KR20110052692A - Creation of high density multidimensional addressable assemblies - Google Patents

Abstract

The present invention is directed to a method and apparatus for a molecular scale assembly of a structure comprising a high density production, a geometrically patterned two- and three-dimensional addressable assembly of a polymer containing nucleobase, and a configuration that is compatible with it. It is about. The present invention provides a method of making a production item comprising covalently attached biopolymers formed from a complementary master shape formed into a defined shape and exhibiting complementary single stranded polynucleobases. The present invention provides a method of manufacturing a production item comprising the step of producing a uniformly concave shape formation in a photosensitive solid substrate matrix and performing metal vapor deposition to attach a self-assembled biopolymer pattern.

Description

CREATION OF HIGH DENSITY MULTIDIMENSIONAL ADDRESSABLE ASSEMBLIES}

The present invention relates to a method and apparatus for a molecular scale assembly of a structure comprising a composition of high density production, geometrically patterned two- and three-dimensional addressable assemblies of polymer-containing nucleobases and shapes that are compatible with them. will be.

This application relates to the following applications, each of which is incorporated herein by reference: US Provisional Application No. 60 / 918,144, filed March 15, 2007; US Provisional Application No. 60 / 969,154, filed August 30, 2007; US Provisional Application No. 60 / 985,961, filed November 6, 2007; US Application 11 / 936,045, filed November 6, 2007; United States provisional application 61 / 032,118, filed February 28, 2008; PCT Application PCT / US08 / 57013, filed March 14, 2008; United States provisional application 61 / 043,981, filed April 10, 2008; US Provisional Application 61 / 047,201, filed April 23, 2008; US Provisional Application 61 / 048,599, filed April 29, 2008; United States provisional application 61 / 061,555, filed June 13, 2008; United States provisional application 61 / 086,633, filed August 6, 2008.

Generation of catalysts based on single stranded dimensions (sometimes referred to herein as "SSDC", a trademark of Incitor LLC of Albuquerque, Mexico, United States) is found in polynucleobases that are assembled in a nanoscale geometrically accurate, reproducible and useful manner. May be dependent on the construction of single-stranded DNA (ssDNA), peptide nucleic acid (PNA), ribonucleic acid (RNA), or other address (any one of the foregoing so far). Such addresses include (i) transbiotic catalysts such as enzymes, (ii) 2D and 3D configurations of useful nanoscale components and devices, (iii) supramolecular assemblies, (iv) other assemblies that rely on hybridization-based localization and assembly, And (v) complementary polynucleobase probes via Watson-Crick hybridization to place physicochemical groups in three-dimensional (3D) space that facilitate the discovery and development of binding of any and all such devices. Localize.

As described herein and in the applications included herein, SSDC is conceived in this manner and will produce a high density 3D addressable array of polynucleobases reproducibly accurate at large scale and at nanometers when applied and performed. You can rely on your skills. Such related techniques include (1) photoelectron beam lithography, (2) soft lithography in which 3D patterns are replicated from liquids or colloids to solids via phase changes, and (3) polynucleobase probes with physicochemical groups attached through linkers. , (4) complementary and "mirror image" polynucleobase pattern duplication, and for this document (5) self and // of polynucleobases initially and ultimately defining the parameters of 3D high density addresses at each stage of production. Or a directed assembly.

As described herein and in the applications included herein, SSDC can begin with the construction of a mold, the components of which are lithographically matched to the 3D foundation of the solid material of positive and negative structures and the contours of the foundation. Polynucleobases. This configuration serves as the basis for the fabrication of many of the fabrication platforms that can be generated by mirror image pattern replication of the foundation shape of the mold and polynucleobase sequences using materials that facilitate scaled amounts of replication. The method of template fabrication may or may not be based on polynucleobase pattern replication from the initial stage master since the purpose of template creation is backbone based lithography of sequence addresses complementary to previous master and subsequent platforms.

1 is a 2D diagram of polynucleobase sequence and geometry based pattern replication. The master foundation (pastel) is contoured with a depression coated with a material (cardinal) that supports lithography and / or assembly of polynucleobase strands. The left permanent template (yellow-on-blue; red mounted polynucleobase) consists of hybridization of the complementary base and replication of the lithographic master pattern. The right platform is made in a manner similar to that of the template from the master in that the polynucleobase sequence & pattern geometry and the 3D contour and shape are reproduced in a "mirror image" format. The molds are hybridized with the required addresses on the platform, the latter with impression molding, thin films (purple, ash, green, overlay of hatched layers, as shown), and other techniques. Is produced through.

SSDC based platform catalysts may rely on precise localization within the 3D space of physicochemical groups that facilitate catalysts in enclosures which are typically fabrication platforms. Such groups are typically at the ends of linkers attached to the backbone of polynucleobase probes that hybridize to complementary polynucleobase addresses in the platform. The geometric pattern of the address (as a function of successful regeneration from the template based on the described fabrication method) determines the 3D orientation and spatial position of the catalyst group. As such, through (i) successful production of scaled catalyst products, (ii) conditions of its activity, (iii) reliable production and replication, and (iv) accurate modeling and computer programming through element and vector analysis Other means undertaken are all highly dependent on the initial stage generation of the pattern of polynucleobases, preferably repeating the geometric pattern as an important aspect, the assembly of which is lithography onto the original master or manufacturing template as described.

