Catalysis via photonics, microelectronics, and temperature

Abstract

A method comprises providing a silicon surface having hydride terminated silicon and / or silicon nitride groups thereon and functionalizing the silicon surface to form an organic monolayer on the silicon surface. The method further includes patterning the organic monolayer to form attachment sites; and coupling an enzyme molecule to a respective attachment site. A microdevice includes a silicon surface with hydride terminated silicon and silicon nitride groups thereon, and a functionalized organic monolayer linked to the hydride terminated silicon and / or silicon nitride groups. The functionalized organic monolayer has a pattern of the attachment sites.

Description

CATALYSIS VIA PHOTONICS, MICROELECTRONICS, AND TEMPERATURECROSS-REFERENCE TO RELATED APPLICATIONS10001} This application claims the benefit of U.S. provisional application Serial No. 63 / 771,233, filed March 13, 2025, the disclosure of which is hereby incorporated in its entirety by reference herein.TECHNICAL FIELD[00021 The present application is directed to a microsystem platform for physics-based control of catalysis, and more specifically, functionalized surfaces of the microsystem platform to support catalysts thereon.BACKGROUND

[0003] Microsystem platforms are generally used in various devices such as biologic devices and microfluidic devices. Such systems incorporate catalysis (e.g., enzyme cascades) on the surface of the microsystem platform via deposition of the catalysts on the surface. Immobilization of the catalysts (e g., enzymes) on the microsystem surface is an important aspect of tunability of control, and nanometer-scale spatial registration is desired for functionality and output yield. While modern biotechnology provides a toolbox of diverse enzymatic catalysts that can be combined to synthesize a vast number of high-impact molecules, optimizing such multi-enzyme production systems is often laborious, bespoke, and expensive, since different enzymes may re-quire conflicting conditions for optimal activity

[0004] Conventional multi-enzyme cascades suggest that immobilizing enzymes in close proximity on self-assembled molecular scaffolds, such as DNA origami, improves catalytic activity, however such scaffolds are bespoke, static, and confound multiple factors that influence catalysis. Furthermore, while immobilized catalysts have been used for conventional reactors,certain immobilization techniques utilized for reactors hinder enzyme activity, are typically focused on a single catalyst type, and a full system for programmable and reconfigurable control of multiple independent catalysts has yet to be demonstrated. The closest existing technologies for microsystems integrated with biology are for biosensing and DNA sequencing; however, in these conventional approaches the system is used for readout, not control.SUMMARY

[0005] According to at least one embodiment, a method includes providing a silicon surface having hydride terminated silicon and / or silicon nitride groups thereon and functionalizing the silicon surface to form an organic monolayer on the silicon surface. The method further includes patterning the organic monolayer to form attachment sites and coupling an enzyme molecule to a respective attachment site.

[0006] In at least one embodiment, the providing may include radically induced hydrosilylation of the silicon surface to form the hydride terminated silicon groups thereon. In one or more embodiments, the functionalizing may include introducing a linker to form a functionalized organic monolayer. In one or more further embodiments, the functionalized organic monolayer may be an alkyne functional monolayer. In at least one further embodiment, the enzyme molecule may be an azide derived molecule. In at least one further embodiment, the method may further include converting surface alkynes in the alkyne functional monolayer to metal free click coupling sites. In one or more embodiments, the patterning may be via electron beam lithography. In at least one embodiment, the coupling may include forming an enzyme cascade on the silicon surface. According to one or more embodiments, the method may further include stripping native oxides from the silicon surface.

[0007] According to one or more embodiments, a microdevice includes a silicon surface with hydride terminated silicon and / or silicon nitride groups thereon, and a functionalized organic monolayer linked to the hydride terminated silicon and silicon nitride groups. The functionalized organic monolayer has a pattern of the attachment sites.

[0008] In at least one embodiment, the functionalized organic monolayer may be an alkyne functional monolayer. In one or more embodiments, the microdevice may further include an enzyme coupled with an attachment site. In at least one further embodiment, the enzyme may be an azide derived molecule. In one or more embodiments, at least a portion of the functionalized organic monolayer may include converted surface alkynes as metal free click coupling sites. According to one or more embodiments, an enzyme cascade may be coupled with the attachment sites per the pattern.

