System and methods for generating dynamic materials having artificial metabolism

Abstract

To provide a system and methods for generating dynamic materials having an ordered structure and artificial metabolism.SOLUTION: The approach disclosed herein allows, in an artificial fashion, autonomous and dynamic generation of materials with structural hierarchy by simultaneously coupling both irreversible synthesis (and optionally decomposition) and dissipative assembly processes. As an exemplary embodiment, DNA-based Assembly and Synthesis of Hierarchical (or "DASH") materials have been generated. Systems, devices, reagents and methods for generating the materials, as well as additional applications of the present methodology, are disclosed.SELECTED DRAWING: Figure 1-1

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority to U.S. Provisional Application No. 62 / 829,702, filed on April 5, 2019, which is hereby incorporated by reference in its entirety.

[0002] Description of Research and Development Sponsored by the Federal Government The present invention was made with government support under Award Numbers EFRI - 1331583 and SNM - 1530522 awarded by the National Science Foundation (NSF) of the United States. The government has certain rights in the invention.

[0003] Incorporation by Reference of a Sequence Listing The sequence listing in the 6KB ASCII text file named 37255PCT_7787_02_PC_SequenceListing.txt, created on March 30, 2020, and submitted to the United States Patent and Trademark Office via EFS - Web, is hereby incorporated by reference in its entirety.

Background Art

[0004] The characteristic properties of life, such as the dynamic self-generation of organisms, are maintained by metabolism. Using the flow of matter and energy, molecules are irreversibly synthesized from components by a series of biological reactions, and then dynamically assembled further into macromolecules and beyond, resulting in the structural hierarchy of the materials of life. Although various approaches to designing dynamic materials by bionics have been reported, the construction of materials by mimicking metabolism from scratch has not been achieved. For example, designed biomaterials enable the generation of materials by life, but the reported approaches rely on external biological systems, such as cells that generate the materials. Similarly, other dynamic biomaterials, such as the active cytoskeleton, directly use the existing metabolism designed by life. In general, the bionics approach has the potential to create new dynamic biomaterials with sophisticated active behavior, but the current approach is based on the existing metabolism of life and is therefore fundamentally limited to the existing metabolism of life. Various chemical approaches, especially dissipative self-assembly, have enabled the construction of dynamic materials from scratch using chemical reactions.

Summary of the Invention

Means for Solving the Problems

[0005] The present disclosure relates to systems and methods for generating dynamic materials having regular structures and artificial metabolisms. Similar to the metabolism found in nature, the approach disclosed herein enables the autonomous and dynamic generation of materials having a structural hierarchy in an artificial manner by simultaneously combining both irreversible synthesis (and optionally degradation) and dissipative assembly processes. The artificial metabolism is designed using molecules and reactions, for example, not limited to life itself, but using biomolecules and biological reactions, by a bottom-up design of synthesis combined with assembly.

[0006] To illustrate the present system and methodology, and as an exemplary embodiment, hierarchical DNA assembly and synthesis (or "DASH: DNA-based Assembly and Synthesis of Hierarchical") materials were generated using a mesoscale approach that uses artificial metabolism to create dynamic materials from building blocks of biomolecules (Figure 1A). Similar to materials in living organisms, materials generated by DASH can synthesize and assemble into pre-coded patterns via assimilation. Further, by integrating assimilation (generation) with dissimilation (degradation), and by combining both generation and degradation regularly in response to innate spatio-temporal feedback, the generated materials can be autonomously degraded and periodically regenerated in situ. DASH materials with various patterns were generated. Also, DASH materials were generated that exhibit an emergent "mobility" behavior similar to that of a slide mold. Further, DASH materials with two locomotive bodies that exhibit emergent competitive behavior were also generated. The dynamic materials disclosed herein can be utilized as a scaffold for further functionalization to form hybrid materials. In embodiments where the material is a DASH material, the material can serve as a platform for providing the function of DNA in a cell-free setting (such as cell-free protein expression). Further, the present system and method for generating the dynamic materials disclosed herein can be applied to pathogen detection. By integrating assimilation (generation) with dissimilation (degradation), and by combining both generation and degradation regularly in response to innate spatio-temporal feedback, the generated materials can be autonomously degraded and periodically regenerated in situ. DASH materials with various patterns were generated. Also, DASH materials were generated that exhibit an emergent "mobility" behavior similar to that of a slide mold. Further, DASH materials with two locomotive bodies that exhibit emergent competitive behavior were also generated. The dynamic materials disclosed herein can be utilized as a scaffold for further functionalization to form hybrid materials. In embodiments where the material is a DASH material, the material can serve as a platform for providing the function of DNA in a cell-free setting (such as cell-free protein expression). Further, the present system and method for generating the dynamic materials disclosed herein can be applied to pathogen detection.

[0007] In one aspect, the present disclosure provides a system for generating materials having a regular structure and artificial metabolism. The system includes an apparatus and a generation mix, where the generation mix is a reagent containing components for forming a polymer, and where the apparatus enables a directed flow of a solution therethrough to initiate and facilitate an assembly of polymers synthesized in the apparatus, thereby forming a material, and is designed to have obstacles of a shape and size that maintain spacing in a predetermined pattern and enable the generation of vorticity in the directed flow of the solution containing the generation mix, including a main chamber.

[0008] In some embodiments, the apparatus is in the form of a flow cell, and the main chamber includes at least one inlet port and at least one outlet port. The solution containing the generation mix is directed to flow from at least one inlet port, through the main chamber, i.e., through the flow paths or spaces between the obstacles, to at least one outlet port. In some embodiments, the main chamber has dimensions in the micron range, such as a microfluidic chamber. In some embodiments, the main chamber has a planar shape.

[0009] In some embodiments, the system further includes a degeneration mix in addition to the generation mix and the apparatus, where the degeneration mix contains reagents for depolymerizing the polymers generated by the generation mix.

[0010] In some embodiments, the main chamber is designed to receive a solution containing a production mix and a solution containing a degradation mix, and to allow for a directed flow. In some embodiments, the main chamber includes at least two inlet ports for separately injecting a solution containing a production mix and a solution containing a degradation mix, and at least one outlet port, where, on the directed flow of the solution through the main chamber, the processes of polymer synthesis and assembly as well as the process of polymer degradation occur autonomously and in combination, leading to the formation of materials with regular structures and artificial metabolisms.

[0011] In some embodiments, the material produced by the system has a static pattern that can take on any shape and form. In some embodiments, the produced material has a dynamic pattern, for example having two movers that exhibit emergent movement behavior or competitive behavior.

[0012] In some embodiments, the apparatus includes a plurality of main chambers that expand the types of patterns of materials that can be produced.

[0013] In some embodiments, the polymer is DNA, and the produced material is also called a DNA SH material. In some embodiments, the production mix includes deoxynucleotides (dNTPs), a template nucleic acid (DNA or RNA), a primer, and a DNA polymerase. In some embodiments, the primer and the template can anneal before being injected into the main chamber. In some embodiments, the template nucleic acid is circular DNA. In some embodiments, the template nucleic acid is circular DNA formed from linear DNA in the presence of a primer and a ligase. In some embodiments, the degradation mix includes a deoxyribonuclease, for example including exonuclease, endonuclease, or a combination thereof.

[0014] In some embodiments, the generation mix includes a reagent that produces a detectable signal (e.g., fluorescence) that facilitates viewing of the generated material.

[0015] In a further aspect, the present disclosure provides a method for generating materials having a regular structure and an artificial metabolism.

[0016] In some embodiments, the method includes providing the apparatus and the generation mix described herein, supplying a solution containing the generation mix to the main chamber of the apparatus, and directing the flow of the solution through the main chamber, thereby enabling the synthesis of polymers and the assembly of the synthesized polymers to form a material. In some embodiments, the method utilizes an apparatus in the form of a flow cell, and the main chamber includes at least one inlet port and at least one outlet port. The solution containing the generation mix can be directed to flow through the main chamber from at least one inlet port, i.e., through the flow path or space between the obstacles, to at least one outlet port.

[0017] In some embodiments, the method includes providing the apparatus, the generation mix, and the degradation mix described herein, supplying a solution containing the generation mix and a solution containing the degradation mix to the main chamber of the apparatus, and directing the flow of the solution through the main chamber, thereby generating a material. In some embodiments, the main chamber includes at least two inlet ports for separately injecting the solution containing the generation mix and the solution containing the degradation mix, and at least one outlet port, where on the directed flow of the solution through the main chamber, the processes of polymer synthesis and assembly and the process of polymer degradation occur autonomously and in combination, leading to the formation of a material having a regular structure and an artificial metabolism. The solution containing the generation mix and the solution containing the degradation mix can be injected into the main chamber simultaneously, continuously, or in a predetermined order.

[0018] The generated material can be visualized by the naked eye, camera, fluorescence microscope, optical microscope, or electron microscope.