1 is a 2D diagram of polynucleobase sequence and geometry based pattern replication.
2 is a schematic diagram of an assembly of a square hatch pattern and a dash address.
3 is a schematic diagram of an assembly of hexagonal patterns and indented addresses.
4 is a schematic diagram of the final template of dashed addresses from a square hatch pattern.
5 is a schematic diagram of a design of a square hatch pattern and a dash address.
6 illustrates a level 1 photomask.
7 illustrates a level 2 photomask.
8 illustrates a coated substrate after exposure with a level 1 photomask.
10 illustrates the same substrate after removal of residual photosensitive material.
11 illustrates the same substrate after treatment with a level 2 photomask.
12 illustrates the same substrate after application of another coating, for example deposition of a coating comprising 5 nm Ti and 15 nm Au.
13 illustrates the same substrate after removal of the photosensitive material.
14 illustrates a plan view of the substrate after the treatment.
Fig. 15 illustrates a 3d view of the substrate after the above treatment.
16 is an example of SSDC scheme for two-dimensional heterogeneous catalyst.
17 shows a geometric approximation of the catalytic site of a single catalytic enzyme.
18 shows the conversion of the geometric approximation to a universal gap for the catalyst.
19 shows the design of a stamp for soft lithography of a universal gap foundation.
20 shows the preparation and preparation of a stamp (mold array) for soft lithography.
21 illustrates platform fabrication via soft lithography.
22 shows DNA weaving based self assembly of 2D addressable arrays.
23 shows a DNA geometry based directed assembly of a 3D addressable array.
FIG. 24 shows stacking conformation of catalytic function from hybridized polynucleobase libraries. FIG.
25 shows an exemplary method of mounting polynucleobases on a base material.
26 illustrates boundary layer flow as a function of assembly fabrication schemes.
27 illustrates an apparatus designed to promote boundary layer flow at the assembly level.

Typically a library of medium length ssDNA oligonucleotides (100-1000 bases; approximately 50-500 nm long in full extension) can be designed, which under certain conditions addressable positions, i.e., unhybridized poly, as a result of self-assembly Spontaneously forms stable assemblies that repeat the geometric pattern of nucleobase sites. Using the dependent techniques described below, nanometer precise 2D and 3D patterns of polynucleobases, so far referred to as "addressable assemblies", can be fabricated with a high degree of uniformity, verifiability and utility for master and mold lithography. have:

1. Watson-Crick hybridization of complementary polynucleobase segments;

2. Bulk solution conditions to facilitate hybridization optimized and managed at each stage of the assembly;

3. standards in art Receptor-Ligand based biochemical technology;

4. Physical conditions and physical components to further facilitate self-assembly and localization;

5. Interfacial, electrostatic and solvation conditions to facilitate lithography of the assembly.

The present invention, as described, includes: (i) nuclear or dippen nanolithography (DPN), (ii) receiving mensicus or other retardation based extension, elongation or stretching of polynucleobases, (iii) iii The use of electrical, magnetic, optical, inertial and other fields and energy for the accomplishment of, such fields being fixed on polynucleobase strands or solid materials—typically at the ends of the strands and acting on beads having optical or magnetic sensitivity a variety of current techniques as appropriately described, such as (iv) extension of the polynucleobase strand in or on a lithopic pattern that facilitates the desired conformation, or (v) any combination or variation with respect to the non-dependent technique. Is compatible with

2 is a schematic diagram of an assembly of a square hatch pattern and a dash address. On the left is shown a diametrically opposed polynucleobase (grey and blue; color refers to orientation rather than sequence) with constant complementarity to adjacent strands. The region of close overlap indicates the site of hybridization. On the right is shown the location of oligobases complementary to the site without hybridization (green for gray polynucleobases; red for blue). All complementary oligobases are in the same orientation.

3 is a schematic diagram of an assembly of a hexagonal pattern and an indented address. On the left is shown a diametrically opposed polynucleobase with differently adjacent strand complementarity to that of FIG. 2. As before, the close overlap represents the site of hybridization. On the right, the position of the oligobase complementary to the site without hybridization (green for gray; red for blue) is shown. All complementary oligobases in the proximal address block (vertical direction) are in the same orientation.

Typically, within a large sequence pool, hybridize to only one address, for example for polymerase chain reaction (PCR) or other primer based techniques such as site directed mutations, site specific targeting, microarrays, etc. The design of the probes required requires a sequence of approximately 21-27 bases in oligobase length. If a 25-mer address is appropriate then the hybridization that occurs in the representative assembly is approximately 12.5 nm in length and is longer than the unhybridized site of 25 base pairs separated by similar or smaller distances based on the sequence design and the intended address pattern. Geometrically precise groupings are shown.

These final addressable assemblies (more 2D patterns and 3D shapes can be created) provide new and potentially innovative improvements in the current art that are appropriate for such technology. Some example applications of the present invention are described below.

Application A. With respect to the microprocessor industry , a platform lithographically lithographically assembled with such assemblies includes 1 / 16th of the differentiated size units of the minimum feature size according to state-of-the-art scaled manufacturing in photolithography. That is, 50 nm on the side features fabricated by deep ultraviolet or immersion-lithography are less than 100 molecules that can be functionalized with components such as carbon nanotubes and the linear backbone of 25-mer (12.5 nm) oligobases. It is 16 times the size of charge transfer or optical element. Briefly, the ability to create 3D addressable assemblies of polynucleobases potentially provides the ability to generate 10 times as many microprocessors as currently available components.

Application B. With respect to the supramolecular assembly and the bottom -to-lob scaled manufacturing industry, the platform is now close to the length scale (in short: horizontal in FIG. 2; vertical in FIG. 3) where the chemical reaction increases the conversion rate. The ability to arrange reactive chemistry by the precision and agility provided by an addressable assembly eliminates stereo non-specific aspects of performing the reaction in bulk or between phases lacking such precision. . For functionalized groups and applicable platform foundations for probes, the proximity of such addresses eliminates the need for much orthogonal protection that is also required for liquid bulk scale synthesis and coupling based assemblies.

Application C. With regard to the interfacial and templated synthesis industry, no "high density addressable arrays" are presently known that present the precision and regularity of the techniques described herein. The absence of such an assembly order in the currently applied molded synthesis, and the absence of its application under stable interfacial conditions, are not only in the form of products that can be produced by the requirements of synthesis in the liquid phase, but also for further dehybridization and reroving. Due to the inability of current techniques to sequester polynucleobases from reactors in a manner that preserves their reusability, they severely limit the range of reactions that can be carried out.

Application D. With regard to industries requiring catalysts, the potential advantages of ultra-high density and geometrically precise addressable assemblies are covered below and in the following documents.

