies in the range for Pt¹¹, which may be attributed either to reduction by PEO or in-situ photoreduction. XPS reveals that the Pt¹¹ has been fully reduced to Pt° after performing the second thermal treatment at high, as indicated by the shift in energy of the Pt 4f_7/2 peak to 70.9 eV, which closely matches that of metallic Pt. This pathway was also corroborated by ex-situ TEM (Figure 14).

[0130] Metals with a much lower reduction potential, such as Fe, follow Pathway 3 (Figure 13b, right panel). The XPS spectra for both the precursor containing ink (prior to the firs thermal treatment) and after performing the first thermal treatment at 7i_ow showed that the Fe 2p_3/2 peak was about 709-710 eV, which is consistent with mixed oxides of iron. After the second thermal treatment is performed, the Fe 2p_3/2 peak shifted in energy to 712.3 eV, which may be attributed to the formation of Fe₂0₃. This was confirmed by HRTEM (Figure 15).

[0131] The method of the disclosure advantageously allows for the formation of nanoparticles from a block-copolymer nanostructure precursor ink or printed feature using the first and second thermal treatments, despite the mechanism by which particle formation is achieved. Figure 23 illustrates representative STEM images for nanoparticle formulation using the methods of the disclosure for high and low reduction potential materials. For example, Ag, like Au forms particles via pathway 1 (Figure 16). The precursor materials for materials proceeding via pathway 1 are reduced easily and can migrate even after reduction at Ti_ow Pd nanoparticles, like Pt, form via pathway 2. Pd is not very mobile in the reduced state and, therefore, ion aggregation must occur prior to reduction to avoid the generation of multiple nucleation sites and many particles within one polymer feature. Co, Ni, and Cu, like Fe, form oxide nanoparticles via pathway 3. The precursors of such nanoparticles must aggregate before the second thermal treatment at Thigh, which facilitates oxide formation and polymer decomposition. As illustrated in Figure 14, the crystallinity and composition of the synthesized nanoparticles was verified by HRTEM images. Figures 17 and 18, illustrate EDS and XPS images further confirming the nanoparticle synthesis. In Figure 17, the Si signal is from the silicon nitride membrane. Al and Cu signals are from the TEM sample holder. Since a Cu signal is always present in the background, an EDX spectrum of Cu-containing nanoparticles is not shown. Figure 18 illustrates XPS spectra of nanoparticles composed of Ag, Pd, C0₂O₃, NiO, and CuO. All core element peak positions in Figure 18 fall within the expected range for the listed compositions, and all compositions were corroborated with results from HRTEM (Figure 14). Many of the particles formed via pathway 3 exist as metal oxides under ambient conditions. Further annealing of the metal oxide nanoparticles in a reducing atmosphere can be performed to obtain metal nanoparticles.

[0132] The method can be further used to form alloy nanoparticles by blending precursors in the ink. For example, 1 : 1 alloys were formed by loading Ag⁺ and Au³⁺ precursors in the polymer in a 1 : 1 molar ratio. Any suitable blending ratios between 0 and 1 can be used depending on the alloy structure to be formed.

[0133] The size of the nano structures synthesized by a method in accordance with embodiments of the disclosure can be controlled, for example, by controlling the volume of the patterned block copolymer containing features and the loading concentration of the nanostructure precursor. For example, increasing the loading concentration of the nanostructure precursor results in nanostructures having an increased size. . Additionally, without intending to be bound by theory, it is believed that increasing the molecular weight of the copolymer block results in a larger micelle cores, and hence, larger structures. The structure precursor determines the local concentration of ions within the polymer micelle. The lower the concentration, the small the synthesized nanostructures. Figure 20, for example, illustrates control of the size of gold nanoparticles in a size range between 3.6 nm and 56 nm by varying the concentration of the gold precursor in the block copolymer- nanostructure precursor ink in a range of about 4: 1 to about 256: 1 (block

copolymenprecursor ink).

[0134] The dwell time (also referred to herein as the tip-substrate contact time) during patterning of the block copolymer-nanostructure precursor inks can be about 0.01 seconds to about 30 seconds, about 0.01 second to about 10 seconds, about 0.05 seconds to about 8 seconds, about 0.1 seconds to about 6 seconds, about 0.5 seconds to about 4 seconds, about 1 second to about 2 seconds, about 10 seconds to about 30 seconds, about 8 seconds to about 26 seconds, about 6 seconds to about 24 seconds, about 15 seconds to about 20 seconds, or about 10 seconds to about 15 seconds. Other suitable dwell times includes, for example, about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30 seconds.

[0135] The size of the nanostructures synthesized by a method in accordance with embodiments of the disclosure can also be controlled by varying the dwell time when patterning by DPN or polymer pen lithography methods. The feature size dependence on tip- substrate contact time (dwell time) exhibited when using DPN or polymer pen lithography methods can be used to control both the size of the printed feature (having the block copolymer and the nanostructure precursor) and the size of the resulting nanostructure. For example, nanostructures synthesized using a method in accordance with embodiments of the disclosure and patterned by DPN can have a diameter that is linearly dependent on the square root of the tip- substrate contact time (dwell time).

[0136] In an exemplary embodiment, metal precursors are mixed with an aqueous solution of the block copolymer poly(ethylene oxide)-W6>cfc-poly(2-vinyl pyridine) (PEO-&-P2VP) and then cast onto arrays of DPN tips. The tips are mounted onto an AFM and subsequently brought into contact with hydrophobic surfaces to deposit the block copolymer loaded with metal precursors at selected sites, yielding large arrays of uniform, domed features that serve as nanoreactors for nanoparticle synthesis in later steps (Figures. 12a, b). After patterning, the metal precursors are homogenously distributed in the polymer nanoreactors, as evidenced by uniform contrast as viewed by scanning electron microscopy (SEM). To effect metal ion aggregation without reduction, the substrate with the nanoreactors was heated to Ti_ow = 150 °C in a tube furnace under a flow of Ar. This temperature is above the glass transition temperature of the polymer (T_g = -76 °C and 78 °C for PEO and P2VP, respectively, Polymer Source, Inc.), but below its decomposition temperature (T^ = 409 °C, Figure 19).

[0137] Generally, after aggregation of the precursor at T_low, a high temperature annealing step at Thigh = 500 °C is performed to decompose the polymer matrix and form the nanoparticle. By annealing at a temperature Thigh that is above the thermal decomposition temperature T^Sa of the metal salt precursor, the precursor decomposes and forms metal nanoparticles. In some embodiments, such as when Au and Ag ions are present in the ink, continued heating at 150 °C results in metal ion reduction and formation of a nanoparticle. Phase separation during the previous step concentrates the precursors into a single region, enabling the formation of a single nanoparticle in each spot. This process also decomposes the polymer, thereby removing the majority of the organic material. Any remaining organic material can be removed, for example, using a cleaning process. For example, the substrate can be immersed in a Piranha solution (3: 1 sulfuric acid : hydrogen peroxide).