[0019] In another aspect, the present disclosure provides systems and devices for detecting nucleic acids of pathogens. In accordance with this aspect, the devices and generation mixes described herein are utilized. However, DNA synthesis and generation of DASH materials occur only when the target pathogen nucleic acid is present in the sample. In some embodiments, the generation mix includes dNTPs, a first linear form of template DNA, primers, and DNA polymerase, where the template DNA circularizes in the presence of a ligase, and the circularized DNA functions as a template for DNA synthesis in the device (e.g., via rolling circle amplification). In these embodiments, the first linear form of template DNA can be contacted with the sample to be tested and a ligase prior to being supplied to the main chamber to enable circularization of the template DNA if the target pathogen nucleic acid is present in the sample. Alternatively, the first linear form of the template DNA, along with other components in the production mix, ligase, and the sample, is supplied to the main chamber, and cyclization, as well as polymer synthesis and assembly, occur in the main chamber. In other embodiments, a production mix containing dNTPs, primers, and DNA polymerase without a template nucleic acid is utilized. The target nucleic acid of the pathogen, if present in the sample, serves as a template for polymer synthesis. In still other embodiments, a production mix containing dNTPs, a template nucleic acid, and DNA polymerase without primers is utilized. The target nucleic acid of the pathogen, if present in the sample, serves as a primer for polymer synthesis. Detection can be achieved by supplying a solution containing the production mix (and, in some embodiments, the sample) to the main chamber of the device and directing the flow of the solution through the main chamber, whereby, if a pathogen nucleic acid is present in the sample, generation and assembly of DNA into a material having a regular structure and artificial metabolism are enabled, where the generation of the material indicates the presence of the pathogen nucleic acid. In some embodiments, the pathogen nucleic acid is DNA. In some embodiments, the pathogen nucleic acid is RNA.

[0020] In a further aspect, the material generated herein is used as a scaffold for generating additional functional materials. In some embodiments, the DASH material is contacted with a DNA-binding reagent that is supplied to the main chamber of the device in which the DASH material is formed. In some embodiments, the DNA-binding reagent can be, for example, avidin, quantum dots, and gold nanoparticles. In some embodiments, the DASH material bound to the DNA-binding reagent can be further functionalized. For example, the DASH material bound to avidin can be contacted with a biotinylated enzyme (e.g., horseradish peroxidase).

[0021] In another aspect, cell-free protein expression is provided using the DASH material.

[0022] In yet another aspect, the present disclosure provides a method for designing obstacles disposed in a main chamber of an apparatus for generating the materials described herein. The method includes defining a main chamber for generating a material having a regular structure, defining a pattern of the material to be generated therein, and determining the size, shape, and position of a plurality of obstacles in the main chamber of the apparatus necessary to direct the flow of a solution along the shortest path within the main chamber and between adjacent obstacles.

Brief Description of the Drawings

[0023]

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Modes for Carrying Out the Invention

[0024] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Any methods and materials similar to or equivalent to those described herein can also be used in the practice or testing of the present invention, and the preferred methods and materials are described below. All publications referred to herein are incorporated herein by reference and disclose and describe the methods and / or materials related to the ones to which the publication is cited.

[0025] Materials The present disclosure is directed to the generation of materials having a regular structure and an artificial metabolism.

[0026] The term "artificial metabolism" is used herein to describe both the constitution of the methodology for generating materials and the properties of the generated materials. Similar to the metabolism found in nature the methodology disclosed herein enables the autonomous and dynamic generation of materials with a structural hierarchy in an artificial manner by simultaneously combining both irreversible synthesis / decomposition and dissipative assembly processes. By integrating anabolism (generation) with catabolism (decomposition), the methodology disclosed herein enables the generation of materials that are autonomously decomposed and periodically regenerated by combining both generation and decomposition regularly in situ in response to innate spatio-temporal feedback. Thus, the generated materials are said to have an "artificial metabolism" in that they have a "metabolism" in the sense that the molecular structure underlying the materials is generated autonomously and dynamically (anabolism) by simultaneously combining irreversible synthesis and dissipative assembly processes, and in embodiments where decomposition is further included, the molecular structure underlying the materials is also decomposed autonomously (catabolism); in other words, the molecular structure underlying the materials is generated, decomposed, and periodically regenerated autonomously and dynamically in situ. Since the processes involved are artificially created, the above-described metabolism is referred to as an artificial metabolism.

[0027] The term "regular structure" is used herein to describe the hierarchical structure mechanism of the materials generated herein. For example, the DASH material can have a fibrous structure composed of bundles of one-dimensional micron-scale DNA molecule networks (as a result of dissipative assembly), and then the DNA molecules are polymers formed from nanoscale monomers (as a result of polymer synthesis).

[0028] The materials generated herein can be of any pattern. When referring to the materials generated herein, the term "pattern" includes both the characteristics of shape and dimension, as well as the behavioral characteristics. For example, as described in FIGS. 1D-K and 42, a wide variety of mesoscale patterns and shaped materials have been generated, from one-dimensional lines of periodic patterns to any two-dimensional shape. Materials with dynamic patterns, such as DASH materials showing emergent locomotion behavior and DASH materials with two moving bodies showing competitive behavior, have been generated. As disclosed herein, the pattern of the material can preferably be designed and achieved based on the positions of obstacles having predetermined sizes and shapes, with the aid of computational fluid dynamics ("CFD") simulations.

[0029] Materials having regular structures and artificial metabolisms can be generated from various types of building blocks, i.e., monomers, dimers, trimers, or oligomers, and can be used to synthesize polymers in situ, where the synthesized polymers can also depolymerize in situ. In some embodiments, the polymer is DNA or RNA.

[0030] Generation mix The process of in situ polymer synthesis is achieved by supplying the components necessary for polymer synthesis to an apparatus designed to generate the materials described herein. The term "generation mix" is used herein to describe the reagents containing the components necessary for polymer synthesis.

[0031] In some embodiments where the polymer is DNA, the reaction mixture can include a DNA template (double-stranded or single-stranded, linear or circular), primers, deoxynucleotides (dNTPs), and a DNA polymerase. In some embodiments, the DNA template and primers can be annealed together before use. In some embodiments, the template is circular DNA that has been circularized in the presence of primers and ligase. DNA polymerases suitable for use herein include, but are not limited to, DNA polymerases derived from prokaryotes (such as DNA Pol I, II, and III from prokaryotes like Escherichia coli) or eukaryotes (such as DNA Pol α, β, γ, δ, and ε), many of which are commercially available. In some embodiments, the DNA polymerase is Phi29 DNA polymerase, which can achieve DNA synthesis by rolling circle amplification (RCA). In some embodiments where the polymer is DNA, the reaction mixture can include an RNA template, primers, deoxynucleotides (dNTPs), and a reverse transcriptase.

[0032] In some embodiments where the polymer is RNA, the reaction mixture can include a DNA template (double-stranded or single-stranded), primers, nucleotides (NTPs), an RNA polymerase, and any transcription factors appropriate for inclusion. RNA polymerases suitable for use herein include, but are not limited to, RNA polymerases derived from prokaryotes or eukaryotes, many of which are commercially available.

[0033] In some embodiments, the generation mix may contain all the necessary components for in situ polymer synthesis. In some embodiments, the generation mix may contain the necessary components for polymer synthesis, but synthesis occurs only if the target nucleic acid is present in the sample being tested. For example, in embodiments where the present system and methodology are applied for the purpose of detecting pathogen DNA or RNA, the generation mix may contain all the necessary components for in situ DNA synthesis, but the template DNA is provided in a linear form and functions as a template only after being circularized by ligase only if the target pathogen DNA or RNA is present in the sample. In other embodiments, the generation mix contains components for polymer (DNA or RNA) synthesis such that when pathogen components for polymer synthesis are present in the sample, they then assemble into a material having a predetermined pattern that can then be visualized, and DNA synthesis does not occur and the material is not formed if the pathogen DNA or RNA is not present in the sample, and may contain components for polymer synthesis except for the nucleic acid template, such that it functions as a template for initiating the synthesis of DNA molecules.

[0034] In some embodiments, the generation mix contains a compound that binds to the generated material to enable viewing of the pattern of the material. For example, the generation mix may contain a dye that binds to DNA, such as SYBR Green I.

[0035] Dissociation mix The process of polymer synthesis depolymerization, or degradation of the synthesized polymer, can be achieved by supplying the components necessary for polymer depolymerization to an apparatus designed to generate the materials described herein. The term "dissociation mix" is used herein to describe a reagent containing the components necessary for polymer depolymerization.

[0036] In some embodiments where the polymer is DNA, the digestion mix may include one or more deoxyribonucleases (DNases), which can be exonucleases or endonucleases (including restriction enzymes), many of which are commercially available. In some embodiments, the digestion mix may include one or more of exonuclease I, exonuclease III, DNase I, and DNase II.