Representative implementation of the invention. The concept, design, fabrication and lithography of addressable assemblies are described herein. Dependent technologies previously mentioned: hard (beam) and soft (polymer) lithography, hybridization solutions, receptor ligands biochemical, physical (electromagnetic, inertial and liquid-gas interfaces) and tribological (IES: solid-) Liquid interfacial, electrostatic and solvent) management is included as an example. Other dependence techniques, such as computer-based programs and algorithms to assist and optimize the design of addressable assemblies, and the needs of nuclear, scanning or transmission electron microscopes (AFM, SEM and TEM, respectively) for quality determination are inherent in this specification. .

Concept- the creation of a permanent template . In this embodiment implementation, it is desirable to create a template suitable for scaled fabrication and 3D shape replication of the platform via polynucleobase sequences and patterns. The template is an array of multiple (eg 1 billion) identical convex extensions each having a generalized shape of a cylinder cut along a longitudinal axis with dimensions approximately 150 nm long, 30 nm wide and 15 nm large. Each unit shape (1) maximizes the useful surface area of the mold unit, i.e., the address area along most of the surface area of the half cylinder, and (2) on the mold unit through surface functionalization, polynucleobase backbone modification and solventization management. "Webbing" of the addressable assembly in such a way as to preserve the ability to lithography the assembly and (3) to initiate complementary pattern replication, i. May be preferred.

The material may be Si (100) and vapor deposited with 10 nm titanium and 5 nm gold, representing most of the height profile, to ensure that the mold is sufficient for elasticity to withstand successful stamping-based platforms that are manufactured to scale. The mold array must be lithographically lithographically 150x30x15nm on every axis to 50nm to maximize productivity. The 100 cm ² template array should then exhibit approximately micromoles with respect to manufacturing capacity per batch of platform stamping.

4 is a schematic diagram of the final template of dash addresses from a square hatch pattern. The contour map of the convex template unit is made of hard cast silicone and coated with [Ti] support [Au] to accommodate (P) thioate skeletoned 25-mer oligo DNA (red and green). ssDNA is located around the template unit via hybridization by the master assembly.

Using an addressable assembly fabricated with a square hatch pattern, as in FIG. 2, an individual 12.5 nm long 25-mer address spaced approximately 12.5 nm apart is 4 per 50 nm = 12 address for a 150 nm long extension or enclosure on a single axis. Indicates an address. As 30 nm in diameter, the semicircle can accommodate approximately four addresses lithographically around it. Thus, the total number of addresses on the simple mold unit is approximately 50 sites following hybridization.

Regarding platform fabrication, polynucleobases complementary to the phosphorothioate backbone ssDNA lithography on the template are transcribed into soft cast polymers to produce impressions, short castings to form negative shapes, heating to separate platform addresses from template DNA, templates Imprinted by removal of the array and hard casting of the platform.

Design - the mold Addressable Assembly. The stable square hatch patterned assembly is designed such that the anchor point is matched to the half cylinder while localizing and orienting the assembly for electrostatic based positioning and final covalent bond mediated lithography by specific solvent conditions. The lithographic product is outlined below.

5 is a schematic diagram of a design of a square hatch pattern and a dash address. At the top is shown a representative simplified assembly (trunk) located on a mold unit-a convex half cylinder. The prefabricated assembly, which is a combination of hybridized and unhybridized regions, is designed to match the fabricated shape of the mold unit in 3D. The edges of the assembly (not shown) are sealed with oligomers to prevent damage to shape integrity and add elasticity to the structure. The 5 'end of one central strand (dark blue) and the 3' end of its complex (light blue) are biotinylated to attach 10 nm streptavidin beads (mustard). At the bottom is a top view of the final platform with the address area lithographic (red and green). The previous location of the lithographic or deposited biotin anchor point is positioned for reference to the contour of the platform unit. The location of each 25-mer addressable site is complementary to the unhybridized site on the assembly around the template.

Generation of proper shape. 6-15 illustrate one method of generating shapes on a nanometer & micron scale that can be activated with ssDNA lines or dsDNA weaves to create various molecular structures. The process shown represents gold as a bonding substrate, but other substrates such as polymers or other materials are possible. This example is not limited by structural shape or substrate, but is used only as an example. Those skilled in the art will appreciate changes in the illustrated process.

6 illustrates a level 1 photomask. The photomask may include, for example, approximately 120 nm wide lines separated by approximately 300 nm. Such lines can be expanded to cover one dimension of the mask, and the line / separation pattern is repeated to cover another dimension of the mask.

7 illustrates a level 2 photomask. The photomask may include, for example, a grid of elements that are approximately 140 nm wide and 200 nm long with 280 nm separation along the width dimension and 200 nm separation along the length dimension. This pattern can be repeated to fill the mask. The level 2 photomask can be designed such that the elements in the level 2 photomask are substantially aligned with the lines in the level 1 photomask.

8 illustrates a coated substrate after exposure with a level 1 photomask. The substrate, for example a silicon substrate, may be coated with a photosensitive material and then the coated substrate may be exposed through a level 1 photomask. After removal of the exposed area, the substrate is left with a coating substantially corresponding to the level 1 photomask.

9 illustrates the same substrate after etching by isotropic etching or the like. The area masked by the level 1 photomask is protected by the photosensitive material; The other area is etched under the initial surface of the substrate.

10 illustrates the same substrate after removal of residual photosensitive material. This substrate has an area that substantially corresponds to a line on a Level 1 photomask that is substantially the same as the original substrate surface. The substrate also has an area substantially corresponding to the separation space in the level 1 photomask etched below the original substrate surface.

11 illustrates the same substrate after processing with a level 2 photomask. This substrate may be coated with a photosensitive material and then the coated substrate may be exposed with a level 2 photomask. After removal of the exposed area, the substrate is left with a coating substantially corresponding to the level 2 photomask. Since the level 1 and level 2 photomasks have the same shape, the etched areas on the original substrate surface may remain exposed after processing with the level 2 photomask.