[0009] According to one or more embodiments, a method includes stripping native oxide from a silicon surface of a substrate; functionalizing the silicon surface via radically induced hydrosilylation to form an organic monolayer on the silicon surface with hydride terminated silicon and / or silicon nitride groups thereon; patterning the organic monolayer to form attachment sites; and coupling at least one enzyme molecule to a respective attachment site.

[0010] In at least one embodiment, the organic monolayer may be an alkyne functional monolayer. According to at least one further embodiment, the method may further include converting surface alkynes in the alkyne functional monolayer to metal free click coupling sites. In one or more embodiments, the functionalizing may be via introduction of a linker. In at least one further embodiment, the linker may be aliphatic or aromatic.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIG. 1 is a schematic diagram of a microsystem platform according to at least one embodiment;

[0012] FIG. 2 is a schematic example of the functionalization of a microsystem surface according to one or more embodiments, depicting Si-H surface formation and attachment of alkene functional organic molecules to form an organic monolayer;[00131 FIG. 3 shows graphs depicting stability data for SiN-alkene monolayers;

[0014] FIG. 4 is a schematic diagram of a method of hydrosilylation on silicon nitride and integration of enzymatic molecules into microsystem platforms;

[0015] FIG. 5 is a schematic example of functionalizing the monolayer, according to an embodiment;

[0001] FIG. 6 is a schematic example of coupling the enzymes to the attachment sites, according to an embodiment;

[0017] FIG. 7 is a flow chart of a method of preparing a microsystem surface for catalysis, according to an embodiment;

[0018] FIG. 8 are images of nanowell device features; and

[0019] FIG. 9 shows an example of a reaction pathway for a microdevice platform according to an embodiment.DETAILED DESCRIPTION

[0020] As required, detailed embodiments of the present invention are disclosed herein; however, it is to be understood that the disclosed embodiments are merely exemplary of the invention that may be embodied in various and alternative forms. The figures are not necessarily to scale; some features may be exaggerated or minimized to show details of particular components. Therefore, specific structural and functional details disclosed herein are not to be interpreted as limiting, but merely as a representative basis for teaching one skilled in the art to variously employ the present invention.

[0021] Except in the examples, or where otherwise expressly indicated, all numerical quantities in this description indicating amounts of material or conditions of reaction and / or use are to be understood as modified by the word "about" in describing the broadest scope of the invention. Practice within the numerical limits stated is generally preferred. As used herein, the term “about” means that the amount or value in question may be the specific value designated or some other value in its neighborhood. The term “about” or “generally” denoting a certain value is intended to denote a range within + / - 5% of the value. As one example, the phrase “about 10” denotes a range of 10+ / - 5, i.e. the range from 95 to 105. When the term “about” or “generally” is used, it can be expected that similar results or effects according to the invention can be obtained within a rangeof + / - 5% of the indicated value. It should also be appreciated that integer ranges (e.g., for measurements or dimensions) explicitly include all intervening integers. For example, the integer range 1-10 explicitly includes 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Similarly, the range 1 to 10 includes 1, 2, 3, 4, . . . 97, 98, 99, 10. Similarly, when any range is called for, intervening numbers that are increments of the difference between the upper limit and the lower limit divided by 10 can be taken as alternative upper or lower limits. For example, if the range is 1.1. to 2.1 the following numbers 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, and 2.0 can be selected as lower or upper limits.