[0037] In some embodiments where the polymer is RNA, the digestion mix may include one or more ribonucleases (RNases). In some embodiments, the RNase includes an endoribonuclease. In some embodiments, the RNase includes an exoribonuclease. In some embodiments, the endoribonuclease is selected from the group consisting of RNase A, RNase H, RNase III, RNase L, RNase P, RNase PhyM, RNase T1, RNase T2, RNase U2, and RNase V. In some embodiments, the exoribonuclease is selected from the group consisting of polynucleotide phosphorylase (PNPase), RNase PH, RNase R , RNase D, RNase T, oligoribonuclease, exoribonuclease I, and exoribonuclease II.

[0038] Apparatus The processes of polymer synthesis and assembly, as well as depolymerization, can be achieved using an apparatus designed to produce materials having specific pre-coded patterns.

[0039] The apparatus includes a main chamber in which the processes of polymer synthesis and assembly, and optionally, depolymerization, occur as desired. Generally speaking, the main chamber allows for the supply of a solution containing a production mix and, optionally, a solution containing a breakdown mix, and the supplied solution has a directed flow through the chamber (e.g., a flow from one end of the chamber with an inlet port to another end with an outlet port) to produce a pre-designed pattern of material, and is not limited to any particular shape or dimension as long as it enables this. In some embodiments, the apparatus is a microfluidic device, where the main chamber has dimensions in the micron range and a substantially planar shape, as shown, for example, in FIGS. 1B - 1C.

[0040] For the production of materials having a regular structure and a specific pattern, the main chamber includes a plurality of obstacles spaced (i.e., arranged) in a predetermined manner based on the pattern of the intended material product, and the obstacles are shaped in a shape and size that causes vorticity in the flow of the solution directed through the main chamber. In some examples, the plurality of obstacles are uniform. In some examples, the plurality of obstacles includes at least one first obstacle and at least one second obstacle, and the at least one first obstacle has a different size and / or a different shape from the at least one second obstacle. In some examples, the spacing between the plurality of obstacles is uniform. In some examples, the spacing between at least some of the plurality of obstacles is different from the spacing between other ones of the plurality of obstacles. In some embodiments, the main chamber does not have a substantially planar shape.

[0041] The design of obstacles, including shape, size, and position, to generate materials with a regular structure having a specific pattern can be achieved by following the guidelines developed by the inventors based on observations from the experiments described herein. Specifically, the inventors have found that the mechanism behind the set of final products is a combination of the dynamic formation of vortex excitation of a new network of in situ synthesized polymers and the redistribution directed by the flow of a pre-formed network. More specifically, the inventors have observed that DNA network formation starts from the side edges of the pillars in the middle of the chamber (i.e., the center of the z-axis), and then, with further generation, these DNA networks begin to connect into one continuous fibrous structure between the pillars. Further, the inventors have found that the sides of the pillars coincide with regions of high vorticity (as illustrated, for example, in FIG. 2H), and that devices having pillar shapes that produce higher vorticity generate the DASH pattern faster (as described, for example, in FIGS. 13 - 15). From these observations, it is shown that flow, particularly vorticity, is important in the formation process by locally and dynamically inducing the physical entanglement of DNA into the network at the sides of the pillars. Greater vorticity across the pillars leads to an earlier start time of generation. The formed network then redistributes along the direction of the flow in the region of highest velocity, forming a continuous and fibrous anisotropic structure along the direction of the flow (FIGS. 2F, 2G). The thickness of the structure then increases, ultimately along with the gap between the pillars that is finally filled with the DNA network.

[0042] The set of final products is a combination of the dynamic formation of vortex excitation of a new network of in situ synthesized polymers and the redistribution directed by the flow of a pre-formed network Based on the recognition of the matching, using computational fluid dynamics (CFD) simulations, the inventors have confirmed that considering the desired pattern of the material, the pattern of obstacles in the chamber (i.e., shape, size, and position) can be designed based on two simple guidelines: the pattern should take the shortest path within the chamber along the direction of the flow, and can be predicted by connecting adjacent pillars (obstacles). As described, a microfluidic device was designed by a simple combination rule using seven types of structural units (Figure 7). By combining the units, the positions of the pillars along the flow path in the device are encoded to satisfy both guidelines, enabling a general design strategy for the DASH pattern. Figure 27 illustrates a number of patterns of the material and the underlying pattern (shape, size, and position) of the obstacles for generating materials having such patterns of the material. Generally speaking, the obstacles can be recognized as a plurality of nodes connected by connections, and the "nodes" are regions with high vorticity and correspond to the "tips or ends" beside the obstacles in terms of geometry (e.g., the points p, q, r, s mentioned in Figures 7A - 7G, the side ends of the square pillars, etc.), and the "connections" are the shortest connections between these points along the direction of the flow.

[0043] In some embodiments, the pattern of the material, once visualized, has a static, i.e., non - dynamic appearance. For example, DASH materials are generated in a wide variety of patterns, from one - dimensional lines having, for example, periodic patterns to two - dimensional arbitrary shapes (Figures 1D - K and Figure 27).

[0044] In some embodiments, the pattern of the material is dynamic (i.e., moving).

[0045] Materials showing emergent locomotion behavior in some embodiments. As described, the inventors demonstrated the generation of DASH materials showing emergent locomotion behavior. The overall behavior of the material is described by using a finite-state automaton (“FSA”) having three states of initiation, growth, and decay with autonomous and periodic state transitions (Figure 3B). This abstract concept with discontinuous states and state transitions enables the interpretation of the overall behavior of the material as a machine, similar to the way it is used in mechanical robots, and thus enables further programming of the behavior. In particular, the state transition between growth and decay was switched by spatio-temporal feedback. A microfluidic device having three inlet ports and one outlet port was used to illustrate this embodiment (Figure 16). A production mix was injected into the device through the middle inlet port, while a degradation mix was injected into the device through the other two outer inlet ports. Initially, all three solutions flowing into the device remained layered (“initiation” state). Thus, the degradation mix remained separated from the production mix, and the process of assimilation started in the center of the device (“growth” state). Gradually, the assembly by the redistributed DNA network began to fill the gaps between the pillars, substantially changing the flow dynamics. This spatio-temporal feedback made it possible to mix both the generation and degeneration solutions, thus inducing a state transition. Next, the process of dissimilation began to dominate, and finally the material was decomposed (“decay” state). Experimentally, an inlet flow path containing the generation and degradation mixes was prepared as a predefined track with gaps between adjacent pillars adjusted from small (downstream) to large (upstream) region by region along the track (Figure 3E). Each region corresponds to each FSA (initiation, growth, and decay). The size of the gap defining the vorticity magnitude indicates the parameter (waiting time) in each unit to induce the state transition from initiation to growth. As programmed, this vorticity gradient started from the downstream region of the track, inducing a spatio-temporal delay in the transition from the initiation to the growth state.In short, the direction of movement was interpreted experimentally as the gradient of the vorticity magnitude under a constant flow velocity. The direction of movement was programmatically planned to oppose the direction of the flow in all cases. Autonomous generation starting in the downstream region and D. After the body constructed by the ASH pattern began to grow, the spatial feedback by the generated pattern also induced a transition to a decaying state starting similarly from the downstream. The transition to the decaying state also propagates to the downstream region by the flow, assuming that the dissimilation action is dominant in these regions. At the same time, the transition from the starting to the growing state continued towards the upstream region (i.e., down the gradient of the vorticity). As a result, the overall movement behavior of the body along the track against the direction of the flow appeared as programmed by a series of FSAs.

[0046] In some embodiments, the material exhibits the spontaneous competitive behavior of two competing bodies. To explain these embodiments, the inventors demonstrated the generation of DASH material with two moving bodies exhibiting spontaneous competitive behavior by programming two consecutive FSAs (FIG. 3G). Each sequence was designed similar to the example of spontaneous movement behavior; also, a simple interference was added between the two moving bodies. Specifically, the state transition signal from growth to decay can also interfere between the tracks; the faster moving body can affect the state of another track and change it to decay, thus "decelerating" the movement of the body on the other track by inducing generation. Experimentally, the design was implemented by simply reversing the type of flow (generation mix on the outer flow path and decomposition mix on the inner flow path). There is no boundary between the two tracks, so the changed flow in one track can also affect the state of the other track. From the results, the competition between the two bodies, in which track number 2 becomes the winner, is explained (FIG. 3I). As programmed, presumably once the symmetry between the two bodies is broken by the flow and the randomness of the bodies, the decaying state caused by the body leading with track number 2 affects the body of track number 1, resulting in the decomposition of the body. After the body of track number 2 reached the goal, finally the behavior ended with the complete decomposition of the body of track number 1.

[0047] Method and apparatus configuration To generate the material, a solution containing a production mix is supplied to the main chamber of the apparatus and directed to flow through the main chamber of the apparatus along the flow path (i.e., space) between the obstacles. In some embodiments, the solution containing the production mix is injected into the main chamber through the inlet port and towards the outlet port. The flow rate can be controlled by various means, for example via a pump connected to the inlet or outlet port. Once the solution is supplied to the main chamber, the simultaneous processes of polymer synthesis and assembly occur and continue autonomously.