12 illustrates the same substrate after application of another coating, for example deposition of a coating comprising 5 nm Ti and 15 nm Au. This coating is attached to the exposed photo surface corresponding to the remaining photosensitive material, the area etched below the original substrate surface, and the portion of the original substrate surface left exposed by the level 2 photomask.

13 illustrates the same substrate after removal of the photosensitive material. The area covered by the photosensitive material does not have any metal, while the area that is not so covered has metal coding.

FIG. 14 illustrates a top view and FIG. 15 illustrates a 3d view of the substrate after the processing. This substrate is free of metal coating except that it remains coated after processing discussed in connection with FIGS. 12 and 13. Thus, the metallized region forms a grid of elements of controllable size (in this example consistent size) with controllable separation between elements (in this example consistent size).

Photomasks, such as those described, can be realized using techniques known in the art. For example, this technique is commonly used for the manufacture of semiconductor devices. Photosensitive materials are known in the art, for example photoresists are used mechanically in the patterning of semiconductor device fabrication. Substrate processing using photomasks and photosensitive materials, and coating processes are commonly used in semiconductor device fabrication. In addition, other processing techniques such as e-beam patterning, focused ion beam lithography, x-ray lithography and molecular imprinting can be used in place of photolithography to produce the desired shape.

Mounted directly to the formed platform Self Assembly Address. In addition, the present invention provides methods for the fabrication of three-dimensional assemblies (referred to as 3D addressable assemblies or 3DAA) of polymer containing nucleobases, and incorporation into solid or colloidal foundations, to make up catalysts and other useful nanoscale products. It includes. The specific emphasis is the design of 3DAA so that many steps in the construction of heterogeneous catalysts based on single strand dimensional configuration (SSDC) as described in some of the above referenced applications are greatly reduced and technically simplified. Lies in the simplification of fabrication and manufacturing. The particular improvement highlighted here is a defined method of stabilizing the geometry and structure of the 3DAA, and the contribution of the solid or stably cast phase, which serves as a foundation or structural scaffold, contributes significantly to the geometric integrity and catalytic activity of the final product. do.

The overall overview produced according to the invention can be generalized by the following steps:

I. Copolymers of previously unhybridized polymers and single-stranded DNA (ssDNA), peptide nucleic acids (PNA) or other polynucleobases are synthesized using computer-assisted design and structure prediction of sequences to design the intended three-dimensional (3D) ) Self-assembly into the structure.

II. Generally concave depressions or gaps in the solid phase are made on a scale in which the number of positions accommodated in the 3DAA coincides with the number of structures intended for mounting. EXAMPLES Manufacturing techniques include industry standard soft lithography processes, industry standard semiconductor etching procedures, and other techniques from hard cast stamps in which cracked, generally convex, negative shapes are used as the template.

III. Self-assembled ssDNAs and PNAs now evolve position-specific interactions in the face of arrays of depressions on previous soft cast polymers that now act as foundations to form consistent electrostatic or covalent bonds. The 3D final hybridization of the polymer, and the contribution compatibility of the foundation, define the SSDC catalyst.

As mentioned in the above referenced application, the concept, design, fabrication and lithography of 3DAA may depend on the application of several techniques. The invention further includes hard (beam) and soft (polymer) lithography, bulk solution conditions, physical (electromagnetic, inertial, and liquid-gas interface) and tribologic (IES: solid liquid interface, electrostatic and solventization) management. Although included, allows the number and extent of dependencies to be reduced. Other techniques, such as computer-based programs and algorithms that aid in the design and optimization of addressable assemblies, and the advantages from nuclear, scanning or transmission electron microscopy (AFM, SEM and TEM) for quality determination are inherent as previously described. It is.

The present invention produces a three-dimensional stable geometric precision pattern under physiological conditions and hybridization-based self-assembly of ssDNA-PNA oligomers that address radially skeletal functionalized PNA segments orthogonal to the assembly and generally towards the focal point or center. Allow the application of Such 3DAA can be designed predictably and accurately with computer software of the present technology and the additional findings described below: (i) improved accuracy of physicochemical functional orientation in three dimensions, and (ii) as described above. Incorporation of copolymerized PNA in an orientated non-linear ("ladder") configuration, and (iii) Integration of the IES factor to promote 3DAA structure placement with destination gaps or depressions.

The preparation of SSDC catalysts can be achieved in a cost effective, fast and accurate manner by the accompanying and repeated application of the following methods. This technique is generally discussed temporarily, although it is not necessary for the full dependence order.

Structural and Dynamic Catalyst Modeling. First, important enzymes or catalytic pathways include (i) structural physiology of the location and dynamic behavior of catalysts and other key amino acid residues, and (ii) transition states, electron transport vectors, dynamic redox states, metastable mediators, transient and Permanent bond formation and cleavage is modeled based on the parametric catalyst profile. This information is then transformed into a 3D polynucleobase format developed for SSDC to define a set of addresses of activated probe hybridization, linker type and orientation, probe backbone and orthogonal physicochemical functions.

16 is an embodiment of the SSDC scheme for a two-dimensional heterogeneous catalyst. Orthogonal chemical function required for mimetization of alpha chymotrypsin enzyme. SER (top left) and HIS (bottom left) are attached to the amide backbone of the PNA segment address via sterol-based linkers. In addition, the residue without catalyst has a sterol linker which leads to stack formation through van der Waals interactions, improving the direct alignment of the catalyst. The accompanying hydraulic layer (yellow) and the cation surface (purple) are hardened soft cast polymer surfaces for electrostatic mounting of phosphodiester-backboned address polynucleobases (DNA, as shown). It exists around (green).

Material design and fabrication of enclosures for catalysts . As defined by the modeling above, the position in the 3D space of the chemical function determined to be required for the catalyst (orthogonal group of amino acid groups when performing the memetization) is determined by the conformation of the chemical function (on the radial axis and the longitudinal axis). Predetermined coordinate positions through address-specific probe and linker molecules that manage typical cone and elliptic ranges. Typically the concave shape is then defined as a foundation that localizes a high density addressable polynucleobase array that successfully localizes and orients the catalyst. The enclosure resembles the size and shape of the DNA assembly congruent with surface modifications, functionalization, linker molecules and other factors that facilitate anchoring, positioning and mounting of the 3DAA within the enclosure gap.