[0022] Also, unless expressly stated to the contrary: percent, “parts of,” and ratio values are by weight; the term “polymer” includes “oligomer,” “copolymer,” “terpolymer,” and the like; molecular weights provided for any polymers refers to weight average molecular weight unless otherwise indicated; the description of a group or class of materials as suitable or preferred for a given purpose in connection with the invention implies that mixtures of any two or more of the members of the group or class are equally suitable or preferred; description of constituents in chemical terms refers to the constituents at the time of addition to any combination specified in the description, and does not necessarily preclude chemical interactions among the constituents of a mixture once mixed; the first definition of an acronym or other abbreviation applies to all subsequent uses herein of the same abbreviation and applies mutatis mutandis to normal grammatical variations of the initially defined abbreviation; and, unless expressly stated to the contrary, measurement of a property is determined by the same technique as previously or later referenced for the same property.[00231 It is also to be understood that this invention is not limited to the specific embodiments and methods described below, as specific components and / or conditions may, of course, vary. Furthermore, the terminology used herein is used only for the purpose of describing particular embodiments of the present invention and is not intended to be limiting in any way.

[0024] Also, unless expressly stated to the contrary: all R groups (e.g. Ri where i is an integer) include hydrogen, alkyl, lower alkyl, Cl -6 alkyl, C6-10 aryl, C6-10 heteroaryl, -NCh, -NH2, -N(R’R”), -N(R’R”R”’)+L , Cl, F, Br, -CF3, -CCI3, -CN, -SO3H, -PO3H2, -COOH, -CO2R’, -COR’, -CHO, -OH, -OR’, -O-M+, -SO3M+, -P03M+, -C00 M+, -CF2H, -CF2R’, -CFH2, and -CFR’R” where R’, R” and R’” are Cl-10 alkyl or C6-18 aryl groups M is a metal atom (e.g., Na, K, Li,etc.) and L’ is a counter anion (e.g., Cl’, Br’, tosylate, etc.); single letters (e.g., "n" or "o") are 1, 2, 3, 4, or 5; in the compounds disclosed herein including compounds described by formula or by name, a CH bond can be substituted with alkyl, lower alkyl, Cl -6 alkyl, C6-10 aryl, C6-10 heteroaryl, -NO2, -NH2, -N(R’R”), -N(R’R”R’”)+L’, Cl, F, Br, -CF3, -CCh, -CN, -SO3H, -PO3H2, -COOH, -CO2R’, -COR’, -CHO, -OH, -OR’, -O’M+, -SO3’M+, -PO3’M+, -COO’M+, -CF2H, -CF2R’, -CFH2, and -CFR’R” where R’, R” and R’” are Cl-10 alkyl or C6-18 aryl groups M is a metal atom (e.g., Na, K, Li, etc.) and L’ is a negative counterion; and percent, "parts of," and ratio values are by weight.

[0025] The term “alkyl” refers to Cl-20 inclusive, linear (i.e., “straight-chain”), branched, saturated or at least partially and in some cases fully unsaturated (i.e., alkenyl and alkynyl) hydrocarbon chains, including for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertbutyl, pentyl, hexyl, octyl, ethenyl, propenyl, butenyl, pentenyl, hexenyl, octenyl, butadienyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, and allenyl groups. “Branched” refers to an alkyl group in which a lower alkyl group, such as methyl, ethyl or propyl, is attached to a linear alkyl chain. “Lower alkyl” refers to an alkyl group having 1 to about 8 carbon atoms (i.e., a Cl-8 alkyl), e.g., 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms. “Higher alkyl” refers to an alkyl group having about 10 to about 20 carbon atoms, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms.

[0026] The term “substantially,” “generally,” or “about” may be used herein to describe disclosed or claimed embodiments. The term “substantially” may modify a value or relative characteristic disclosed or claimed in the present disclosure. In such instances, “substantially” may signify that the value or relative characteristic it modifies is within ± 0%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5% or 10% of the value or relative characteristic.

[0027] It should also be appreciated that integer ranges explicitly include all intervening integers. For example, the integer range 1-10 explicitly includes 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Similarly, the range 1 to 100 includes 1, 2, 3, 4. . . . 97, 98, 99, 100. Similarly, when any range is called for, intervening numbers that are increments of the difference between the upper limit and the lower limit divided by 10 can be taken as alternative upper or lower limits. For example, if the range is 1.1. to 2.1 the following numbers 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, and 2.0 can be selected as lower or upper limits.