[0048] In some embodiments, a solution containing a production mix and a solution containing a decomposition mix are both supplied to the main chamber of the apparatus and directed to flow through the main chamber. The two solutions can be supplied to the main chamber simultaneously, continuously, or in a predetermined order, enabling various patterns of generation. In some embodiments, the two solutions are injected through separate inlet ports arranged in various manners, for example through three inlet ports where the middle port is used for the production mix and the outer ports are used for the decomposition mix; or vice versa. Once the solutions are supplied to the main chamber, the simultaneous processes of polymer synthesis / decomposition and assembly occur and continue autonomously.

[0049] The generated material can be visualized by various means including, for example, the naked eye, a camera, a fluorescence microscope (where the polymer is bound to a fluorescent compound, for example), an optical microscope, or an electron microscope.

[0050] Further applications of DASH materials In a further aspect, the methodology for generating DASH materials is applied to pathogen detection. According to this aspect of the disclosure, a production mix is prepared to provide selective amplification and generation of DASH materials only if a target pathogen DNA or RNA sequence is present in the sample. .

[0051] In some embodiments, the generation mix comprises dNTPs, a first linear form of template DNA, primers, and a DNA polymerase, where the template DNA circularizes in the presence of a pathogen nucleic acid and a ligase, and in the apparatus, the circularized DNA serves as a template for DNA synthesis (e.g., via rolling circle amplification). In these embodiments, the first linear form of template DNA may be contacted with the sample to be tested and a ligase prior to being supplied to the main chamber to enable circularization of the template DNA if a target pathogen nucleic acid is present in the sample. Alternatively, the first linear form of template DNA, a ligase, and the sample are supplied to the main chamber together with the other components of the generation mix, and circularization, as well as polymer synthesis and assembly, occur in the main chamber. In some embodiments, the generation mix may comprise template DNA, dNTPs, and a DNA polymerase without primers necessary to initiate DNA synthesis; if a target pathogen DNA is present in the sample, it serves as a primer necessary to initiate DNA synthesis when combined with the generation mix. In some embodiments, the generation mix may comprise primers, dNTPs, and an RNA-dependent DNA polymerase (or reverse transcriptase) without a template necessary to initiate DNA synthesis; if a target pathogen RNA is present in the sample, it serves as a template necessary to initiate DNA synthesis when combined with the generation mix.

[0052] A solution comprising the generation mix and the sample is injected into the main chamber of the apparatus described above herein, and DASH material is formed only if a target pathogen DNA or RNA is present in the sample. A control test may be performed in parallel in which a solution comprising the generation mix and the target pathogen DNA or RNA is supplied to the apparatus.

[0053] Using the present aspect of the disclosure, it is possible to detect DNA or RNA of any pathogen, including, for example, bacteria, fungi, or viruses. Suitable samples for use include any sample suspected of containing a pathogen, including environmental samples (e.g., soil, water), agricultural or food products (e.g., fruits, vegetables, and poultry), and samples obtained from human or non-human animals (e.g., oral or nasal swab samples, blood samples, urine or fecal samples, etc.). The sample can be a processed sample, for example, by subjecting the original sample to centrifugation, cell lysis, fractionation, or any other procedure that can facilitate the release, purification, and / or concentration of the target pathogen DNA or RNA before injection into the device.

[0054] To illustrate the present aspect of the disclosure, the inventors selected a target sequence taken from the Cucumber Mosaic Virus (CMV) as a model pathogen and demonstrated the success of target detection at concentrations of 500 and 50 pM by recognizing the self-generated DASH pattern (Figures 4A and 19 - 21). In contrast, a negative control target sequence having a mismatch of only 2 base pairs did not generate a pattern, demonstrating the specificity of the detection method.

[0055] In yet another aspect, the material generated is used as a scaffold for generating further functional materials. For example, the DASH material can act as a versatile mesoscale scaffold for generating a diverse range of functional nanomaterials beyond DNA.

[0056] In some embodiments, the DASH material once formed is contacted with a reagent that binds to DNA. The reagent can be injected into the main chamber of the device in which the DASH material is formed and can be in the range of materials including inorganic nanoparticles such as avidin, quantum dots, and gold nanoparticles. The DASH material bound to any of these reagents can be further functionalized. For example, the DASH material bound to avidin can be contacted with a biotin-conjugated enzyme (e.g., horseradish peroxidase).

[0057] In some embodiments, DNA molecules within the DASH material are used to generate proteins encoded by the DNA molecules in a cell-free manner and in a spatiotemporally controlled manner. This can be achieved by supplying a solution containing a cell-free protein expression system to an apparatus in which the DASH material is formed. Cell-free protein expression systems are known in the art and are also commercially available. In some embodiments, the cell-free protein expression system includes a lysate containing the components necessary for protein synthesis. In some embodiments, the components necessary for protein synthesis include tRNA, ribosomes, amino acids, initiation, elongation, and termination factors. In some embodiments, the cell-free protein expression system includes an E. coli lysate. In some embodiments, the cell-free protein expression system includes a wheat germ lysate. In some embodiments, the cell-free protein expression system includes a rabbit reticulocyte lysate. In some embodiments, the cell-free protein expression system includes a HeLa-based lysate.

[0058] In some embodiments, the cell-free protein expression system also includes primers designed to bind to a promoter in the DNA of the DASH material and activate it, thereby initiating transcription of the gene encoding the desired protein and subsequent protein production.

[0059] The specific examples listed below are merely examples and are in no way limiting.

Example

[0060] The DASH assimilation pathway consists of two important simultaneous and autonomous processes that represent the concept of artificial metabolism: 1) the biochemical synthesis of DNA molecules as precursors of materials, achieved by in situ enzymatic reactions, and 2) the dissipative assembly of precursors to form materials with pre-coded patterns and shapes by flow. Specifically, in the process of precursor synthesis, in situ DNA synthesis was achieved by rolling circle amplification (RCA) using Phi29 DNA polymerase in a reaction mix that also included seeds (DNA templates with primers) and building blocks (Figure 1B and Figure 5). During the process of dissipative assembly, flow was used in a microfluidic device to directly assemble specific patterns from precursor DNA. Specifically, the reaction mix was continuously injected into a microfluidic device with precisely spaced obstacles to assemble the precursor DNA into specific pre-coded patterns (Figure 1C, Figure 6, and Figure 8). DASH thus achieved the aforementioned assimilation pathway by autonomously generating materials with structural hierarchies that span scales: starting from nanoscale building blocks through all simultaneous processes, to polymer precursors, micron-scale networks (hydrogels), and finally mesoscale patterns and shapes.

[0061] Through experiments, a variety of mesoscale patterns and shapes were generated, from one-dimensional lines with periodic patterns to arbitrary two-dimensional shapes, and the material assimilation pathway was demonstrated (Figs. 1D - K and 27A - 27H). The pathway enabled the autonomous generation of patterned materials by organizing one-dimensional, micron-thick fibrous DNA networks. Using computational fluid dynamics (CFD) simulations, it was found that these DASH patterns could be deterministically designed based on two simple guidelines: the pattern takes the shortest path within the flow channel and is predicted by connecting adjacent pillars (obstacles), both of which follow the direction of the flow. To simplify the process, a microfluidic device was designed by simple combinatorial rules using seven types of structural units (Figs. 7A - 7G). The combination of units codes the positions of the pillars along the flow path in the device to satisfy both guidelines, enabling a general design strategy for DASH patterns.

[0062] The detailed morphology of the generated material was revealed by confocal fluorescence microscopy observation and scanning electron microscopy (SEM) observation. Confocal microscopy observation of the two-dimensional network pattern showed that the material was formed in the middle of the microfluidic chamber (away from the upper and lower ends of the chamber) with a fibrous morphology (Figs. 2A and 2B). SEM observation revealed a more detailed morphology (Figs. 2C and 2D): the fiber structure was composed of bundles of anisotropic DNA networks with the orientation consistent with the direction of the flow. Here, a device with a one-dimensional line pattern was selected because the transition was easier for observation. Most of the material was localized at the side edges of the pillars parallel to the direction of the flow and the spaces between them, and the distinguishable DNA only covered a little around the pillars. Also, SEM observation revealed spherical structures with an average diameter of about 0.3 μm embedded inside the network, similar to the previous report on physically entangled DNA hydrogels (J.B. Lee et al., Nature Nanotechnology. 7, 816 - 820 (2012)). However, the anisotropic network was obvious here between the spherical structures but was not obvious in the DNA hydrogel, probably due to the directed flow.