17 shows a geometric approximation of the catalytic site of a single catalytic enzyme. Approximate half-cylindrical depression of the binding, transition, stabilization and release sites of A. Cellulolyticus Endoglucanse 1. Radius design is meant to integrate the lithography and directional orientation of ssDNA and PNA of a group of catalysts generally towards the central axis that mimics the active site location.

Design of Addressable and Geometrically Regular Polynucleobase Assemblies. In terms of shape as the basis of the design and taking into account factors other than those of the catalytic design that are important for hydrodynamic forces, mass transport on the interface, and catalyst, the actual SSDC gap is the minimum that the end product platform geometry is optimized to address physicochemical functionalities. It can be designed to act as a catalyst of complexity. The geometrically repeating pattern is designed to (i) design and fabricate 3DAA to clarify computer modeling of dynamic catalysts through the application of predictable address positions, and (ii) to maintain address positions and orientations and to mount solid or colloidal foundations. It can be used for increased structural strength of 3DAA to promote.

In addition, subassemblies of polynucleobases that are designed for folding, reinforcing assembly, and other 2D and 3D coincidence determinations can be synthesized and integrated through covalent or non-covalent linkages. Such subassemblies may include terminals and intervening units that bend, fold, brace, and otherwise match a flat addressable assembly typically in a geometry that is congruent with the intended catalyst model and the fabricated enclosure. .

18 shows the conversion of the geometric approximation to a universal gap for the catalyst. Linker and PNA-based physicochemical functions determine nearly cylindrical gaps. The inclined ends augment the end product from the mass transport and gap sites of the substrate (eg, small peptides and polysaccharides) that are soluble into the gap sites. The hemispherical end wall (tilt) and the centrally located removable hemispherical brace (vertical to plane, not shown) are blue. The honeycomb pattern (yellow) is not scaled.

Design of molds for mass production based on soft lithography. Foundations for hard cast and metallized shapes that are convex and negative in the catalytic gap can be fabricated by focused ion beam (FIB), light / electron beam lithography, hard cast lithography processes or laser ablation. . It can be used as a mold, mold or stamp for the repeated creation of concave shapes onto a receiving soft cast polymer or other material that forms a foundation or enclosure upon final hard casting.

In view of the polymer dynamics during and after stamping of the soft cast polymer, the mold can be grooved, textured or otherwise surface modified to increase the IES factor, eg lubricity or friction, which optimizes the soft lithography based generation of the enclosure. Effective and cost-effective manufacture of enclosures requires rapid and consistent manufacturing in such a way as to produce the intended shape through scaled iterations without damaging or losing the shape integrity of the mold or product.

19 shows the design of a stamp for soft lithography of a universal gap foundation. On the left, the negative and reverse orientation images of the catalyst gap described in FIG. 18 are shown. This serves as a starting point for the unit design of the mold mold. On the right side, the slope of both ends of the mold unit is shown, which creates a gap through soft lithography. Starting materials are generally silicon derivatives (eg, Si (100)) and arrays of “inclined end quantum” shapes spaced approximately 150 nm apart on both (x, y) axes are FIB, photolithography or more advanced. It can be produced by the method. Lines represent contours and may indicate lubricity “grooves” for increased tribological management and ease of repeated production of products.

Fabrication of mold arrays for mass production. Multiple stamps, or mold arrays, may be prepared for multiple rounds of soft lithography. This may include the application of a thin but elastic physical vapor deposition layer of stamping metal (eg titanium) that cooperates with the desired lubrication option without adversely affecting the geometry or shape of the mold unit. The latter may depend on the polymer material intended as the enclosure product. Previously, a backing metal backing that produces a galvanic effect (eg, aluminum) is applied through a conductive adhesive (eg, graphite carbon doped polyacrylate). The mold assembly can be introduced into a manufacturing process such as a roller, stamp and peel, thin film, interlayer assembly, or spin coater.

20 illustrates the manufacture and preparation of a stamp (mold array) for soft lithography. On the left is an enlarged negative shape of the catalyst gap defined for lithography on fuscia. In the center are shown individual unit shapes. A layer of conductive adhesive (olive) is applied to the bottom of the mold. On the right is shown an assembly against [Al] plate (light blue) mounted to the mold via adhesive, and PVD of approximately 10 nm [Ti] on the stamping surface. A “metallized lip” residual material from the PVD is expected to define the limits of impression molding and pressure application (more defined in FIG. 21).

Performing Productivity Soft Lithography . Thin layers of low volatility, high surface tension solvents can be applied to shapes and soft cast polymers or molds formed by stamping. Solvents (1) lubricants that manage the dynamic friction of metal-to-polymer interactions, and (2) (i) accept and transfer the forces of stamping, (ii) insulate the polymer from direct contact with the mold, and (iii) It can act as an uncompressed thin film layer that balances inductive forces over the entire mold surface encased in a polymer. These factors contribute to repetitive and accurate shape duplication and minimize damage to the mold and product. Current technical methods for soft lithography and thin film fabrication (eg, Langmuir-Blodgett, interlayer electrostatics, physicochemical self-assembly, etc.) are inherent to be performed for this purpose.

21 illustrates platform fabrication via soft lithography. On the left are the components of the mold and platform described previously. I. L., Impression Restriction; A.D., applicable distance. In the center is shown the lubrication layer at the maximum compression point and the geometry of the compression limitations of the dynamic polymer (mold removed for ease of viewing even if inherent). Hydrodynamic thermal compression limits are shown (cherry). The right side shows the shrinkage change in the polymer geometry due to post-casting relaxation (curve in green) beyond the compression limit defined by the mold.