[0028] When referring to a numeral quantity, in a refinement, the term “less than” includes a lower non-included limit that is 5 percent of the number indicated after “less than.” For example, “less than 20” includes a lower non-included limit of 1 in a refinement. Therefore, this refinement of “less than 20” includes a range between 1 and 20. In another refinement, the term “less than” includes a lower non-included limit that is, in increasing order of preference, 20 percent, 10 percent, 5 percent, or 1 percent of the number indicated after “less than.”

[0029] “ One or more” includes a function being performed by one element, a function being performed by more than one element, e.g., in a distributed fashion, several functions being performed by one element, several functions being performed by several elements, or any combination of the above.[0030J In the examples set forth herein, concentrations, temperature, and reaction conditions (e.g., pressure, pH, flow rates, etc.) can be practiced with plus or minus 50 percent of the values indicated rounded to or truncated to two significant figures of the value provided in the examples. In a refinement, concentrations, temperature, and reaction conditions (e.g., pressure, pH, flow rates, etc.) can be practiced with plus or minus 30 percent of the values indicated rounded to or truncated to two significant figures of the value provided in the examples. In another refinement, concentrations, temperature, and reaction conditions (e.g., pressure, pH, flow rates, etc.) can be practiced with plus or minus 10 percent of the values indicated rounded to or truncated to two significant figures of the value provided in the examples.

[0031] For all compounds expressed as an empirical chemical formula with a plurality of letters and numeric subscripts (e g., CH2O), values of the subscripts can be plus or minus 50 percent of the values indicated rounded to or truncated to two significant figures. For example, if CH2O is indicated, a compound of formula C(o.8-i.2)H(i.6-2.4)0(o.s-i.2). In a refinement, values of the subscripts can be plus or minus 30 percent of the values indicated rounded to or truncated to two significant figures. In still another refinement, values of the subscripts can be plus or minus 20 percent of the values indicated rounded to or truncated to two significant figures.

[0032] Interfacing molecular components (e.g., catalytic components) within microsystem platforms, such as, but not limited to, microelectronic systems, benefits from several critical factorsgoverned by the chosen functionalization chemistry of the surfaces involved. Of note, microelectronic systems benefit from robust covalent coupling, high spatial precision, multiple options for conjugation chemistries, and linking structures that enhance transduction between the microelectronic system and function of the components supported on the surfaces. By optimizing the immobilization and surface loading of the components (e.g., catalysts), the molecular components can be controlled through various physical mechanisms, including photonic, electronic, and thermal control. These mechanisms provide distinct realms of control for the microelectronic device. If a specific enzyme is targeted for photonic control and loaded on the appropriate surface, electronic control methods will not influence that specific enzyme. The importance of the non-influence of enzymes across the various control mechanisms is due to the small distance between enzymes in the microsystem. The short distances (e.g., on the nanometer scale, in some embodiments under 10 nm) are important for effectiveness in schemes such as catalysis, however the control mechanisms may overlap multiple enzymes. Thus, according to various embodiments and examples provided herein, the microsystem exerts control over distinct molecular components using an immobilized design supported on the surface, rather than precisely targeting the control physics themselves to nanometer-scale accuracy.

[0033] According to at least one embodiment, a microsystem is provided with components, such as, and interchangeably hereinafter enzymes, supported on at least one surface of the microsystem, where the components may be controlled by various mechanisms, including, but not limited to physics, resulting in tunable photonic, electronic, and thermal control of the components that are immobilized on the chip surface.