[0063] To better understand the mechanism behind pattern generation by DASH, time-lapse videos were recorded and quantified (Fig. 2E). The existence of the elapsed time between the start of the flow and the start of pattern generation indicated the minimum molecular weight of the synthesized precursor DNA (e.g., for the two-dimensional network pattern using a 5 nM production mix, 3.3×10 7Network formation was suggested to depend on the (putative molecular weight). Interestingly, we observed that network formation started from the side edges of the pillars in the middle of the chamber (i.e., the center of the z-axis), and then there was further generation and these DNA networks began to connect into one continuous fibrous structure between the pillars. If the dominant mechanism were to cover around the pillars with DNA networks, the fibrous morphology should have started from the upstream end instead of the side edges of the pillars, and the overall pattern generation should have started from the upstream region of the device. From the observations disclosed herein, it was shown otherwise; the side edges of the pillars were the main locations where the aggregation started. Thus, the inventors hypothesized that the mechanism of the DASH pattern aggregation is a combination of two processes: the formation of DNA networks induced at the side edges of the pillars and the redistribution (both in situ and in the flowing solution) of the pre-formed networks into a continuous fibrous anisotropic structure along the direction of the flow. From the time-lapse images, it was explained that the thickness of the structure increased at a later stage and finally the gap between the pillars was filled with the DNA network. This further thickening strongly suggested that the redistribution of the excess DNA networks formed in the solution occurred not at an earlier time point but at a later time point in the process of pattern formation.

[0064] To investigate the mechanisms underlying the assembly process, the inventors first performed CFD simulations and then confirmed them experimentally in two aspects: the formation of new networks and the redistribution of pre-formed networks into fibrous forms (Figs. 2F - 2H). Due to geometric simplicity, a DASH pattern with a one-dimensional line was selected for comparison. For formation, it was found that the side of the pillar coincides with the region of high vorticity (Fig. 2H). Together with sensitivity measurements (Figs. 10A - 10C, Figs. 11A - 11C, and Fig. 12) and comparative experiments on the shape of the pillar (Figs. 13A - 13B, Figs. 14A - 14B, and Fig. 15), these results suggest that flow, particularly vorticity, is important in the formation process by locally and dynamically inducing the physical entanglement of DNA into the network on the side of the pillar. In short, greater vorticity on the side of the pillar leads to an earlier onset time of generation. Similar vorticity-induced structure formation observed in biofilms and proteins also supported this hypothesis. For redistribution, it was clearly shown from the overlay of the time-lapse video and the simulation of the flow velocity that a fibrous structure was actually formed along the direction of the flow in the region of the highest velocity, which was consistent with the mechanism of redistribution (Figs. 2F - 2G). Thus Therefore, the most likely mechanism behind assembly is a combination of the dynamic formation of vortex excitation of new networks and the flow-induced redistribution of pre-formed networks.

[0065] The metabolic pathway of artificial metabolism was further extended by integrating the above anabolic generation process with the catabolic degradation process via DNA hydrolase. First, both anabolic and catabolic pathways were used to induce continuous generation and degradation of patterns at a static position. Here, the DASH pattern was autonomously generated by a combination of enzymatic reactions and flows and then decomposed synchronously and autonomously (Figs. 3A–3C). Reagents required for both generation and degradation were simultaneously introduced into the microfluidic device. Importantly, once the flow was initiated, both the generation and degradation processes were carried out without any external operation. The microfluidic device with three inlets was used with a central inlet containing a generation mix with DNA polymerase, while the inlets on either side contained a degradation mix with DNA hydrolase, DNase I. The overall behavior of the material was described by using a finite state automaton (FSA) with three states: initiation, growth, and decay, with autonomous and periodic state transitions (Fig. 3B). This abstract concept with discontinuous states and state transitions enabled the interpretation of the overall behavior of the material as a machine, similar to the method used in mechanical robots, and enabled further programming of the behavior described below. In particular, the state transition between growth and decay was switched by spatio-temporal feedback. Hydrodynamics during the process was explained from CFD simulations (Figs. 16A–16D). First, all three solutions flowing into the device remained laminar (initiation). Thus, the degradation mix remained separated from the generation mix, and the anabolic process started in the center of the device (growth). Gradually, aggregation by the redistributed DNA network began to fill the gaps between the pillars, substantially changing the flow dynamics. This spatio-temporal feedback made it possible to mix both the generation and degradation solutions and thus induce a state transition. Next, the catabolic process began to dominate, and finally the material was decomposed (decay).From further experimental tests, it has been shown that the continuous generation and decomposition (periodic regeneration) can be autonomously repeated at least twice when the DNA synthesis time is constant (Figure 17), and it has been demonstrated that both the anabolic and catabolic pathways can be seamlessly integrated and regulated in a regenerative manner without any external interference.

[0066] Based on the dynamic generation and decomposition behavior of materials in a static position, the movement behavior driven by artificial metabolism using DASH was programmed (Figures 3D - 3F). Inspired by the shape and movement behavior of the pseudomorph (slug) of the cellular slime mold Dictyostelium discoideum (J.T. Bonner, American Journal of Botany. 31, p. 175 (1944)), the behavior was programmed such that a slug-like body was first generated by the autonomous growth of the DASH pattern, followed by the autonomous movement of the body along the track against a certain flow. The movement was realized as an emergent behavior based on continuous and polarized regeneration: the front end generates the body, and the rear end decomposes itself. At an abstract design level, the behavior was programmed by extending the FSA introduced above in a continuously connected manner (M1 - M6), and each FSA was regarded as an autonomous module unit (Figure 3D). Each unit (M n ) can receive a "changed flow" signal from an adjacent unit (M n+1 ) that induces a state transition from growth to decay, and can also propagate the signal to the next (M n-1 ). The movement behavior was programmed by setting different waiting times (t1 < t2 <... < t6) until a state transition between start and growth was induced. The growth of each FSA starts from M1 according to the waiting time. Once the unit M n changes its state to decay by its internal feedback, the signal of the changed flow is sent to the adjacent unit M n-1and propagates to the next one, ensuring the state transition at the rear end of the body. As a result, the direction of movement is represented as a spatio-temporal delay in the transition to growth and subsequent decay. Experimentally, a flow path with a plurality of similar inlets including generation and decomposition mixes as pre-determined tracks for behavior was prepared (Fig. 3E). Here, the gap between adjacent pillars was , adjusted to be small (downstream) to large (upstream) by region along the track. Each region corresponds to each FSA. The size of the gap defining the vorticity magnitude indicates the parameter (waiting time) in each unit so as to induce the state transition from start to growth. As programmed, this vorticity gradient started from the downstream region of the track and induced a spatio-temporal delay in the transition from start to the growth state. In short, the direction of movement was interpreted experimentally as the gradient of the vorticity magnitude under a constant flow velocity. The inventors also emphasize here that the direction of movement was deliberately programmed to oppose the direction of flow in all examples. After the self-generated body constructed by the autonomous generation and DASH pattern starting in the downstream region began to grow, the spatial feedback by the generated pattern also induced the transition to the decay state starting from the downstream. The transition to the decay state was also propagated to the downstream region by the flow (represented as the signal of the "changed flow" sent to M n-1 ). At the same time, the transition from start to the growth state continued towards the upstream region (i.e., down the vorticity gradient). As a result, the overall movement behavior of the body along the track against the direction of flow appeared as programmed by a series of FSAs. The behavior was observed experimentally in both straight (wide and narrow widths) and curved tracks, thereby explaining the design flexibility of the trajectory. The moving speed was measured as 2.3 mm / hour in the narrow track (Fig. 3F).

[0067] To further demonstrate the application of materials as machines, the design was extended by harnessing the power of abstract programming methods, and the emergent competitive behavior of two competing bodies was achieved by two consecutive FSAs (Figure 3G). Similar to the previous emergent movement behavior, each consecutive (M 11 ~M 61 、M 12 ~M 62 ) was designed; also, here, a simple interference was further added between the two moving bodies. Specifically, the state transition signal from growth to decay can also interfere between the tracks (indicated by the arrows between the two consecutive FSAs); the faster moving body can affect the state of the other track and change it to decay, thus inducing decomposition to "decelerate" the movement of the body on the other track. This program can be interpreted as two tracks representing two consecutive FSAs arranged side by side without any physical boundaries between them (Figure 3H). Experimentally, in the wide tracks introduced in the previous section, the design was implemented by simply reversing the type of flow (outer generation mix and inner decomposition mix). Since there is no boundary between the two tracks, the changed flow in one track can also affect the state of the other track. The results showed a successful competition between the two bodies where track number 2 became the winner (Figure 3I). As programmed, probably due to the flow and body randomness, once the symmetry between the two bodies is broken, the decay state caused by the body led by track number 2 affects the body on track number 1, resulting in the decomposition of the body (see snapshot at 127.5 minutes). After the body on track number 2 reached the goal (135 minutes), finally the behavior ended with the complete decomposition of the body on track number 1 (180 minutes).