[0138] After patterning, the substrate was loaded into a tube furnace and annealed in an argon stream. The annealing conditions were programmed as follows: for the first thermal treatment the furnace was ramped to 150°C in 1 h, soak at a temperature Ti_ow of 150°C for 4- 24 h, cool down to room temperature in 1 h. For the second thermal treatment the furnace was ramped to 500°C in 1 h, soak at a temperature Thi_gh of 500°C for 2-4 h, and cool down to room temperature in 1 h. The soaking time of the first and second thermal treatments was varied to ensure full phase separation between the metal compound and the polymer at 150°C and full decomposition of all materials at 500°C, respectively. Passivation Agents

[0139] In various embodiments, the substrate and optionally the nanostructures patterned on the substrate can be passivated to prevent non-specific adsorption of the biological species onto unpatterned areas. In embodiments in which the nanostructure is passivated, the nanostructure can be subsequently selectively functionalized with a material to allow for site- specific binding of the biological species onto the nanostructure.

[0140] Passivation agents include any material that does not bind to the biological species being immobilized. Exemplary agents include, but are not limited to silicon oxide, silicon dioxide, silicon nitride, silicon oxy-nitride, an organic film such as polyamide, a metal having a thin layer of oxidation (e.g., oxidized aluminum) and compounds containing sulfur groups (e.g., thiols, sulfides). Still other passivation agents used in methods provided alkanethiols as described above but without a substituted with a carboxylic acid functional group. In methods using an alkanethiol as a passivation agent, the alkanethiol can be linear or branched, having a carbon chain length of from C 8 to C 22. Linear alkanethiols have, in certain aspects, a chain length of from C8, C9, CIO, Cl l, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21 or C22. Alkanethiols which may be mentioned are carboxylic acid substituted forms of n- decanethiol, w-dodecanethiol, ieri-dodecanethiol, w-tetradecanethiol, n-pentadecanethiol, n- hexadecanethiol, w-heptadecanethiol, ra-octadecanethiol, ra-nonadecanethiol, w-eicosanethiol, n-docosanethiol. Use of alkanethiols in mixtures is also contemplated for use in the methods provided. Also, alkanethiols contemplated for use include poly- or oligo- ethyleneglycol thiol (Pale-Grosdemange et al., J. Am. Chem. Soc. (1991) 113: 12-20). In one embodiment, the passivation agent is polyethylene glycol functionalized silane.

Nanostructure Functionalization

[0141] The patterned nanostructure can either be functionalized in itself or can be functionalized with a material for allowing site specific binding or for purposes of coordinating metal ions, for example. The nanostructure can be, for example, silver, gold, platinum, palladium, cadmium, zinc, iron, cobalt, nickel, and copper.

[0142] In various embodiments, the nanostructure can be functionalized with a material that allows site specific binding of the protein to the functionalized nanostructure. For example, the nanostructure can be functionalized with biotin, an alkylthiol, such as an alkylthiol with a nitrilotriacetic acid (NTA) group, an N-succinimidyl ester group , afarnesyl pyrophosphate, coenzyme A, trimethoprim, and glutathione. In one embodiment, the nanostructure can be functionalized by forming self-assembled monolayers of biotin- alkylthiols on the nanostructures. The self-assembled monolayers of biotin-alkylthiols can be formed for example by immersing the nanostructures in a solution containing a biotin- alkylthiol. In another embodiment, the nanostructure can be functionalized with self assembled monolayers of an alkylthiol with a nitnlotriacetic acid group, which can allow, for example, for binding of unmodified IgG by metal affinity since these antibodies possess a histidine rich sequence near the C-terminal of the F_c region.

Biological Species

[0143] The biological species can be, for example, proteins, polypeptides, polysaccharides, oligonucleotides, and combinations thereof. For example, the biological species can be a protein, for example, a globular protein. For example, the biological species can include streptavidin, avidin-horseradish peroxidase, IgG, and/or decarboxylase enzymes, racemase enzymes and luciferase that have been suitably modified for immobilization.

[0144] The biological species can be immobilized on the surface of the nanostructure using site specific binding of the biological species to the nanostructure itself or to a nanostructure that is functionalized to allow for the site-specific binding. For example, the site-specific binding can be metal-ion binding in which the immobilized biological species associates with the metal ion surface. Other known binding interactions can also be used. For example, if the biological species to be immobilized is streptavidin, biotin can be used for site specific binding.

[0145] Biological species of interest that are conjugated with the appropriate fusion proteins can also be used, for example, for site-specific binding to small molecule ligands on the nanostructure. For example, fusions of dihydrofolate reductase to trimethoprim, glutathione 5-transferase to glutathione, biotin carrier protein to biotin and peptidyl carrier proteins to coenzyme A.

[0146] In various embodiments, the biological species is provided in solution and thus, hydrodynamic radius can be used to relate determine a suitable size of the nanostructure to control immobilization of the biological species thereon. In various embodiments, the radius of gyration can alternatively be used as a suitable parameter to determine a suitable size of the nanostructures to control immobilization of the biological species thereon. As noted above, the diameter of gyration as used herein refers to two times the radius of gyration.

[0147] The biological species can have a hydrodynamic radius, for example, of less than about 80 nm, less than about 60 nm, less than about 50 nm, less than about 40 nm, less than about 30, less than about 20 nm, less than about 15 nm, less than about 10 nm, less than about 9 nm, less than about 8 nm, less than about 7 nm, less than about 6 nm, or less than about 4 nm. For example, the biological species can have a hydrodynamic radius of about 1 nm to about 20 nm. The hydrodynamic radius can be measured conventionally, for example, by dynamic light scattering with the biological species being provided in a lx PBS buffer solution. As noted above, the hydrodynamic diameter as used herein refers to two times the hydrodynamic radius. Thus, the biological species can have hydrodynamic diameter of less than about 160 nm. In one embodiment, the biological species has a hydrodynamic diameter of less than 20 nm.

[0148] The biological species can have a radius of gyration, for example, of less than about 80 nm, less than about 60 nm, less than about 50 nm, less than about 40 nm, less than about 30, less than about 20 nm, less than about 15 nm, less than about 10 nm, less than about 9 nm, less than about 8 nm, less than about 7 nm, less than about 6 nm, or less than about 4 nm. For example, the biological species can have a radius of gyration of about 1 nm to about 20 nm. The biological species can retain its activity after immobilization on the nanostructure. In one embodiment, streptavidin is immobilized on a nanostructure. For example, streptavidin can be immobilized on a gold nanostructure functionalized with biotin. In another embodiment, avidin-horseradish peroxidase can be immobilized on a nanostructure, for example, a gold nanostructure. In yet another embodiment, IgG can be immobilized on a nanostructure, for example, a gold nanostructure.

[0149] The biological species can include a marker or label, for example, for identification of the biological species, confirmation of immobilization of the biological species on the nanostructure, and/or quantification of the biological species. The marker or label can be, for example, a quantum dot, for example a CdSe/ZnS core- shell quantum dot. In various embodiments, the biological species can include a nanoparticle as a label or marker. For example, NANOGOLD^® streptavidin (Nanoprobes) is a commercially available streptavidin that is labeled with a 1.4 nm Au nanoparticle. Any known labels or markers can be used and selected based on their compatibility with the biological species. Examples include, but are not limited to, organic fluorophores, radioisotopes, quantum dots, nanoparticles, dyes, and combinations thereof.