[0034] According to one or more embodiments, an example of a microsystem is provided in FIG.1. The microsystem includes a substrate with a surface, having an organic monolayer functionalized thereon. The organic monolayer is functionalized with immobilized molecular components on the surface. The immobilized components may be one or more of catalysts, enzymes, proteins, and the like, as suitable for microsystems, that are controllable through mechanisms such as, but not limited to, photonic, electronic, and thermal control. Thus, in the example shown in FIG. 1, an example microsystem is shown for catalysis, however any other microsystem is contemplated and reference to catalysts and catalysis hereinafter is not intended tobe limiting and is interchangeable for other molecular components with other reaction schemes. In the example of FIG. 1, substrates (e.g., ethanol or other suitable solvent) flow over the surface of the microsystem platform and to the immobilized components supported on the surfaces via a microfluidic channel (A). Independent components are then activated by the following physicsbased control mechanisms as depicted in FIG. 1: (1) the photonic integrated circuit (PIC) routes light (B) to immobilized components, implementing photo-switchable dimerization or caging, thus activating / deactivating specific components; (2) calibrated voltages, applied to electrodes on the PIC via control electronics, implement charge injection for redox cycling or electrostatic guidance (C); (3) integrated heaters (D) implement thermal control through localized or global temperature. The final product exits via the microfluidic channel (E).[0035| In at least one embodiment, an organic monolayer may be functionalized on the microsystem surface by immobilizing molecular components (e.g., catalysts such as enzymes, or proteins) thereon and to the microsystem surface, allowing for the substrate(s) to flow thereover. Thus, according to at least one embodiment, the functionalization strategy may be surface directed growth of organic monolayers on silicon hydride (Si-H) surfaces in order to support the molecular component, such as the enzyme, protein, or catalyst, thereon. In this process, after oxide stripping on the surface of a silicon substrate, molecules are attached in successive fashion to hydride terminated atoms of a silicon crystal surface of the silicon substrate, typically Si(l 11), Si(100) or Si(l 10). This approach is conducive to microelectronic incorporation of organic molecules as it employs standard silicon substrates as assembly templates.[0036| As such, in one or more embodiments, one or more surfaces of the microsystem may be functionalized by surface-directed growth of organic monolayers on hydride terminated silicon (Si-H) and / or silicon nitride surfaces. While hydride terminated silicon and silicon nitride surfaces are described herein, these are examples of representative backbones based on the specific component or enzyme cascade for the example application described herein, and is not intended to be limiting. In that regard, other support functionalization for organic monolayers are contemplated for the surface as based on the selected component and mechanism desired, such as the selected enzyme and / or enzyme cascades (or, similarly selected protein) and the desired output of the microsystem. According to at least one embodiment, surface functionalization may beachieved by hydrosilylation of hydride terminated surfaces. In this process, unsaturated groups on organic molecules (e.g., alkynes, alkenes) react with surface bound Si-H groups to form a direct covalent link to the underlying substrate. This chemistry is especially conducive to microelectronic incorporation of organic molecules as it directly modifies common device substrates such as silicon and silicon nitride. An example of the functionalization of the microsystem surface is schematically shown in FIG. 2, depicting Si-H surface formation and attachment of alkene functional organic molecules to form the organic monolayer.

[0037] According to one or more embodiments, grafting to Si-H surfaces can occur through multiple chemistries including, but not limited to, heat, light and chemical activation, producing monolayers with notable advantages over other chemistries without such flexible activation. Namely, the process of grafting is self-limiting, produces thermally stable bond strengths comparable to condensed silanes and substantially higher than thiol-Au assemblies, and yields monolayers resistant to hydrolysis even at extreme pH ranges (as a non-limiting example, below 3 and above 12) beyond that survivable by DNA constructs. For example, stability data for SiN-alkene monolayers is provided in FIG. 3, depicting hydrolytic and thermal stability for a 1-octadecene monolayer coupled to silicon nitride as monitored by XPS. The monolayer example of FIG. 3 was stable outside of basic conditions and maintained integrity up to -228 degrees C. Molecules grafted to Si-H surfaces may be arranged with atomic precision as dictated by the underlying arrangement of crystalline silicon atoms, periodically spacing molecules by, in some examples, 1-2 nm. Accordingly, this form of surface grafting enables high precision in molecular arrangements especially with respect to other molecules on the surface.