Hybridization Based Assembly of Addressable Polynucleobase Structures . As previously described, polynucleobase sites addressing probes that contribute to the catalyst can be fabricated in a geometric manner. Current technical methods such as DNA Weaving, Origami and Self Assembly Scaffolds can be used to construct 3DAA; The knowledge of those skilled in the art combined with the above referenced application describes the techniques. A new and primary evolution of this technique is the design and fabrication of typically rectangular "sheets" of hybridized polynucleobases that incorporate PNA and other modified parts that are absolutely necessary for the structure of assembled DNA. Sequences of individual oligonucleotide strands are designed such that the PNA and other portions are addressed as required and preferentially and regularly repeat portions of the final assembly.

22 shows DNA weaving based selfassembly of a 2D addressable array. On the left is shown a representative 3DAA consisting of hybridization of oligonucleotides to produce a regular hexagonal array: each 100 address blocks are weaved into 2D rectangular sheets. In the center is shown a representative pattern of geometrically repeating PNA sites weaved into address blocks via hybridization (off color). On the right is shown an address representing a representative PNA sequence in a ladder (non-helical configuration) with orthogonal function of SER and HIS addressed via a sterol-based linker facing the 3D axis center radially (see FIG. 16).

Variation of 3 DAA structures to match the geometry . The subassemblies of the weaved and otherwise self-assembled polynucleobases may be made necessary or cleavable from the main addressable assembly represented by the yellow hexagonal sheets in FIGS. B7 and B8. Representative strategies for (1) addressing orthogonal physicochemical functions in radial directions and (2) matching 2D sheets into shapes following mounting to the enclosure are: (i) identical or forming a terminal end or intervening center segment "wall"; Similar geometric patterns, (ii) one or polynucleobase “brases” which are absolutely necessary for the addressable structure, and (iii) for example, through the solarene-mediated generation of cyclobutane-pyrimidine dimmers between special adenosyl and thymidine bases. Site-specific “reinforcement” of portions of 3DAA.

Figure 23 illustrates a DNA geometry based directed assembly of a 3D addressable array. On the left, a representative option is shown for folding a 2D sheet with the desired half cylinder configuration: full weaved vertical "wall" (aqua), and double stranded "brace" (blue + yellow). On the right is a radial orientation of the catalytic function depending on the geometry of the 3DAA and mounted to the enclosure. The example implies an "complete" orientation of the address (green) and the orthogonal function (solid color) to the target area (blue bullseye). Incomplete consideration of the "incomplete" orientation of the other addresses (purple and teal) is shown. Reorientation considerations are inherent in linkers and other designs.

3 Design, functionalization and assembly of catalytic functions for DAA structures. Part of the design of the SSDC catalyst is to address physicochemical functions that contribute to the catalyst in previously modeled mechanisms. Given the subassembly facilitating 3D conformation and the successful fabrication of addressable assemblies and the mounting of those on fabricated enclosures that preserve and facilitate the desired configuration, addressing and orientation of orthogonal functions can now be designed. have.

After individual addresses and address blocks having physicochemical functions are determined by modeling, for example, the synthesis of polynucleobase strands consisting of copolymers of ssDNA and PNA as well as pure DNA oligonucleotides can be synthesized. Base composition can be determined by the desired (regular hexagon as representatively described) geometric pattern, packing distance, stiffness and overall size of the initial 2D “sheet” prior to folding the conformation. Orthogonal functions, eg, amide backbone-functionalized PNA segments, that define addresses can be copolymerized into structural ssDNA elements, where the last part of the strand serves as a scaffold for the orientation and placement of PNA-based addresses. .

FIG. 24 depicts stacking of the conformation of catalytic function from hybridized polynucleobase libraries. At the top, a representative ladder (non-helix) configuration of the alpha-chymotrypsin catalyst function described in FIGS. 16 and 22 is shown. Sterol / alkaloid / polycyclic linker elements contribute to directional integrity through hydrophobic interactions that bridge SER (hydroxyl) and HIS (indole / imidazolic) functions in certain residues and limit the range of motion of the function. . At the bottom is shown an exemplary polynucleobase library consisting of pure structure (yellow) and mixed structure and functional scaffolding members (blue, colored blue + green).

Solid foundation for the design, functionalization and mounting of assemblies . There are a number of standard technical methods for modifying DNA and other polynucleobases each focusing on phosphodiester and amide backbones that facilitate more efficient anchoring and mounting of 3DAA to conformation accommodating and protective enclosures. Likewise, various surface and / or enclosure functionalization options exist for physical, chemical and biochemical modification of materials for anchoring and mounting of "plain" (phosphodiester backbones) or modified DNA.

USPTO 61 / 086,633 discloses lithographic streptavidin (SA), for example, via a linker to be anchored directly to an enclosure fabricated on top, or indirectly via lithographic biotin and SA beads at 10 nanometer scale. The ability of 3DAA to be biotin functionalized is detailed. This method can be reasonably extended to other receptor ligand systems including digoxigenin and antibody (anti-DIG), other antibody based systems, as well as less specific electrostatic systems such as poly-L-lysine and anionic ligands. have. Therefore, this specification highlights mounting options that are more congruent with 3DAA geometry preservation, eg, polynucleobase backbones, for solid phases. Since the latter outline is well documented, improvements through the state of the art can be achieved using existing methods and protocols that enable the design, advancement, activation, qualification, optimization and manufacture of the scale of SSDC catalysts through the dependencies described here and now. Include specific applications.

Representative methods of modifying 3DAA and materials for direct and indirect anchoring of portions of the addressable assembly, and for mounting the entirety of the addressable assembly include those described below.

1. Epoxide Phosphorothioate for polymer thio benzoate - Indirect mounting assembly of the modified DNA. Metastable epoxide groups on the polymeric material can be condensed (surface end cured) with alkyl diamines. Residual free amine terminations are attached to a bifunctional crosslinker (e.g. SSMCC as described in 60 / 918,144) comprising succinimide groups for covalent linkages to amines-sulphhydryls. The latter can be covalently linked to the phosphorothioate residues of dsDNA.

2. Epoxide Force on polymer Fora midayi agent - directly mounting assembly of the modified DNA. In bulk, and interfacially, the solvation of (P) -amidite on the polymer can be modified to enhance and promote amine mediated epoxide reduction with the final covalent linkage of the assembly backbone directly to the polymer.