[0150] In various embodiments, the biological species can be immobilized by contacting the nanostructure with the biological species. For example, the nanostructure can be contacted with the biological species by immersing the nanostructure in a solution containing the biological species. Biological species can also be printed onto the nanostructures, for example, using dip pen nanolithography, polymer pen lithography, and hard tip spring lithography, as is known in the art. As discussed above, the biological species can be immobilized on the nanostructure using known methods and site- specific binding. For example, metal affinity binding can be used, such as described in U.S. Patent Application Publication No. 2010/0160182, the entire disclosure of which is incorporated herein by reference. Functionalization of the nanostructure can also be used to induce site-specific binding of the biological species. Non-site specific binding methods may also be employed for the functionalization of the nanostructures, including the use of N-succinimidyl active esters, epoxides, vinyl sulfones. Biological species can also be immobilized through nonspecific hydrophobic or electrostatic interactions.

EXAMPLES

Example 1: Patterning using Dip Pen Nanolithography

[0151] PEO-&-P2VP was dissolved in an aqueous solution at a concentration of 0.5% w/w. The PEO had a molecular weight of 2.8 kg/mol, and the PVP had a molecular weight of 1.5 kg/mol. HAuCl₄"3H₂0 was added to the solution at a 2: 1 molar ratio of P2VP to Au. The copolymer-gold salt solution was stirred for 24 hours. A DPN twelve pen tip array (available from Nanolnk, Skokie, IL) was dipped into the ink solution and then dried with nitrogen. The DPN experiment was performed on an Nscriptor system (Nanolnk) equipped with a 90 μηι closed loop scanner and commercial lithography software. The ink tips were brought in contact with a hexamethyldisilazane (HDMS) coated Si/SiO_x surface. Dots of uniform size were produced with a tip dwell time of 0.01 s at 70% relative humidity. Facile transport of PEO under high humidity environments allows for rapid deposition of PEO-£>-P2VP. The process was repeated 1600 times for a total patterning time of less than about 2 minutes to generate a 40 by 40 array of dot features, as shown in Figure 1C. The distance between features was 500 nm. In a representative 20-dot line generated by a single pen, each feature diameter was approximately 90 nm with a size deviation below 10%, as measured by AFM topography (Figure ID).

[0152] Referring to Figure 2A, the incorporation of AuCLf in the polymer micelle cores provided enough Z-contrast for observation by transmission electron microscopy (TEM), revealing the existence of spherical micelles in a bulk aqueous solution. The spherical micelles had a diameter of about 2 nm. When the PEO-Z?-P2VP/AuCi₄ inked pen array was brought in contact with the sample surface, micelles were transported to the substrate through the meniscus formed at the tip end, wherein interactions take place between the pyridine units due to tip-induced higher local concentration of the block copolymers, resulting in the coalescence of multiple micelles loaded with AuCl₄ ^~ ions, as shown in Figure 2B.

[0153] Referring to Figure 3, the pattern was then reduced by oxygen plasma, resulting in the formation of Au nanoparticles within the aggregated micelles. The surrounding polymer matrix was removed by the oxygen plasma, leaving square arrays of sub- 10 nm Au nanoparticles on the Si substrate (Figure IE). Referring to Figure 4A, scanning electron microscopy indicated that the method achieved 100% yield of single Au nanoparticles per spot in the 11 x 8 array. Figure 4B is a registry analysis of 400 particle features over different areas of the formed pattern. The distribution error is defined as the ratio of the distance of the particle away from the center of the block copolymer feature to the feature diameter.

[0154] The PEO-¾-P2VP/AuCl₄ ^~ ink was also patterned on a 50 nm Si₃N₄ TEM membrane followed by oxygen plasma reduction. Referring to Figure IF, TEM images revealed that the mean diameter of the Au nanoparticles in the array was 8.2 nm + 0.6 nm. The clear lattice fringes with an interplanar spacing of 0.24 nm corresponding to the (111) plane in face- centered-cubic Au. The spherical Au nanoparticles were highly crystalline. The

characteristic electron diffraction pattern also confirmed the single crystal nature of the Au nanoparticles (see inset of Figure IF).

Example 2: Varying the Feature Size

[0155] The time-dependent ink transport characteristics of DPN provide a facile route for controlling the size of the nanomaterials synthesized within the deposited block copolymer nanoreactors. It was observed that the diffusive characteristics of the block copolymer ink are similar to previous reports of feature size dependence on tip-substrate contact time. It is believed that the nanoparticles synthesized using this DPN-based approach have dimensions that are linearly dependent on the square root of the tip-substrate contact time.

[0156] Referring to Figure 5A, DPN was used to produce Au nanoparticles of different diameters in an environment of saturated humidity. Tip dwell times of 0.01, 0.9, 0.25, 0.49, and 0.81 seconds were used to generate the nanoparticles. The Au nanoparticles of various sizes without removal of the block copolymer matrix were confirmed by SEM and TEM images, as shown in Figures 5C, and 5D. The dimensional variation in the spot sizes deposited by DPN was measured by the height profile in topographical AMF (Figure 5B) and are graphically summarized in Figure 5E. The spot sizes increased from about 170 nm to about 240 nm as the dwell time increased from 0.01 seconds to 0.81 seconds, following the linear growth rate and square root dependence. Referring to Figure 5E, an increase in the diameter of the Au particles of from about 16 nm to about 24 nm was observed with increasing tip dwell time. Within the range of dwell times performed, a near linear relation between the dot size of the parent block copolymer matrix and the diameter of the synthesized Au nanoparticle at a fixed ratio of about 10. This demonstrates that the DPN- generated nanoparticles can have a dimension ten times smaller than that of the directly patterned original material, which is a significant advantage of embodiments of the method of the disclosure.

[0157] Referring to Figure 6, Au nanoparticles were also synthesized with varying features using a PEO-&-P2VP/HAuCl₄ ink by varying the dwell time. The features were patterned on S1₃N₄ substrates using DPN with dwell times of 25, 16, 9, 4, and 1 second (from the top to bottom of Figure 6). After reduction with oxygen plasma, single Au nanoparticles were formed within the block copolymer matrix. The circled features of Figure 6 illustrate features wherein multiple Au nanoparticles formed. Without intending to be bound by theory, it is believed that when the block copolymer features are large enough (for example, about 450 nm in diameter), more than one Au nanoparticle can form within the original printed feature.

Example 3: Patterning of sub-5 nm Au Nanoparticles

[0158] Sub-5 nm Au nanoparticles were synthesized by decreasing the salt concentration while using the same block copolymer as the synthetic nanoreactor. HAuCl₄ was added to the PEO-&-P2VP micelle solution to obtain a 4: 1 molar ratio of 2-vinylpyridine to gold. After stirring for one day, a pen array was loaded with the block copolymer-gold salt ink. The ink was then patterned on a S1₃N₄ membrane, followed by oxygen plasma exposure for Au reduction. Referring to Figure 8A, SEM images illustrated the formation of an array of Au nanoparticles having sub-5 nm diameters. The size of the Au nanoparticles was measured using the Z-contrast TEM image shown in Figure 8C. Referring to Figure 8B, the average diameter of the Au nanoparticles was 4.8 nm + 0.2 nm, a 4% variation.