[0038] Molecules grafted according to the embodiments and examples provided herein have very high surface densities (e.g., of at least greater than 4 molecules / nm2) enabling high proximity for immobilized molecular arrangements, for example, which can support enzymatic cascades for catalysis. Finally, Si-H coupling enables ideal device dynamics due to the lack of interfering interfaces typically associated with silane condensation on oxide surfaces. Both high electron transport for redox processes and efficient molecular photonic coupling are supported by silicon organic monolayer systems. As such, according to at least one embodiment, prior to thelithographic patterning, radically induced hydrosilylation is provided and may improve the integration ability of Si(100)-H surfaces.[0039J Furthermore, with reference to the method of FIG. 4 and the proposed hydrosilylation of the surface, Si-H functionalization can also be site specific and dictated by the microelectronic system through electrografting of diazonium molecules, providing further control over molecular localization. Thus, not only can hydride-coupled monolayers achieve high immobilization accuracy, but they are also extremely robust, as they can survive extreme pH (1 to 13), do not hydrolyze, and are a versatile platform for conjugating biomolecules such as enzymes. The method of FIG. 4 supports various outcomes, including, but not limited to, consistent molecular placement with the intended specifications, dense packing, specific-orientation and atomically arranged results, and well-defined molecular domains in a scalable fashion.[0040| Further advantages of Si-H coupling are realized in the effectiveness of bio-conjugation demonstrated for SiNW-FETs, Bio-FETs, and amperometric biosensors. Multiple conjugation pathways have been utilized including carbodiimide, Biotin / Strepativin, Michael addition, and click chemistries through linkers like 1,8-nonadiyne. A high degree of catalytic incorporation may be employed in microdevices as previously demonstrated for proteins and peptides based on the attachment methodology described herein. Finally, Si-H coupling enables ideal device dynamics of molecules directly bonded at the semi-conductor interface. Fine control over redox interactions can be realized from high electron transport across an oxide free interface affording lOx increases in sensitivity and dynamic range. Additionally, organic monolayers on Si-H have remarkably low recombination velocities in the range of 102versus 106cm / s for thin native oxides. Consequently, sophisticated photo-electrodynamics can be employed where focused illumination on an electrode can stimulate charge transfer by a localized increase in charge carrier density allowing electron transfer through the interface into the silicon substrate. This provides a unique mode of coupling photo and electrical control over molecular function that can only be realized though catalytic integration on the Si-H surface.[00411 As such, according to at least one embodiment and with reference to FIG. 4, hydrosilylation on silicon nitride features is provided to integrate molecules (e.g., enzymatic molecules) into microsystem platforms. As shown in FIG. 4, the surface of the microsystem device will be strippedof native oxide, and reacted with linker molecules to form a functional monolayer. In this process, after oxide stripping, molecules are attached in successive fashion to hydride terminated atoms of the substrate surface as shown in the example of FIG. 2.

[0042] Electron-beam (e-beam) lithography, or other suitable lithography, may then be utilized to form a pattern that will define attachment sites within the monolayer. Enzymes are then conjugated for attachment at the attachment sites. Generally, the process may be monitored by x-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM).

[0043] For the initial monolayer step, as shown in FIG. 4, functionalizing the SiNx device features with multiple linkers is also contemplated. Notably, utilizing multiple linkers provides options to identify an optimal linker structure. Moreover, certain linkers may be more suitable for certain enzymes. For example, the linkers may include, but are not limited to, commercially available 1,4-diethynylbenzene (DEB) and / or 1,8-nonadiyne. These examples of linkers are molecules that are fairly short in length, and may support good optical coupling from the SiNx waveguide of the microsystem device. DEB is a fully conjugated aromatic molecule while 1,8-nonadiyne provides an aliphatic alternative allowing tuning at redox electrodes. Both of these example linkers provide alkyne functionality for efficient subsequent click coupling of azide functional enzymes, however other functionality for click coupling is also contemplated as dependent on the functionality of the enzymes selected. Thus discussion of alkyne functionality for click coupling of azide functional enzymes is not intended to be limiting.