[0068] Finally, in addition to the use of DASH materials in mechanical applications, several other applications were developed. One of the applications was nucleic acid detection (Figure 18). The goal of this application was to demonstrate the advantages of the self-generating properties of the material. Generating species that could be amplified only when the target pathogen DNA / RNA sequences were present in the sample were prepared. The assimilation properties of the material were thus converted to act as a selective amplification process only for the targeted DNA / RNA. The generated DASH patterns were then read by naked-eye observation or by a Fourier transform-based pattern recognition algorithm, and a mechanism was adopted as a binary readout method (Figure 9). Experimentally, target sequences collected from cucumber mosaic virus (CMV) were selected as model pathogens. By recognizing the self-generated DASH patterns, the targets were successfully detected at concentrations of 500 and 50 pM (Figure 4A, Figures 19A - 19B, Figures 20A - 20B, and Figure 21). Control targets with a mismatch of only 2 base pairs did not generate a pattern, demonstrating the specificity of the detection method. Then, the self-generated material To demonstrate the potential use of the material, various hybrid functional materials were created from the DASH patterns. The DASH material served as a versatile mesoscale scaffold for a wide range of functional nanomaterials beyond DNA, ranging from proteins to inorganic nanoparticles, such as avidin (Figure 4B, Figures 22A - 22B, Figures 23A - 23B), quantum dots (Figure 4C, Figures 24A - 24D), and DNA-conjugated metal nanoparticles (Figure 4D, Figure 25). The generated patterns also became functional by catalytic activity when bound to enzymes. It was shown that the DNA molecules within the DASH pattern retained the genetic properties of DNA and that the material itself successfully generated green fluorescent protein (GFP) cell-free by incorporating a reporter gene for sfGFP (Figure 4E, Figures 26A - 26B). The protein-generating ability of the material established the basis for future cell-free generation of proteins, including enzymes, that are spatiotemporally controlled.

[0069] In conclusion, the present disclosure is directed to dynamic materials driven by artificial metabolism using simultaneous processes of biochemical synthesis and dissipative assembly. Through the implementation of the DASH concept, various applications of the materials have been successfully demonstrated. In particular, the inventors have succeeded in constructing machines from this new dynamic biomaterial with emergent regenerative, locomotive, and competitive behaviors by programming it as a series of FSAs. The bottom-up design based on the fundamentals of life-unconstrained bioengineering has enabled these active and programmable behaviors in a fundamental way. This material can be integrated as a mobility element in biomolecular machines and robots. The DASH pattern can be easily recognized by the naked eye or a smartphone, leading to better detection techniques that are more feasible in point-of-care settings. DASH can also be used as a template for other materials to create, for example, dynamic waves of protein expression or nanoparticle assemblies.

Examples

[0070] Materials and Methods Materials RepliPHI (trademark) Phi29 DNA polymerase, 10x RepliPHI (trademark) buffer (400 mM Tris-HCl (pH 7.5), 500 mM KCl, 100 mM MgCl2, 50 mM (NH4)2SO4, and 40 mM DTT), and deoxynucleotides (dNTPs) were obtained from Epicentre (Madison, WI). T4 DNA ligase, exonuclease I, and exonuclease III were obtained from New England Biolabs (Ipswich, MA). Adenosine triphosphate (ATP) was obtained from Teknova (Hollister, CA). Oligonucleotides were chemically synthesized and purified using standard desalting methods by Integrated DNA Technologies (IDT) (Coralville, IA). GelRed (trademark) nucleic acid gel stain and nuclease-free water were obtained from VWR (Radnor, PA). SYBR Green I, 40% acrylamide / Bis (19:1), ammonium persulfate (APS), and polydimethylsiloxane (PDMS) silicone elastomer kit (Sylgard 184, Dow Corning) were obtained from Thermo Fisher Scientific (Waltham, MA). Tetramethylethylene diamine (TEMED) was obtained from Sigma-Aldrich (St. Louis, MO).

[0071] Generation mix preparation The seeds of generation were prepared by circularizing the template DNA together with the primer DNA (Figure 5). First, the chemically synthesized template and primer DNA were mixed at an equimolar concentration of 1 μM final in the final 1x RepliPHI reaction buffer, and then thermally sa Annealed from 95 °C to 4 °C by Ikler (-1 °C / min). Added 200 units of T4 DNA ligase and ATP (final 1.25 mM), and then incubated overnight at 4 °C for the reaction (total 20 μL scale, final seed concentration 0.5 μM). The resulting seed solution ligated at a final concentration of 5 nM (or as otherwise described) was then mixed on ice, for the resulting mix, in final 1x RepliPHI reaction buffer, with dNTPs at final 1 mM each, SYBR Green I at final 1x concentration, and Phi29 at 5.7 units / μL.

[0072] Microfluidic device design The device was designed by following three steps. First, the layout of the final DASH pattern was roughly determined. Next, obstacles were assigned by following a pattern using an abstract method based on the connection diagram. A total of seven types of standard structural units were used for the design. Finally, the main chamber design was connected to the inlet / outlet flow channels.

[0073] All devices were designed by LayoutEditor (Juspertor GmbH, Germany) and KLayout (Klayout website) and exported to the GDSII format. Except for the initial tests at Cornell NanoScale Science and Technology Facility (CNF) (Ithaca, NY), Chrome photomask processing was performed by an external vendor (Suzhou Mask-Fab Corp., China). At CNF, a Heidelberg DWL2000 was used for mask writing; a Hamatech-Steag Mask Processor for development and post-processing.

[0074] The glass wafers (4-inch diameter) were washed with water and then immersed in acetone with ultrasonic treatment for 5 minutes. Then, they were transferred to isopropanol with ultrasonic treatment for an additional 5 minutes. After that, the wafers were washed with deionized water and dried in a clean air stream. All glass wafers were pretreated with hexamethyldisilazane before photoresist coating. AZ P4620 photoresist (MicroChemicals GmbH, Germany) was dipped in the center of the wafer and spun on a spin coater at 1000 r.p.m. for 2 minutes to achieve a thickness of approximately 16 μm. Then, the wafer was baked on a hot plate at 95 °C for 8 minutes and gradually cooled to room temperature. The coated wafer was exposed to UV light for 30 seconds on a MA / BA6 mask / bonder aligner (SUSS MicroTec, Germany) using a quartz mask and then placed in a developer solution composed of az 400K and deionized water in a ratio of 1:3 for 2 minutes. The developed wafer was rinsed with deionized water and dried by an air stream. Finally, the wafer was baked on a hot plate at 100 °C for 30 minutes to improve photoresist adhesion. The glass wafers were placed in Petri dishes (Greiner Bio-One, Austria) and fixed by taping the four sides at the edges for the molding process. The microfluidic devices were molded with polydimethylsiloxane (PDMS) silicone elastomer at a 10:1 Base Curing Agent ratio (Sylgard 184, Dow Corning, Corning, NY). After baking at 70 °C for 1 hour, the individual devices were cut out of the Petri dishes and then the inlet and outlet ports were punched out. Finally, the devices were covalently bonded to PDMS-coated microscope slides (VWR, Radnor, PA) via oxygen plasma treatment.

[0075] Device design process Based on the experimental results and CFD simulations, two empirical guidelines were found: the patterns were formed by 1) following the flow direction inside the device and 2) taking the shortest path inside the flow channels that connect between the pillars. Based on these guidelines, the devices were designed according to the following deterministic method.

[0076] Device layout First, the overall size of the device including the flow path between the inlet / outlet and the main chamber was designed. In this document, the length of the flow path was set to avoid interference with the objective lens of a fluorescence microscope (BX51, Olympus, Japan) when connected to the piping; based on the image size of the microscope, a typical main chamber length (2 mm) was set. The flow path width between the main chamber and the inlet / outlet was fixed at 50 μm; the typical main chamber width (excluding complex geometric shapes such as the letters "D, N, A" and the "double helix" figure, the device with three chambers for vorticity control experiments, and the narrow straight track for movement) was set at 500 μm throughout the design for consistency (Figure 6). The overall size of the device was limited by the size of the glass wafer (7 cm square) to include additional margin for cutting out individual devices and to seal the device during the processing process.

[0077] Main chamber design The layout of the main chamber was designed by following three steps. First, the layout of the final DASH pattern was roughly determined. Next, obstacles were assigned by following the lines drawn in the first step. Finally, the obstacles were merged with the flow path and main chamber design.

[0078] Barriers were designed based on combinations of boundaries and / or pillars. A total of seven types of standard structural elements were used in the design (Figs. 7A - 7G). The structural elements were classified into three classes such as "linear", "divided", and "merged" according to the morphological characteristics of the abstract patterns of the connection diagrams. The connection represents the final redistribution form of the DASH structure, and the node represents the point where the DASH structure was generated. The basic geometric shape was based on a solid boundary with triangular barriers (Figs. 7A, 7B, 7F, and 7G). The solid boundary (the gray area in Fig. 7) defines the overall laminar flow direction in the device (the blue line in Fig. 7). Since the DASH structure takes the shortest straight-line path between the vertices (points p, q) of the barrier, a straight line connecting the two points (the green line in Fig. 7) was generated. Typically, due to limitations in the processing process, the flow channel width was designed to be wider than 20 μm. In the case of "divided" and "merged" layouts, the flow direction defines the overall design of the side channels. In order for the flow to redistribute along with the generated DASH structure in the flow direction, the inner corners (points r, u) of the curvature of the branch must always be acute angles so that the corners become the points of generation and fixation (i.e., the DASH structure merges / divides at the exact position). The angle between adjacent generation points (between r - s, t - u) defines the angle of the generated branch structure. Also, rectangular barriers can also be used instead of triangular barriers (Fig. 7B). Furthermore, in addition to physical boundaries, this strategy can be extended to "virtual" boundaries by utilizing the symmetry of the laminar flow inside the device (Figs. 7C, 7D, and 7E). Once a line-symmetric pillar structure (blue dashed line) is designed, the laminar flow also becomes axisymmetric (CFD simulation is required to confirm the symmetric flow); as a result, the boundary structure can be basically eliminated, and the design of the barriers can be greatly simplified by the pillars. In this document, three types of elements with virtual boundaries were used: a zero lateral distance (c) between the pillars, a positive distance (+x) (d), and a negative distance (-x), (e). The positive distance defines the maximum width of the DASH structure (Fig. 1D); the zero (Fig. 1E) and negative (Fig. 1F) distances enable the generation of DASH with a minimum width.Note that in the case of an axisymmetric design such as "Character D", the design process can be reduced by simply replicating most of the upper half of the geometric shape to the lower half.