Example 4: Patterning Using Polymer Pen Lithography

[0159] A 1 cm² polymer pen array (about 15,000 PDMS pens) with 80 μιη spacing between tips was inked with the PEO-Z>-P2VP/AuCl₄ ^~ ink by spin coating at a rate of 2000 rpm for 2 min. Using a Park AFM platform (XEP, Park Systems Co., Suwon, Korea) at 80% humidity, each pen in the PPL array was used to make a 20 x 20 dot array with 2 μηι spacing between the dots (Figure 9A). The deposition time for each dot was 0.5 seconds. Thus, an array of approximately 25 million dots (400 dots/pen) was generated in less than 5 minutes. Referring to Figure 9B, the block copolymer matrix was removed by oxygen plasma, resulting in the formation of an array of single Au nanoparticles.

Example 5: Immobilization of Stre tavidin on Gold Nanoparticles

[0160] Square arrays of about 10 nm Au nanoparticles with a 2 μιη pitch were formed on a silicon wafer. Chloroauric acid (HAuCl₄ was added to an aqueous solution of PEO-&-P2VP. After stirring for 24 hours, the metal ion-coordinated block copolymer mixture was coated onto scanning probes, which were then used for patterning. The block polymer precursor spots were deposited (as illustrated in Figure 24a), plasma treated to reduce the metal ions, and then the organic polymer was subsequently removed (as illustrated in Figure 24b). As illustrated in Figures 24c and 24d, nanoparticles can be formed to have diameters between 10 nm and 40 nm.

[0161] The silicon wafer having the Au nanoparticles patterned thereon was then modified with a polyethylene glycol (PEG)-functionalized silane in order to prevent non-specific adsorption of the protein on unpatterned areas. Self-assembled monolayers of biotin- alkythiols were formed on the Au nanoparticles by immersing the nanoparticles in a biotin- alkylthiol solution (Figure 25a). The biotin-bearing Au nanoparticle arrays were then used to immobilize CdSE/ZnS core-shell quantum dots that were modified with streptavidin. The quantum dots provided an easily observable label that was used to confirm binding of the streptavidin to the biotin-Au nanoparticles.

[0162] Transmission electron microscopy images of the substrate after immobilization demonstrated the binding of the quantum-dot labeled streptavidin on every patterned Au nanoparticle functionalized with biotin (Figure 25b). In the TEM images of Figure 25b, the white dots represent the Au nanoparticles and the grey rods are the CdSe/ZnS quantum dots. Energy-dispersive X-ray spectroscopy (EDX) elemental mapping of Au and Cd also confirmed the identity of the quantum dots immobilized onto patterned Au nanoparticles (Figure 30). No quantum dots were found on the PEG-passivated surface, confirming that streptavidin was exclusively attached to the Au pattern and there was no significant nonspecific adsorption.

Example 6: Controlling the Number of Biological Species Immobilized

[0163] Au nanoparticles (patterned features) were produced as described in Example 5 and treated with a solution of streptavidin that had been separately labeled with 1.4 nm Au nanoparticles. Commercially supplied Au nanoparticle- streptavidin conjugates were purified such that the material was predominately a 1 : 1 ratio of nanoparticles to streptavidin

(NANOGOLD^® streptavidin, Nanoprobes). These conjugates enabled the quantification of the number of streptavidin molecules immobilized onto the arrays by observation and counting of the smaller streptavidin bound Au nanoparticles that were bound to the Au nanoparticle patterned features. As illustrated in Figure 26 the smaller streptavidin- conjugated 1.4 nm Au nanoparticles were distinguishable from the larger about 10 nm Au nanoparticles patterned on the substrate by scanning probe block copolymer lithography. On average, each of the about 10 nm Au nanoparticles was associated with three of the smaller streptavidin-conjugated 1.4 nm Au nanoparticles. This demonstrates that the number of molecules attached to the nanoparticle can be controlled by controlling the size of the Au nanoparticle patterned feature to substantially corresponded to the hydrodynamic radius of the biological species and the number of biological species to be immobilized on the nanostructure, since the hydrodynamic radius of the streptavidin molecule (which is about 4 nm) and the number of molecules attached to the nanoparticle.

[0164] The relationship between the size of the Au nanoparticle patterned feature and the number of protein molecules attached to the nanoparticle was further investigated by deliberately adjusting the dwell time of the inked tip to produce arrays of Au nanoparticles with different diameters (Figure 27a). As described above, the patterned Au nanoparticles were treated with a solution of streptavidin labeled with 1.4 nm Au nanoparticles. The larger patterned Au nanoparticles had more streptavidin molecules attached to the nanoparticles (Figure 27b). When the printed Au nanoparticles was reduced to about 4 nm, which correlated with the hydrodynamic diameter of the streptavidin molecule, approximately 80% of the printed Au nanoparticles had only a single bound streptavidin. Thus, it has advantageously been demonstrated that the number of protein molecules attached to a nanoparticle can be controlled by controlling the patterned feature size. Scanning probe block copolymer lithography allows for precise control over feature size and consequently over the number of protein molecules attached to a patterned feature (i.e., nanostructure).

Example 7: Retention of Biological Activity by the Immobilized Biological Species

[0165] To demonstrate that a biological species immobilized on a nanostructure in accordance with the embodiments of the disclosure, a uniform array of 10 nm Au nanoparticles was generated as described in Example 5. The nanoparticles were

functionalized with biotin and used to immobilize avidin-horseradish peroxidase (HRP) conjugates. The enzymatic activity of the HRP was detected by a 3, 3', 5, 5' tetramethylbenxidine (TMB) assay with hydrogen peroxide. The enzymatic reaction resulted in a blue color which could be quantified by UV-Vis absorbance measurements of the test solution at 652 nm.

[0166] The substrates bearing avidin-HRP immobilized on the printed nanostructures functionalized with biotin were immersed in the assay solution. An increase in absorbance was observed overtime, which confirmed the activity of the immobilized protein (Figure 28).

[0167] Control experiments were performed using substrates that were not patterned, substrates in which avidin-HRP was not immobilized on a nanostructure, and with substrates having avidin-HRP immobilized on nanostructures, but with the TMB omitted from the assay solution. In each of these control experiments, no increase in absorbance above the baseline was observed.