[0044] With reference to FIG. 5, monolayer formation by hydrosilylation of the SiNx-H surface is shown according to certain examples. As shown on the left side of FIG. 5, aliphatic alkyne 1,8-nonadiyne is coupled at 170°C, and on the right side of FIG. 5, aromatic alkynel,4-diethynylbenzene is coupled at 25°C. Thus, in the example shown in FIG. 5, DEB provides the ability to form dense monolayers under mild conditions (e.g., under 50°C), as compared with 1,8-nonadiyne. As shown on the right side of FIG. 5, SiNx-H surfaces may be reacted with acetonitrile solutions of DEB at 25°C in the dark which has been found to produce improved and well oriented functional monolayers. In the example on the left side of FIG. 5, 1,8-nonadiyne requires employing heating at 170°C to drive the reaction. The formation of the monolayers may be characterized by XPS and AFM to verify linker presence and determine surface density.

[0045] Referring again to FIG. 4, the patterning of the grafted organic monolayer may be conducted via e-beam lithography in order to define attachment sites. While e-beam lithography is discussed herein, this is not intended to be limiting, and other types of lithography for patterning is also contemplated. For example, nanoimprint lithography may also be used for accurate patterning on the monolayer. Generally, the lithography used may at least achieve < 50 nm placement accuracy. In this process, after oxide stripping, molecules are attached in successive fashion to hydride terminated atoms of the substrate surface, as shown in the example of FIG. 2.

[0046] With reference to FIG. 6, after e-beam patterning, the defined monolayer attachment sites are conjugated with cascade molecules, such as cascade enzymes, which is referred to hereinafter interchangeably. In at least one example, and as shown in FIG. 6, enyzme conjugation may be by click coupling to alkyne functional monolayer surfaces. In the example of FIG. 6, direct coupling of azide functional enzyme is shown on the left, and conversion to metal free click coupling is shown on the right. Alkyne functional monolayers are generally paired with azide derivatized molecules which demonstrates improved coupling. However, while alkyne functional monolayers are shown as paired with azide derivatized molecules, other molecules are also contemplated as based on the selected monolayer and / or functionality of the selected, and discussion of alkyne coupled with azide derivatized molecules is not intended to be limiting.

[0047] In the example shown in FIG. 6, enzymes are coupled to defined attachment sites through copper-catalyzed azide-alkyne cycloaddition (CuAAC). This form of conjugating proteins may occasionally incur difficulties from enzymes predisposed to ligate Cu(I) cations. As a mitigation strategy, if necessary, the surface alkynes may be converted to metal free click functional groups such as tetrazines through a hetero-bifunctional intermediate as shown in FIG. 6 on the right side in order to improve the linker structure and coupling chemistry of the attachment sites.

[0048] As shown in FIG. 7, a method of preparing a microdevice is provided. The method includes providing a silicon surface having hydride terminated silicon (Si-H) and / or silicon nitride groups. In at least one embodiment, providing the silicon surface having hydride termination silicon and / or silicon nitride groups includes radically induced hydrosilylation of the surface. In at least one embodiment, the method further includes stripping native oxide from the silicon surface. The method further includes functionalizing the silicon surface by forming an organic monolayerthereon. The functional monolayer surface may be formed by providing linkers to the surface. In at least one embodiment, the functional monolayer surface may be an alkyne functional monolayer. The method further includes patterning the monolayer to form attachment sites. The method further includes coupling an enzyme to the attachment sites. In an embodiment, the enzyme may be an azide-derivatized molecule for coupling with the alkyne functional monolayer. In at least one example, the enzyme may be an enzyme cascade. In at least one example, the patterning may be via a suitable lithography, such as e-beam lithography or nanoimprinting lithography.

[0049] In at least one embodiment, monolayers and conjugated enzymes at prescribed attachment sites may be characterized by AFM. In at least one example, as shown in FIG. 8, AFM may be utilized to visualize small molecule and protein surface coupling at specific nanoscale sites in devices. FIG. 8 depicts an individual nanowell device feature with AFM scans depicting the surface functionality from planar substrate to DNA oligo monolayer to surface anchored protein. Specifically, the example of FIG. 8 shows the AFM characterization of an individual 200 nm platinum nanowell on SiOx device feature. The full nanowell relief is shown on the left while height magnified scans of the well floor are shown on right, depicting improvement in surface functionalization from oxide surface, to assembled DNA oligo monolayer to subsequent DNA complexed protein. Thus, AFM may be employed to assess attachment site metrics for spatial precision and yield on microsystems prepared by the steps provided herein.