[0079] Finally, the obstacles were incorporated into the flow channel and main chamber designs. The entire process was repeated to improve the pattern by examining the actual DASH pattern formation or CFD simulation results. After repetition, the final optimized design was determined and tested in an actual DASH generation experiment.

[0080] Device Design Catalog Using the described method, various types of DASH devices and tracks were designed for pattern generation (Figs. 27 and 28 - 42). In this document, a total of 15 types of designs were used. The catalog summarizes the pillar / obstacle designs, the overall geometric shape of the device / track, and the configuration.

[0081] Measurement of the Cross - Sectional Height of the DASH Device The height of the chamber was confirmed by sampling an actual PDMS device cut by a blade. A total of 42 fragments were measured for analysis. From the results, an average height of 17.4 μm with a standard deviation of 1.1 μm was shown, which is within a reasonable range (approximately 8% difference) compared to the ideal thickness of 16 μm.

[0082] Experimental Configuration of the Device Simultaneous synthesis and assembly using microflows were realized by a combination of microfluidic devices connected to tubing and syringes (Fig. 8). The prepared generation mix solution was introduced into a 1 mL BD Medical Tuberculin Syringe (Franklin Lakes, NJ) and Cole - Parmer Microbore Puri - Flex Autoanalysis Tubing (Vernon Hills, IL) connected with a short Microgroup subcutaneous catheter (Medway, MA) as the insertion tip. Immediately after preparing the solution, the syringe was then placed on a Harvard Puri - Flex Autoanalysis Tubing (Vernon Hills, IL). Attached to an Apparatus PHD-2000 syringe pump (Holliston, MA) and injected. Before the experiment, the DASH device was pre-filled with nuclease-free water; both the inlet and the outlet were also covered with water. Once the generated mix appears at the tip, then immediately insert the tip into the DASH device. Both the device and the tip were covered with solution to ensure that no air bubbles entered the device during the process. Typically, the generated mix was injected into the DASH device at 0.1 μL / min.

[0083] For the generation-degradation experiment, a design with three inlets (Figure 36, #14-2) was designed. The central inlet was connected to the generation solution (final species concentration 0.1 nM). The lateral inlets were connected to the degradation solution (DNase I (1 unit / μL) in the final 1x Phi29 reaction buffer). Both the generation and degradation solutions were injected at 0.1 μL / min. For the creative movement experiment, tracks with two and three inlets having gradient vorticity regions (Figures 40, 41, 42, #22-3, 23-3, 23-4) were used. Both solutions were injected at 0.15 μL / min. For the creative competition experiment, a track with three inlets having a gradient vorticity region (Figure 41, #23-3) was used. Both solutions were injected at 0.15 μL / min.

[0084] Fluorescence microscopy Fluorescent microscopy images used for morphological studies and quantitative analysis were taken with an Olympus BX-61 microscope (Japan) equipped with a Sutter Instrument Lambda LS Xenon light source (Novato, CA). Green fluorescence (excitation light 484 nm, emission 520 nm), red fluorescence (excitation light 555 nm, emission 605 nm), and red quantum dot (excitation light 420 nm, emission 605 nm) filters were purchased from Chroma Technology Corporation (Bellows Falls, VT). 4x and 10x objective lenses from Olympus Corporation (Tokyo, Japan) were used. The exposure time of the bright-field channel was set to 100 milliseconds; throughout all experiments, the fluorescence channels were set to 2000 milliseconds. Time-lapse movies were taken with a 4x objective lens using 150 seconds / frame (except for the short observation interval movie (15 seconds / frame) in Supplementary Movie S6). Images containing raw data were captured by Intelligent Imaging Innovations SlideBook (D enver, CO). The raw data (16-bit tiff files) were imported and processed by software within the tissue for detailed observations.

[0085] Confocal laser scanning microscopy (CLSM) images and z-stack movies were taken with two confocal laser scanning microscopes (ZEISS LSM710 (Germany), Olympus IX-81 (Japan)). For time-lapse recording (Supplementary Movie S2), device #9-1 (Figure 30) was selected along with the final 5 nM production mix. Depending on the focal length, a 10x objective lens was selected for observation. For the green fluorescence channel, a filter with excitation light 488 nm and emission 520 nm was used. The capture interval was set to 110 seconds; a total of 30 frames were recorded. 30 layers (z-axis) were taken for each stack.

[0086] SEM After the generation of the DASH pattern, a 4% paraformaldehyde fixative solution (Electron Microscopy Science, Hatfield, PA) was flowed into the device at 0.1 μL / min for 10 minutes. After fixation at 4 °C for 24 hours, the device was opened and the pattern was fixed on the PDMS substrate. When rinsing with nuclease-free water, the pattern was dehydrated by immersion in a series of stepwise ethanol (10%, 25%, 50%, 75%, 90% and 100%) and then in 100% ethanol. Subsequently, the pattern was dried by a critical point drying process using Baltec (Leica) CPD 408 (Germany), and then investigated by LEO (Zeiss) 1550 FESEM (Germany).

[0087] CFD Simulation A two-dimensional CAD file was exported from the original CAD design (GDSII) to the DXF format, then imported into Rhinoceros 3D (Robert McNeel & Associates, Seattle, WA) and simulated using Autodesk Simulation CFD (San Rafael, CA). Inside Rhinoceros 3D, the original two-dimensional CAD file was shaped into a three-dimensional volume with a height corresponding to the actual DASH device. Then, the model was exported as a STEP file for import into the creation software within the CFD software. For the fluid flowing through the geometry, an initial set water profile was used for simplification. For the solid structure, the same properties as the existing initial set material of Silicone Rubber were applied. Then, the simulation was run for up to 500 iterations or until the results converged (automatically detected and stopped by the Autodesk CFD software). Three methods were used for visualization of the simulation results: heat map, vector field, and particle trace. The heat map and vector field were normalized between all results to maintain uniformity and placed 8 μm from the bottom of the volume (midpoint).

[0088] CFD Simulation (Detailed Protocol) Introduction To find small-scale trends in the flow through various geometric patterns, a simple computational fluid dynamics (CFD) model was constructed and executed. The aim of this simulation was to establish a simple pipeline (along with many generalizations) to estimate the flow behavior inside the DASH device. This information was then used for the design of the new DASH device and as an aid for the estimation of the generation mechanism. Note that the scope of this simulation is at the microscale; details such as the flow and the nanoscale behavior of the polymer including entanglement and network formation, which are not considered in this simulation. Further estimations were not considered in this simulation.

[0089] Preparation of Geometric Shapes The 2D CAD file was exported from the original CAD design (GDSII) to the DXF format and then imported into Rhinoceros 3D (Robert McNeel & Associates, Seattle, WA) and simulated using Autodesk Simulation CFD (San Rafael, CA). Inside Rhinoceros 3D, the original 2D CAD file was shaped into a 3D volume with a height corresponding to the actual DASH device. Some simplifications were made in this 3D model compared to the actual physical device, including corners that were physically slightly rounded by the machining process but remained square as designed in the model. Similarly, any rounding at the "columnar walls" in the actual physical DASH device caused by the device machining process was designed as straight walls. The inlet / outlet flow paths of the model were extended to a lesser extent as in the case of the physical device. These extensions of the flow paths provide very little change in the flow behavior and only increase the number of meshes and thus the computational time for each iteration.

[0090] File Transfer and Configuration Next, the Rhino model was exported as a STEP file for import into the creation software within the CFD software. Materials were applied within the CFD. For the fluid flowing through the geometry, an initial water profile was used for simplification. For the solid structure, the following properties similar to the existing initial material of Silicone Rubber were applied. These materials do not have exactly the same material properties as the experiment, but these were acceptable approximations for the current purposes.