Example 8: Immobilization Using Metal Affinity Binding

[0168] The use of metal affinity binding is extensively used both for the purification and immobilization of proteins and other biological species that posses an innate metal binding sequence, or a peptide sequence that is capable of binding divalent metal ions such as polyhistidine "His-tag." The ability to attach a protein to a nanostructure formed using scanning probe block copolymer lithography by metal affinity binding was confirmed. Au nanoparticles having a diameter of about 10 nm were formed on a substrate as described in Example 5. Self-assembled monolayers of an alkylthiol with a nitrilotriacetic acid (NTA) group was formed on the Au nanoparticles. The functionalized Au nanoparticles were used for site- selective binding of native (unmodified) IgG by metal affinity. This antibody possesses a histidine rich sequence near the C-terminal of the F_c region. The IgG was labeled with a 1.4 nm Au nanoparticle (at a 1:1 ratio of IgG:AuNP) to allow for quantification of the number of IgG molecules immobilized on a single patterned nanoparticle. The substrates having the IgG molecules immobilized on the nanoparticles was rinsed with PBS buffer and imaged by TEM. TEM imaging showed a single 1.4 nm Au nanoparticle label per about 10 nm nanoparticle (Figure 29b). IgG has an approximate size of 15 nm x 9.5 nm x 6.5 nm and a hydrodynamic radius of about 6 nm (a hydrodynamic diameter of about 12 nm). The foregoing demonstrates that when utilizing metal affinity binding, the number of biological species attached to a nanoparticle formed using scanning probe block copolymer lithography can be controlled by controlling the size of the nanostructure to correspond with the hydrodynamic radius of the biological species and the number to be attached. [0169] It was further demonstrated that the anti-goat IgG retained its biological activity after immobilization. The arrays were treated with a solution of goat-derived F_a antibody fragments that were conjugated with quantum dot rods. Figure 29b is a representative TEM image illustrating that each of the 10 nm patterned nanoparticles had a single 1.4 nm particle label and at least one quantum dot rod. EDX analysis was used to further confirm the identity of the 1.4 nm particle label and the quantum dot rods (Figure 31).

Example 9: Scanning Probe Block Copolymer Lithography with Precursor Aggregation

[0170] Metal precursors were mixed with an aqueous solution of the block copolymer poly(ethylene oxide)-Wocfc-poly(2- inyl pyridine) (PEO-Z?-P2VP) and then cast onto arrays of DPN tips. The tips were mounted onto an AFM and subsequently brought into contact with hydrophobic surfaces to deposit the block copolymer loaded with metal precursors at selected sites, yielding large arrays of uniform, domed features. The patterned features can serve as nanoreactors for nanoparticle synthesis in later steps (Figs. 12a, b). After patterning, the metal precursors were homogenously distributed in the polymer nanoreactors, as evidenced by uniform contrast as viewed by scanning electron microscopy (SEM). To effect metal ion aggregation without reduction, the substrate with the nanoreactors was heated to Ti_ow = 150 °C in a tube furnace under a flow of Ar. This temperature is above the glass transition temperature of the polymer (r_g = -76 °C and 78 °C for PEO and P2VP, respectively. Polymer Source, Inc.), but below its decomposition temperature ( ^ = 409 °C, Figure 19). After aggregation of the precursor at 7i_ow, a high temperature annealing step at = 500°C was performed to decompose the polymer matrix and form the nanoparticle. By annealing at a temperature T_hi_gh that is above the thermal decomposition temperature _d of the metal salt precursor, the precursor decomposed and formed metal nanoparticles. Phase separation during the previous step was observed to concentrate the precursors into a single region, enabling the formation of a single nanoparticle in each spot (Fig. 1 la). This process also decomposed the polymer, thereby removing the majority of the organic material.

[0171] The block copolymer poly(ethylene oxide)-block-poly(2- inyl pyridine) (PEO-b- P2VP, Mn=2.8-b-1.5 kg^»mol-l, polydispersity index, PDI=1.11) was purchased from

Polymer Source, Inc. and used as received. The glass transition temperatures Tg for PEO and P2VP of the block copolymer are -76 °C and 78 °C, respectively (Polymer Source, Inc.). Metal precursor compounds, HAuCl₄ ^»3H20, AgN0₃, H₂PtCl₆ ^»6H₂0, Na₂PdCl₄,

Fe(N0₃)₃ ^«9H20, Co(N0₃)₂ ^«6H20, Ni(N0₃)₂'6H20, and Cu(N0₃)₂ ^«3H₂0, were purchased from Sigma- Aldrich, Inc. HC1 and HN0₃ were purchased from Sigma- Aldrich and diluted before use. Hexamethyldisilazane (HMDS) and hexane were purchased from Sigma- Aldrich and used as received. DPN® pen arrays (Type M, no gold-coating) were purchased from Nanolnk, Inc. Hydrophobic silicon nitride membranes (membrane thickness=15 nm or 50 nm) were purchased from Ted Pella, Inc. Silicon wafers were purchased from Nova Electronic Materials.

[0172] PEO-&-P2VP and metal compounds were dissolved in water, respectively. After blending the solutions of polymer and metal compound, the pH of the solution was controlled to be between 3 and 4 by adding HC1 or HNO₃, for CI^" or NO₃ ^" containing metal compound, respectively. Figure 21 illustrates the effect of protonation of PEO-&-P2VP on the loading of precursors. The TEM images of Figure 21 are patterned arrays of nanoreactors of PEO-b- P2VP on a silicon nitride window after the first thermal treatment at a temperature Ti_ow of 150°C. Phase separation of Na₂PdCl₄ is only observed when HC1 is mixed in the aqueous solution of PEO-6-P2VP.

[0173] In the exemplified embodiments, the final solution had a PEO-&-P2VP

concentration of 5-100 mg'mi^"1. The ratio of 2VP: Mn+ was varied between 2: 1 and 256: 1 to control the size of the nanoparticles. After stirring rigorously overnight, the solution was dip- coated onto the DPN® pen array. After drying in a nitrogen stream, the pen array was brought in contact with a substrate to generate arbitrary arrangements of printed features using an NScriptor (Nanolnk, Inc.) in a chamber with controlled humidity. The relative humidity was in the range of 75%-95 to control the dimensions of polymer nanoreactors of the printed features delivered from the pen array to the substrate. Both hydrophobic silicon nitride membranes and silicon wafers treated with HMDS were used. Silicon wafers were kept in a desiccator with two vials of HMDS and hexane mixture for 24 h to ensure their hydrophobicity.

[0174] Atomic Force Microscopy (AFM): AFM measurements were performed on a Dimension Icon (Bruker, Inc.) to obtain three-dimensional profiles of the patterned nanoreactors, which were delivered on a surface using dip-pen nanolithography.

[0175] Scanning Electron Microscopy (SEM) and Energy-Dispersive X-ray spectroscopy (EDX): Samples prepared on hydrophobic silicon wafers were imaged with a Hitachi S-4800 SEM at an acceleration voltage of 5 kV and a current of 20 μΑ. Probe current was set to high, and focus mode was set to ultrahigh resolution (UHR). Only the upper second electron detector was used. To determine the elemental composition, INCA (Oxford Instruments INCA 4.15) was used to obtain EDX spectra. [0176] Scanning Transmission Electron Microscopy (STEM), High Resolution

Transmission Electron Microscopy (HRTEM) and EDX: After annealing, samples prepared on 50-nm-thick silicon nitride membranes were imaged with a Hitachi STEM HD-2300A in Z-contrast mode at an acceleration voltage of 200 kV and a current of 78 μΑ. EDX spectra were obtained with Thermo Scientific NSS 2.3. Samples prepared on 15-nm-thick silicon nitride membranes were imaged with a JOEL 21 OOF at an acceleration voltage of 200 kV.

[0177] Thermogravimetric Analysis (TGA): The polymer decomposition temperature was measured on a TGA/DSC (Mettler Toledo International Inc.) by heating from room temperature to 600°C at a ramping rate of 10°C/min. The measurement was performed under an N2 atmosphere.