3. Epoxide Indirect mounting of (P) -thioate -modified DNA or sulfhydryl PNA assemblies to polymers . Metastable epoxide groups on polymeric materials can be stabilized with hydroxyl residues and converted by SNI-type reactions to primary halides. The latter can be condensed into alkyl diamines which are subsequently functionalized via SSMCC to provide surface maleimide groups. For example, the latter covalent linkage to the cisyl residue to the PNA tertiary amine residue can achieve mounting.

4. Indirect mounting of phosphorothioate -modified DNA assembly to gold . A pure (> 99.99997%) gold surface is a bifunctional alkan (eg, 60 / 918,144) containing sulfhydryl groups, maleimide groups, or other free reduced thiol groups, and amine groups for covalent bonds to gold. MUAM as described). After sulfidation of the gold surface, the preamine can be covalently bound to the SSMCC providing the premaleimide group. For example, the latter covalent linkage of dsDNA to phosphorothioate residues can achieve mounting.

5. Direct mounting of (P) -thioate -modified DNA assembly to gold . In bulk, and interfacially, the solvation of (P) -thioate on the polymer can be modified to enhance and promote oxidation of sulfhydryl groups on DNA with respect to final covalent bonds and gold.

25 shows an exemplary method for mounting polynucleobases for the base material. Chain motifs indicate that complex (indirect / horizontal) and single (direct / vertical) covalent bonds between the skeletal-modified residues of the DNA-PNA assembly and the molomer material are produced by soft lithography. Options 4 and 5 imply that the thin gold film is PVD onto the enclosure.

Design of material flow mode for increasing catalyst. Many fabrication options are suitable for shaping foundation enclosures, the ultimate shape of which is catalyzed by transport of solutes (substrate, intermediates and products) into and out of the enclosure, as well as the location and orientation of orthogonal physicochemical functions. Determine the speed. As noted above, the inclined termination can be used to promote such flow adjacent to the boundary layer of the actual assembly scaffolding (where net flow rate = 0). Alternatively, the subassembly can be omitted as a whole (optionally, such as an incised end or intermediate after proper anchoring and mounting of the 3DAA into the receptacle) to remove continuous grooves by the flow closest to the constantly adjacent boundary layer in the laminar flow section. Where the Reynolds number (Re) is very small (generally less than 10).

In addition, the foundation enclosure option includes confined areas where the laminar flow is intentionally disrupted to increase mixing. The latter typically increases the intrinsic catalyst flow rate except for the risk of lowering the overall rate of subsequent conversion because of the possibility of impeding the flow to subsequent assemblies. As inherent in the right side of FIG. B11, turbulence successfully increases the catalyst in a defined region and complicates the flow downstream of the next enclosure where laminar flow is initially required, such as the indicated trough.

Typically, the increased volumetric flow rate (VFR) is transformed into the enclosure containing the catalyst assembly-such as dissolved solute-at an increased rate of catalyst due to the increased rate of the substrate mass transfer. This increase is equivalent to the finite residence time required for the known and designed rate of the catalyst, in particular for the complete catalyst profile (as defined by the dissociation constant, Kd), and other excess VFRs are orthogonal chemistry and / or performed as designed. Or lowers the catalyst rate due to the inability of physical and chemical functions. However, since most of the time scale of Kd of biotic enzymes is on the order of nanoseconds, maximized mixing is usually required for maximum catalyst speed.

Inherent tradeoffs exist to increase the flow immediately adjacent to the boundary layer. As previously described, a "wall", "brase", or other subassembly may be required for proper folding of a differently flat 2D addressable assembly in a shape consistent with a soft lithographic enclosure similar to the active site of the enzyme being mined. have. For example, continuous grooves maximize the available flow and do not provide any ready options for folding the catalyst assembly. Weaved walls can provide the option of matching assemblies to impede flow to and from the catalytic site. A brace made up of a limited number of integral and hybridized “fences” can form a compromise by providing an option for assembly shaping with minimal impact near the boundary layer.

Figure 26 illustrates boundary layer flow as a function of an assembly fabrication scheme. On the left is the movement of supra-vicinal volumetric flow above in successive grooved (light blue), beveled, segmented (purple), partially brazed (red) foundations supporting each 3D assembly. Predicted and generalized All adjacent flow modes become laminar due to the assembly design (Re <10). As described in the text, continuous grooves preserve an almost constant Re, while segmented or braceed grooves interfere with different degrees of flow. The right side shows the predicted boundary layer flow into and out of the constrained trough type enclosure. 3DAA (having orthogonal function indicated in dark color) is preferentially mounted in the restricted area at the end of the trough, where turbulence (Re> 25) is promoted to enhance mixing and catalytic activity.

Design of 3D bulk flow systems for increased catalyst . A number of micro-to-meter scale fabrication options are the final 3DAA-based manufacturing process that increase the catalyst and catalyst speed of turnover by optimizing the flow method at nanoscale as described previously. It is suitable for the inclusion of SSDC catalysts. Many of these options are standard techniques and are only described here in a limited manner, and are typically in the region of confined volume bulk flow where boundary layer laminar flow is promoted on a submicron scale. Two elements dominate in this strategy. [1] The net bulk flow rate is maximized to increase the mixing and processing efficiency of performing the catalyst at or near the theoretical maximum Kd. [2] The limited bulk flow, which facilitates low Re boundary layers, is designed to reduce the mass transfer rate in proximity to adjacent layers to provide adequate Tr for the intrinsic catalyst. Such flow modes are negative or, in other words, inappropriate for biotic enzymes due to the high shear forces derived from the limited flow space and geometry.

When Kd and Tr are optimized in a processing system under biotic conditions, biotic parameters including increased temperature, maximum pH and pi, high flow rates, and more defined geometries, and protein-based enzymes (eg, heavy metals) , Ionic liquids, and emulsification of polyphase and water soluble liquid organic solvents) and the presence of known abiotic cofactors to maximize catalysts in other systems may lead to enclosure support catalyst assembly. It can be performed predictably without excessive damage.