[0050] In at least one embodiment, a microsystem platform is provided which demonstrates all three control mechanisms working in concert to control a six-enzyme reaction cascade, which yields polyhydroxybutyrate. Polyhydroxybutyrate (PHB) is a biodegradable bioplastic produced by microorganisms that can be used in a broad range of applications, such as packaging materials, agricultural fdms, and medical devices. PHB is also a high-impact product for bioplastic and biofuel production. A synthetic catalytic pathway that utilizes ethanol as the substrate may be used to synthesize PHB with high productivity and specificity. This cascade pathway may involve any number of enzymes, and undergo a reaction pathway as shown in the example cascade pathway of FIG. 9. For example, six enzymes and five biocatalysts may impact the cascade reaction (alcohol dehydrogenase, acetaldehyde de-hydrogenase, acetyl-CoA acetyltransferase, acetoacetyl-CoAreductase, polyhydroxyalkanoate synthase). A sixth oxidase biocatalyst recycles the cofactor NADH. Accordingly, a functionalized polymer can be obtained.[0051J While exemplary embodiments are described above, it is not intended that these embodiments describe all possible forms of the invention. Rather, the words used in the specification are words of description rather than limitation, and it is understood that various changes may be made without departing from the spirit and scope of the invention. Additionally, the features of various implementing embodiments may be combined to form further embodiments of the invention.

Claims

WHAT IS CLAIMED IS:

1. A method comprising:providing a silicon surface having hydride terminated silicon and / or silicon nitride groups thereon;functionalizing the silicon surface to form an organic monolayer on the silicon surface;patterning the organic monolayer to form attachment sites; andcoupling an enzyme molecule to a respective attachment site.

2. The method of claim 1, wherein the providing includes radically induced hydrosilylation of the silicon surface to form the hydride terminated silicon groups thereon.

3. The method of claim 1, wherein the functionalizing includes introducing a linker to form a functionalized organic monolayer.

4. The method of claim 3, wherein the functionalized organic monolayer is an alkyne functional monolayer.

5. The method of claim 4, wherein the enzyme molecule is an azide derived molecule.

6. The method of claim 4, further comprising converting surface alkynes in the alkyne functional monolayer to metal free click coupling sites.

7. The method of claim 1, wherein the patterning is via electron beam lithography.

8. The method of claim 1, wherein the coupling includes forming an enzyme cascade on the silicon surface.

9. The method of claim 1, further comprising stripping native oxides from the silicon surface.

10. A microdevice comprising:a silicon surface with hydride terminated silicon and silicon nitride groups thereon; anda functionalized organic monolayer linked to the hydride terminated silicon and silicon nitride groups,wherein the functionalized organic monolayer has a pattern of attachment sites.

11. The microdevice of claim 10, wherein the functionalized organic monolayer is an alkyne functional monolayer.

12. The microdevice of claim 10, further comprising an enzyme coupled with an attachment site.

13. The microdevice of claim 12, wherein the enzyme is an azide derived molecule.

14. The microdevice of claim 10, wherein at least a portion of the functionalized organic monolayer includes converted surface alkynes as metal free click coupling sites.

15. The microdevice of claim 10, wherein an enzyme cascade is coupled with the attachment sites per the pattern.

16. A method compri si ng :stripping native oxide from a silicon surface of a substrate;functionalizing the silicon surface via radically induced hydrosilylation to form an organic monolayer on the silicon surface with hydride terminated silicon and / or silicon nitride groups thereon;patterning the organic monolayer to form attachment sites; andcoupling at least one enzyme molecule to a respective attachment site.

17. The method of claim 16, wherein the organic monolayer is an alkyne functional monolayer.

18. The method of claim 17, further comprising converting surface alkynes in the alkyne functional monolayer to metal free click coupling sites.

19. The method of claim 16, wherein the functionalizing is via introduction of a linker.

20. The method of claim 19, wherein the linker is aliphatic or aromatic.

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