[0178] X-ray Photoelectron Spectroscopy (XPS): To monitor the reduction of metal compounds, aqueous solutions of PEO-b-P2VP with the corresponding metal compound were drop-cast on silicon wafers. After annealing at 150°C and 500°C, the samples were loaded into a vacuum chamber for XPS measurement (Omicron, ESCA probe).

[0179] The foregoing describes and exemplifies aspects of the invention but is not intended to limit the invention defined by the claims which follow. All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the materials and methods of this invention have been described in terms of specific embodiments, it will be apparent to those of skill in the art that variations may be applied to the materials and/or methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved.

[0180] All patents, publications and references cited herein are hereby fully incorporated by reference. In case of conflict between the present disclosure and incorporated patents, publications and references, the present disclosure should control.

Claims

WHAT IS CLAIMED:

1. A substrate comprising an array of nanostructures and one or more biological species immobilized on the nanostructures, wherein at least about 70% of the nanostructures have a single biological species immobilized thereon.

2. The substrate of claim 1, where at least about 80% of the nanostructures have a single biological species immobilized thereon.

3. The substrate of claim 1, wherein at least about 90% of the nanostructures have a single biological species immobilized thereon.

4. The substrate of any one of the preceding claims, wherein the nanostructures have a diameter (or line width) that is substantially equal to a hydrodynamic diameter of the biological species.

5. The substrate of claim 4, wherein each nanostructure has a diameter (or line width) that is within a tolerance of about 0% to about 30% the hydrodynamic diameter of the biological species.

6. A substrate comprising an array of nanostructures and one or more biological species immobilized on the nanostructures, wherein at least about 70% of the nanostructures have a preselected number of biological species immobilized thereon.

7. The substrate of claim 6, where at least about 80% of the nanostructures have the preselected number of biological species immobilized thereon.

8. The substrate of claim 6, wherein at least about 90% of the nanostructures the preselected number of biological species immobilized thereon.

9. The substrate of any one of claims 6 to 8, wherein the nanostructures have a diameter (or line width) that is substantially equal to a hydrodynamic diameter of the biological species times the number of biological species preselected to be immobilized on each nanostructures.

10. The substrate of claim 9, wherein each nanostructure has a diameter (or line width) that is within a tolerance of about 0% to about 30% of the hydrodynamic diameter of the biological species times the number of biological species preselected to be immobilized on each nanostructure.

11. The substrate of any one of the preceding claims, wherein the substrate comprises at least about 10,000 nanostructures.

12. The substrate of any one of the preceding claims, wherein the biological species is selected from the group consisting of proteins, polypeptides, polysaccharides, oligonucleotides, and combinations thereof.

13. The substrate of claim 12, wherein the biological species is a protein.

14. The substrate of claim 13, wherein the protein is streptavidin, IgG, or avidin- horseradish peroxidase.

15. The substrate of any one of the preceding claims, wherein the biological species retains its biological activity after being immobilized on the nanostructure.

16. The substrate of any one of the preceding claims, wherein the biological species has a hydrodynamic diameter of less than about 160 nm.

17. The substrate of any one of the preceding claims, wherein the biological species has a hydrodynamic diameter of less than about 20 nm.

18. A method for controlling a number of biological species immobilized on a nanostructure, comprising:

selecting a number of biological species to be immobilized on a nanostructure, the biological species having a hydrodynamic diameter;

contacting a substrate with a tip coated with an ink comprising a block copolymer and a nanostructure precursor to form a printed feature comprising the block copolymer and the nanostructure precursor on the substrate, wherein the printed feature is formed to have a diameter (or line width) to form a nanostructure having a diameter (or line width) corresponding to (a) the hydrodynamic diameter of the biological species and (b) the number of biological species selected to be immobilized on the nanostructure;

reducing the nanostructure precursor of the printed feature to form the nanostructure having a diameter (or line width) of less than 1 μηι; and immobilizing the biological species on the nanostructure, wherein the selected number of biological species are immobilized on the nanostructure.

19. The method of claim 18, wherein a single biological species is immobilized on the nanostructure.

20. The method of claim 19, wherein the selected number of biological species is a single biological species and the nanostructure has a diameter (or line width) that is substantially equal to the hydrodynamic diameter of the biological species.

21. The method of claim 19, wherein the nanostructure has a diameter (or line width) that is within a tolerance of about 0% to about 30% of the hydrodynamic diameter of the biological species.

22. The method of claim 18, wherein the nanostructure has a diameter (or line width) that is within a tolerance of about 0% to about 30% of the hydrodynamic diameter of the biological species times the number of biological species selected to be immobilized on the nanostructure.

23. A method for forming a sub-micron sized nanostructure comprising a single biological species immobilized thereon on a substrate surface, comprising:

contacting a substrate with a tip coated with an ink comprising a block copolymer and a nanostructure precursor to form a printed feature comprising the block copolymer and the nanostructure precursor on the substrate;

reducing the nanostructure precursor of the printed feature to form a nanostructure having a diameter (or line width) substantially equal to a hydrodynamic diameter of the biological species and having a diameter (or line width) of less than 1 μιη; and

immobilizing a single biological species on the nanostructure.

24. The method of claim 23, wherein the nanostructure has a diameter (or line width) that is within a tolerance of about 0% to about 30% of the hydrodynamic diameter of the biological species.

25. The method of any one of claims 18 to 23, wherein the biological species is selected from the group consisting of proteins, polypeptides, polysaccharides,

oligonucleotides, and combinations thereof.

26. The method of claim 25, wherein the biological species is a protein.

27. The method of claim 26, wherein the protein is streptavidin, IgG, or avidin- horseradish peroxidase.

28. The method of any one of claims 18 to 27, wherein the biological species retains its biological activity after being immobilized on the nano structure.

29. The method of any one of claims 18 to 28, wherein the biological species has a hydrodynamic diameter of less than about 160 nm.

30. The substrate of any one of claims 18 to 28, wherein the biological species has a hydrodynamic diameter of less than about 20 nm.

31. The method of any one of claims 18 to 30, further comprising passivating the substrate to prevent non-specific binding of the biological species.

32. The method of claim 31, wherein the substrate is passivated with a polyethylene glycol functionalized silane or an alkanethiol.

33. The method of claim 32, wherein the alkanethiol is a poly or oligoethylene glycol thiol.

34. The method of claim 32, wherein the alkanethiol is 11-mercaptoundecyl- penta(ethylene glycol) (PEG-SH).

35. The method of any one of claims 18 to 34, further comprising coating the nanostructures with a biotin-alkylthiol prior to immobilizing the biological species on the nanostructure.

36. The method of any one of claims 18 to 35, wherein the nanostructure has a diameter (or line width) of about 10 nm or less.

37. The method of claim 36, wherein the nanostructure has a diameter (or line width) of less than 5 nm.

38. The method of any one of claims 18 to 37, wherein the block copolymer matrix is selected from the group consisting of PEO-&-P2VP, PEO- -P4VP, and PEO-b- PAA.

39. The method of any one claims 18 to 38, wherein the block copolymer comprises a first polymer for concentrating the nanostructure precursor and a second polymer to facilitate ink transport.