Unlike the prior art, the present invention is directed to the specifics of existing technologies and protocols that enable and enable the design, advancement, activation, qualification, optimization and manufacture of the scale of SSDC catalyst through the flow geometry and devices described herein and hereafter. It can provide an application. Exemplary engineering methods include any device or bulk scale fabrication that maximizes the catalyst rate of the 3DAA array through flow rates, Re and flow profiles approaching the theoretical maximum Kd under conditions where processing occurs.

In addition to augmentation of the catalyst by the laminar flow method in general, the limited flow geometry, turnover speed is such that an array of enclosed assemblies of beads, packed columns of such, mixing fins and processing devices It may be increased by the inclusion of the wall of. These options can augment the catalyst by maximizing the surface area available for the substrate promoting the catalyst and the resulting mass transfer. These options can be (i) separately in-line as parallel or in-line parts of the entire manufacturing process, (ii) repeatedly, or (iii) sequentially as separation units that perform any aspect or portion of the catalytic process, or variations thereof. It can be integrated into a limited flow system.

27 illustrates a device designed to augment the boundary layer at the assembly level. On the left, a bulk grooved symmetrical wavy coil is shown to force fluid flow in the downward direction and to sequentially facilitate the defined flow geometry at the point of reduced distance between the coil and the wall (both dark grey). In the center is shown a bulk grooved asymmetrical screw in a helical manner that forces fluid flow down. Radial asymmetry increases mixing by facilitating longitudinal movement as well as torsional movement of the boundary layer and the bulk liquid. On the right is a standard inline, sequential turbine-type mixing device.

The present invention has been described as a method of single strand dimensional construction. It is to be understood that the above description has only exemplified the application of the principles of the invention, the scope of which is to be determined by the claims shown in the context of the specification. Other variations and modifications of the invention will be apparent to those skilled in the art.

Claims (27)

A method for producing and producing co-attached biopolymers formed from a complementary master shape formed into a defined shape and exhibiting complementary single stranded polynucleobases. The method of claim 1,
Wherein said biopolymer is selected from the group consisting of deoxyribonucleic acid, ribonucleic acid, and peptide nucleic acid. The method of claim 1,
The production manufacturing method includes the step of forming a thin film. The method of claim 1,
The covalent attachment comprises at least one of a thiol-gold interaction, or an amine-electrophile alkylation or condensation reaction; A production manufacturing method comprising at least one of direct attachment to a biopolymer backbone or direct attachment through an inertial linker molecule. The method of claim 4, wherein
Wherein said electrophile comprises at least one of an epoxide, an alkyl halide, an anhydride, or a bond acceptor. In a production manufacturing method comprising a limited orientation of addressed biopolymers,
Producing a production using a template foundation comprising a pre-organized pattern of complementary polynucleobases. The method according to claim 6,
Wherein the mold foundation comprises a square hatch pattern of the complementary polynucleobases and produces a dashed pattern of addresses in the production. The method of claim 5, wherein
The template foundation comprises a hexagonal pattern of the complementary polynucleobases and produces an indented pattern of addresses in production. Producing a uniformly concave shape formation in the photosensitive solid substrate matrix to effect metal vapor deposition to enable attachment of self-assembled biopolymer patterns. The method of claim 9,
And said substrate matrix comprises silicon coated with a photosensitive material. The method of claim 9,
The providing of the substrate comprises the step of etching a constant cylindrical trough into the substrate using an isotropic method and a protective photomask. The method of claim 9,
Wherein said metal vapor deposition comprises depositing a 5 nm titanium layer, depositing a 15 nm gold layer, and then removing a photomask. 13. A method of manufacturing an electronic circuit using any one of the methods described in claims 1 to 12. A method of making an assembly that mimics enzymatic conversion comprising any of the methods described in claims 1-12.
Wherein said assembly provides excellent characteristics to at least one similar naturally occurring enzyme in stability, pH, temperature, and the presence of additives known to accelerate the catalyst. A process characterized by molding a shape representing a covalently attached biopolymer produced from a complementary master shape representing itself as a complementary single stranded polynucleobase, as defined by Watson-Crick hybridization pairing. The method of claim 15,
Suitable biopolymers include deoxyribonucleic acid, ribonucleic acid, and peptide nucleic acids. The method of claim 15,
The replication technique includes a thin film. The method of claim 15,
Suitable covalent bonds between the single-stranded biopolymer and the base material include thiol-gold interactions, or amine-electrophilic alkylation or condensation reactions, either through direct attachment of the biopolymer backbone or else via bifunctional linker molecules. And such electrophiles include, but are not limited to, epoxides, alkyl halides, anhydrides, or bond acceptors. A pre-organized pattern of complementary polynucleobases attached to the template foundation resulting in a defined orientation of the addressed biopolymer. The method of claim 19,
Wherein the dashed pattern of addresses originates from a square happy pattern of complementary polynucleobases attached to the template foundation. The method of claim 19,
An indented pattern of addresses is generated from a hexagonal pattern of complementary polynucleobases attached to the template foundation. Process to form a concave shape formation in the photosensitive solid substrate matrix prior to metal vapor deposition to enable attachment of a self-assembled biopolymer pattern. The method of claim 22,
Said substrate being silicon coated with a photosensitive material. The method of claim 22,
Wherein the constant cylindrical trough can be etched into the substrate using an isotropic method and a protective photomask. The method of claim 22,
Uniform metal vapor deposition is formed of 5 nm titanium, followed by 15 nm gold, followed by removal of the photomask. In the application consisting of claims 20 or 21 for the microprocessor industry,
Wherein the 3D addressable assembly of polynucleobases is calculated to be 1 / 16th of the current feature size resulting from scaled fabrication by photolithographic techniques. In an application consisting of claims 20 or 21 for industrially required catalysts,
An assembly that mimics known enzyme conversion can demonstrate superiority to similar natural systems with regard to stability with respect to pH, temperature, and the presence of known additives to accelerate the catalyst.

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