40. The method of any one of claims 18 to 39, wherein nanostructure precursor comprises a metal salt.

41. The method of claim 40, wherein the metal salt comprises a metal selected from the group consisting of silver, gold, platinum, palladium, cadmium, zinc, iron, nickel, copper, and combinations and metal alloys thereof.

42. The method of claim 41, wherein the metal salt is selected from the group consisting of HAuCl₄, H₂PtCl₆, Na₂PdCl₄, Co(N0₃)₂, CdCl₂, ZnCl₂, FeCl₃, AgN0₃,

Cu(N03)₂, Ni(N03)_¾, and combinations thereof.

43. The method of any one of claims 18 to 42, wherein the block copolymer matrix comprises PEO-&-P2VP, the nanostructure precursor comprises HAuCl₄, and the ink comprises an about 1: 1 to about 10: 1 molar ratio of P2VP: Au.

44. The method of any one of claims 18 to 43, comprising reducing the metal salt by performing a plasma treatment.

45. The method of claim 44, wherein the plasma treatment is an oxygen plasma treatment or an argon plasma treatment.

46. The method of any one of claims 18 to 45, comprising contacting the substrate with a tip array comprising a plurality of tips, with each tip being coated in the ink.

47. The method of any one of claims 18 to 46, comprising contacting the substrate with the tip for a period of time of about 0.01 seconds to about 30 seconds.

48. The method of any one of claims 18 to 47, comprising contacting the substrate for a first contacting period of time to form a first printed feature and further comprising moving the tip, the substrate, or both, and repeating the contacting step for a second contacting period of time to form a second printed features, the first and second printed features each having a diameter (or line width) to form first and second nanostructures each having a diameter (or line width) corresponding to (a) the hydrodynamic diameter of the biological species and (b) the number of biological species to be immobilized on the printed feature; and

immobilizing the biological species on the first and second printed features.

49. The method of claim 48, comprising selecting the number of biological species to be immobilized on the first nanostructure to be different than on the second nanostructure, and wherein the first and second contacting periods of time are different.

50. The method of any one of claims 18 to 49, wherein the printed feature comprises block copolymer matrix micelles having the nanostructure precursor contained therein.

51. The method of any one of claims 18 to 50, wherein the printed features have a diameter (or line width) of about 20 nm to about 1000 nm.

52. The method of any one of claims 18 to 51, further comprising removing the block copolymer matrix after reducing the nanostructure precursor in the printed feature.

53. The method of any one of claims 18 to 52, comprising removing the block copolymer matrix by performing a plasma treatment.

54. The method of any one of claims 18 to 53, wherein the nanostructure is a nanoparticle.

55. The method of any one of claims 18 to 54, wherein the tip is a tip for dip pen nanolithography.

56. The method of any one of claims 18 to 55, wherein the tip is disposed on a cantilever.

The method of claim 56, wherein the tip is an atomic force microscope tip.

58. The method of any one of claims 18 to 57, comprising contacting the substrate with at least one tip from a tip array comprising a plurality of tips fixed to a common substrate layer, the tips and the common substrate layer being formed from an elastomeric polymer or elastomeric gel polymer, and the tips having a radius of curvature of less than about 1 μιη.

59. A method for controlling a number of biological species immobilized on a structure, comprising:

contacting a substrate with a tip coated with a composition comprising a block copolymer and a structure precursor to form a printed feature comprising the block copolymer and the structure precursor on the substrate, wherein the printed feature is formed to have a diameter (or line width) to form a structure having a diameter (or line width) corresponding to (a) the hydrodynamic diameter of the biological species and (b) the number of biological species selected to be immobilized on the nanoparticle;

heating the printed feature to a temperature below a decomposition temperature of the block copolymer to aggregate the structure precursor and form a structure precursor aggregated printed feature;

heating the structure precursor aggregated printed feature to a temperature above the decomposition temperature of the structure precursor to decompose the polymer, thereby forming the structure; and

immobilizing the biological species on the structure, wherein the selected number of the biological species are immobilized on the structure.

60. The method of claim 59, comprising selecting a single biological species is immobilized on the structure.

61. The method of claim 60, wherein the structure has a diameter (or line width) that is substantially equal to the hydrodynamic diameter of the biological species.

62. The method of claim 61, wherein the structure has a diameter (or line width) that is within a tolerance of about 0% to about 30% of the hydrodynamic diameter of the biological species.

63. The method of claim 59, wherein the structure has a diameter (or line width) that is within a tolerance of about 0% to about 30% of the hydrodynamic diameter of the biological species times the number of biological species selected to be immobilized on the structure.

64. A method for forming a sub-micron sized nanostructure comprising a single biological species immobilized thereon on a substrate surface, comprising:

contacting a substrate with a tip coated with a composition comprising a block copolymer and a nanostructure precursor to form a printed feature comprising the block copolymer and the nanostructure precursor on the substrate, wherein the printed feature is formed to have a diameter (or line width) to form a nanostructure having a diameter (or line width) corresponding to (a) the hydrodynamic diameter of the biological species and (b) the number of biological species selected to be immobilized on the nanostructure;

heating the printed feature to a temperature below a decomposition temperature of the block copolymer to aggregate the nanostructure precursor and form a nanostructure precursor aggregated printed feature;

heating the nanostructure precursor aggregated printed feature to a temperature above the decomposition temperature of the nanostructure precursor to decompose the polymer and form the nanostructure, the nanostructure having a diameter (or line width) substantially corresponding to the hydrodynamic diameter of the biological species and having a diameter (or line width) of less than 1 μιη; and

immobilizing the biological species on the nanostructure, wherein the selected number of the biological species are immobilized on the nanostructure.

65. The method of claim 64, wherein a single biological species is immobilized on the nanostructure.

66. The method of claim 65, wherein the nanostructure has a diameter (or line width) that is substantially equal to the hydrodynamic diameter of the biological species.

67. The method of claim 66, wherein the nanostructure has a diameter (or line width) that is within a tolerance of about 0% to about 30% of the hydrodynamic diameter of the biological species.

68. The method of claim 64, wherein the nanostructure has a diameter (or line width) that is within a tolerance of about 0% to about 30% of the hydrodynamic diameter of the biological species times the number of biological species selected to be immobilized on the nanostructure.

69. The method of claim 59 to 68, wherein the biological species is selected from the group consisting of proteins, polypeptides, polysaccharides, oligonucleotides, and combinations thereof.

70. The method of claim 69, wherein the biological species is a protein.

71. The method of claim 70, wherein the protein is streptavidin, IgG, or avidin- horseradish peroxidase.

72. The method of any one of claims 59 to 71, wherein the biological species retains its biological activity after being immobilized on the nanostructure.

73. The method of any one of claims 59 to 72, wherein the biological species has a hydrodynamic diameter of less than about 160 nm.

74. The method of any one of claims 59 to 72, wherein the biological species has a hydrodynamic diameter of less than about 20 nm.

75. The method of any one of claims 59 to 74, further comprising passivating the substrate to prevent non-specific binding of the biological species.

76. The method of claim 75, wherein the substrate is passivated with a polyethylene glycol functionalized silane or an alkanethiol.

77. The method of claim 76, wherein the alkanethiol is a poly or oligoethylene glycol thiol.

78. The method of claim 77, wherein the alkanethiol is 11-mercaptoundecyl- penta(ethylene glycol) (PEG-SH).

79. The method of any one of claims 59 to 78, further comprising coating the nanostructures with a biotin-alkylthiol prior to immobilizing the biological species on the nanostructure.

80. The method of any one of claims 59 to 79, comprising contacting the substrate for a first contacting period of time to form a first printed feature and further comprising moving the tip, the substrate, or both, and repeating the contacting step for a second contacting period of time to form a second printed features, the first and second printed features each having a diameter (or line width) to form first and second nanostructures each having a diameter (or line width) corresponding to (a) the hydrodynamic diameter of the biological species and (b) the number of biological species to be immobilized on the printed feature; and

immobilizing the biological species on the first and second printed features.

81. The method of claim 80, comprising selecting the number of biological species to be immobilized on the first nanostructure to be different than on the second nanostructure, and wherein the first and second contacting periods of time are different.

82. A method for forming a nanostructure on a substrate surface, comprising: contacting a substrate with a tip coated with a composition comprising a block copolymer and a nanostructure precursor to form a printed feature comprising the block copolymer and the nanostructure precursor on the substrate;

heating the printed feature to a temperature below a decomposition temperature of the block copolymer to aggregate the nanostructure precursor and form a nanostructure precursor aggregated printed feature; and

heating the nanostructure precursor aggregated printed feature to a temperature above the decomposition temperature of the nanostructure precursor to decompose the polymer and form the nanostructure.

83. The method of any one of claims 59 to 82, comprising contacting the substrate with a tip array comprising a plurality of tips, with each tip being coated in an ink.

84. The method of claim 83, wherein the plurality of tips are coated in a combinatorial set of inks.

85. The method of any one of claims 59 to 82, wherein the tip is a tip for dip pen nanolithography.

86. The method of claim any one of claims 59 to 85, wherein the tip or each tip of the plurality of tips is disposed on a cantilever.

87. The method of any one of claims 59 to 82, wherein the tip is an atomic force microscope tip.

88. The method of any one of claim 59 to 82, comprising contacting the substrate with at least one tip from a tip array comprising a plurality of tips fixed to a common substrate layer, the tips and the common substrate layer being formed from an elastomeric polymer or elastomeric gel polymer, and the tips having a radius of curvature of less than about 1 μιη.

89. The method of any one of claims 59 to 88, comprising contacting the substrate with the tip for a period of time of about 0.01 seconds to about 30 seconds.

90. The method of any one of claims 59 to 89, comprising contacting the substrate for a first contacting period of time and further comprising moving the tip, the substrate, or both, and repeating the contacting step for a second contacting period of time.

91. The method of claim 90, wherein the first and second contacting periods of time are different.

92. The method of any one of claims 59 to 91, wherein the printed feature comprises block copolymer matrix micelles having the nanostructure precursor contained therein.

93. The method of any one of claims 59 to 92, wherein the printed features have a diameter (or line width) of about 20 nm to about 1000 nm.

94. A method of forming a nanostructure on a substrate surface, comprising: heating a substrate comprising a composition comprising a block copolymer and a nanostructure precursor to a temperature below the decomposition temperature of the block copolymer to aggregate the nanostructure precursor to form a nanostructure precursor aggregated composition; and

heating the nanostructure precursor aggregated composition to a temperature above the decomposition temperature of the nanostructure precursor to decompose the polymer and form the nanostructure.

95. The method of claim 94, comprising applying the composition comprising the block copolymer and the nanostructure precursor under conditions sufficient to allow phase separation of the block copolymer.

96. The method of claim 94 or 95, comprising applying the composition comprising the block copolymer and the nanostructure precursor to a substrate by micro contact printing.

97. The method of claim 94 or 95, comprising applying the composition comprising the block copolymer and the nanostructure precursor to the substrate by one or more of dip coating, spin coating, vapor coating, spray coating, and brushing.

98. The method of any one of claims 82 to 97 further comprising immobilizing a biological species on the nanostructure.

99. The method of claim 98, wherein the biological species is selected from the group consisting of proteins, oligonucleotides, polypeptides, polysaccharides, and combinations thereof.

100. The method of any one of claims 59 to 99, wherein the nanostructure has a diameter (or line width) of less than 10 nm.

101. The method of any one of claims 59 to 100, wherein the nanostructure has a diameter (or line width) of less than 5 nm.

102. The method of any one of claims 59 to 101, wherein the block copolymer matrix is selected from the group consisting of PEO-&-P2VP, PEO-^-P4VP, and PEO-b- PAA.

103. The method of any one of claims 59 to 102, wherein the block copolymer comprises a first polymer for concentrating the nanostructure precursor and a second polymer to facilitate ink transport.

104. The method of any one of claims 59 to 103, wherein nanostructure precursor comprises a metal salt.

105. The method of claim 104, wherein the metal salt comprises a metal selected from the group consisting of gold, silver, platinum, palladium, iron, cadmium, cobalt, nickel, copper, and combinations and metal alloys thereof.

106. The method of any one of claims 59 to 105, wherein the nanostructure precursor is selected from the group consisting of HAuCl₄, AgN0₃, H₂PtCl₆, Na₂PdCl₄, Fe(N0₃)₃, Co(N0₃)₂, Ni(N0₃)₂, Cu(N0₃)₂, Na₂PtCl₄, CdCl₂, ZnCl₂, FeCl₃, NiCl₂, and combinations thereof.

107. The method of any one of claims 59 to 106, wherein the composition comprises an about 1: 1 to about 256: 1 molar ratio of block copolymer to nanostructure precursor.

108. The method of any one of claims 59 to 107, wherein the nanostructure is a metal oxide.

109. The method of any one of claims 59 to 108, wherein the nanostructure is a metal nanoparticle.

110. The method of any one of claims 59 to 109, wherein the nanostructure is a metal alloy nanoparticle.

111. The method of any one of claims 59 to 110, wherein the nanostructure is a single nanoparticle.

112. The method of any one of claims 59 to 111, comprising heating the printed feature or the substrate comprising the composition comprising the block copolymer and nanostructure precursor for about 2 hours to about 24 hours.

113. The method of any one of claims 59 to 112, comprising heating the nanostructure precursor aggregated printed feature or the nanostructure precursor aggregated composition for about 2 hours to about 10 hours.

114. The method of any one of claims 59 to 113, comprising heating the printed feature or the substrate comprising the composition comprising the block copolymer and the nanostructure precursor at a rate of about l°C/min to about 10°C/min.

115. The method of any one of claims 59 to 114, comprising heating the printed feature or the substrate comprising the composition comprising the block copolymer and the nanostructure precursor to a temperature above a glass transition temperature of the block copolymer and below a decomposition temperature of the block copolymer.

116. The method of any one of claims 59 to 115, comprising heating the nanostructure precursor aggregated printed feature to a temperature above the decomposition temperature of the nanostructure precursor to decompose the polymer and below a melting temperature of the nanostructure to be formed.