[0091] Simulation Next, the simulation was run for up to 500 iterations or until the results converged (automatically detected and stopped by the Autodesk CFD software). Three methods were used for visualization of the simulation results: heat map, vector field, and particle trace. The heat map and vector field were normalized between all results to maintain uniformity and placed at 8 μm from the bottom of the volume (midpoint). Particle traces were performed using particles with a radius of 13.8 μm and a density of 1.34 g / cm 3 ³. These particles were seeded on the inlet face of the geometry.

[0092] DASH Data Import and Analysis Software Using MATLAB (Natick, MA), DASH data analysis software based on the discrete Fourier transform was developed (Figure 9). The software uses raw intensity images or videos with multiple channels captured by fluorescence microscopy (fluorescent channels including DASH patterns, brightfield channels including the overall device overview) as inputs, and quantitatively converts and analyzes the "intensity" of the DASH patterns appearing in the images by fast Fourier transform (FFT). The software was mainly used for quantitative measurement of "binary" detection of DASH patterns for pathogen detection (known as "naked eye" detection to distinguish the presence / absence of patterns), but it should be noted that the overall process can be easily applied as a general quantitative analysis method for DASH patterns with one-dimensional lines or periodic two-dimensional patterns regardless of the staining method, type of generation mix, and spatial frequency.

[0093] Here, the overall process used in this document is briefly described. First, the raw images (videos) were imported and preprocessed. Since the device was recorded at random positions at random angles in the original raw images, in addition to subtracting the background value in the fluorescent channel, the rotation and position of the device were corrected, and then the images were cropped. Thus, the first step is to normalize the data. The background intensity was subtracted from the average intensity of 100 pixels in the image (selected from the area inside the chamber not including the DASH pattern). The process was repeated for all frames in the video. In some cases, due to the elastic properties of the PDMS-based device, the device itself moved slowly during the observation. In these cases, an additional image stabilization process was applied before preprocessing to ensure consistency throughout all frames. Throughout the document, the imported images and videos were used.

[0094] ​Next, FFT was applied to the image for each frame. In the case of CMV pathogen detection, since a zigzag geometry with a mesh pattern (Figure 30, #9-1) was used in the experiment, two-dimensional FFT was selected as the conversion method. A square region (310 px × 310 px, corresponding to 500 μm × 500 μm of the original size) was selected from the sample image and converted to the frequency domain. The same position was selected throughout all the frames in the video. (In the case of one-dimensional FFT, each slice of a column (1 px × 310 px) was converted one by one.) After the conversion, the spatial frequency peaks that match the DASH pattern were selected. In the case of the two-dimensional zigzag geometry, the fundamental frequency of f = 10 (Hz) with the corresponding angle was selected. This process can be interpreted as the "reverse" of a typical notch filtering process. Usually, a notch filter removes specific peaks in the frequency domain image to eliminate periodic noise in the spectrum. However, in the case of this DASH pattern, these periodic patterns with specific spectral peaks are signals (and vice versa) instead of noise. The strength of this method is that once the DASH pattern is designed, then the spatial frequency and the angle of the pattern are deterministically defined without any arbitrary parameters for adjustment. This method can greatly simplify the overall quantitative analysis and ensure accuracy. For example, since this two-dimensional FFT method can select a specific spatial frequency with a specific angle, noise with other spectra and / or angles such as weak DNA attachment to the column (including frequencies with the same or similar but different angles), as well as random high background, can be automatically distinguished from the actual DASH pattern (signal) and counted as noise. Finally, the signal-to-noise ratio (SNR) of the DASH pattern signal in the image was calculated for each frame (the higher, the stronger the DASH pattern generation) and used as a quantitative indicator of the generated pattern (Figure 33).

[0095] Pathogen Detection Based on DASH For the target, sequences taken from cucumber mosaic virus (CMV) were used. For the non-target, a total of two base pair mismatches in the sequence change (one base pair on each side of the ligation site) were created. For the simplification of the experiment, the total target sequence length was shortened to 33 bases; instead of RNA, chemically synthesized single-stranded DNA was used. Recognition was performed by adding the target DNA to a solution containing the template and primer DNA in the final 1x RepliPHI Phi29 buffer. According to the annealing method (from 95 °C to room temperature at -1 °C / min), the final 10 units / μL of T4 DNA ligase was added together with 1.19 mM ATP, and the reaction was left at 4 °C overnight. Then, a production mix for amplification (DASH pattern generation) was prepared according to a standard method using the corresponding concentration of the ligated template-primer mixture; then, the solution was injected into the device at 0.1 μL / min for a maximum of 4 hours (Figure 30, #9-1). During the process, a time-lapse video was recorded; then, the results were imported using the software in the tissue.

[0096] DASH-avidin / streptavidin hybrid material The standard protocol using the zigzag pattern device (Figure 30, #9-1) was used for 1 hour to 1 hour and 20 minutes to generate the DASH pattern. To confirm that the DASH pattern was accurately formed, SYBR green I at a final concentration of 1x was included in the production mix. Immediately after DASH generation, a 50 μg / mL solution of either Texas Red-conjugated avidin or Texas Red-conjugated streptavidin in the 1x RepliPHI reaction buffer was flowed through the device at 0.1 μL / min for 1 hour. Then, fresh 1x RepliPHI reaction buffer was flowed through the device for 30 minutes to remove any unbound proteins before imaging.

[0097] To ensure that the DASH pattern is formed uniformly and reliably throughout the device, the DASH generating solution was simultaneously pumped through all inlets (at 0.1 μL / min each) for the two-color avidin binding. To avoid spectral overlap with FITC-conjugated avidin, SYBR Green I was not included in the generation mix. After DASH formation, 50 μg / mL Texas Red-conjugated avidin and 50 μg / mL FITC-conjugated avidin were simultaneously pumped into the device (one avidin conjugate per inlet) at 0.1 μL / min each for 1 hour. Before imaging, the device was flushed with 1x Phi29 reaction buffer for 15 - 30 minutes to reduce background.

[0098] DASH - Quantum Dot Hybrid Material Without SYBR Green I, a standard protocol was used to generate the DASH pattern over 1 hour 30 minutes. Immediately after DASH generation, a 250 μg / mL solution of FITC-conjugated avidin (Thermo Fisher Scientific, Waltham, MA) in 1x RepliPHI reaction buffer was flowed through the device at 0.1 μL / min for 1 hour, and then the device was flowed through with the final 0.2 μM biotinylated Qdot605 nanocrystals (Thermo Fisher Scientific, Waltham, MA) in 1x RepliPHI reaction buffer for 10 - 30 minutes. Control samples were tested without the FITC-conjugated avidin binding process.

[0099] DASH - AuNP Hybrid Material Citrate-coated 40 nm and 5 nm gold nanoparticles were purchased from Ted Pella (Redding, CA). The oligonucleotides used were regularly synthesized and conjugated with a 5’ thiol group from Integrated DNA Technologies and activated prior to attachment by deprotection using tris(2-carboxyethyl)phosphine hydrochloride (TCEP). The oligonucleotides were incubated at a 1:5 (DNA:TCEP) ratio. The deprotected DNA was then added to the AuNPs at a DNA:AuNP ratio of 80:1 for the 5 nm and 4200:1 for the 40 nm to ensure maximum surface coverage and then shaken overnight at 500 rpm at room temperature. NaCl was then slowly added to a final concentration of 500 mM over an 8-hour period to reduce DNA-DNA repulsion and further increase DNA coverage. The nanoparticles were then purified by five centrifugations in nuclease-free water to remove salts and excess DNA.

[0100] A DASH pattern was generated using a standard protocol with device #9-1 (Figure 30). After 70 minutes of generation, a DNA-binding 5 nm or 40 nm AuNP solution with the final 1x RepliPHI buffer was flowed into the device interior for 45 minutes (0.1 μL / min). The process was constantly monitored by microscopy to ensure sufficient nanoparticle attachment.

[0101] Cell-free protein expression DASH pattern generation was performed according to a protocol similar to the standard protocol using 8 mM mixture of dNTPs, 4 units / μL of RepliPHI Phi29 DNA polymerase, and 0.5 μg / mL of Hoechst 33342 at a final seed concentration of 15 nM in the final 1x RepliPHI buffer. SYBR Green was used to avoid overlap with the green emission wavelength of sfGFP for the subsequent protein expression step. Instead of I, the blue Hoechst dye was used to stain DNA for confirmation of DASH pattern generation. The generation process was monitored by time-lapse observation until pattern generation was completed.

[0102] After the DASH pattern generation process, protein expression primers were injected at 0.1 μL / min for 60 minutes. The primer sequences were designed to bind to the T7 promoter region present in the DASH pattern to activate protein expression. Then, for protein expression, the Promega (Madison, WI) S30 T7 High-Yield Protein Expression System was used. Nuclease-free water, S30 Premix Plus, and S30 T7 Extract (both supplied with the kit) were mixed at a ratio of 2.4:4:3.6 and injected at 0.1 μL / min. For direct observation of CFPE in the DASH device, the pump was programmed to stop every 20 minutes to increase the residence time, and the fluorescence in the microfluidic device was directly observed. For quantitative measurement (Figure 4E), the solution was collected from the CFPE in the device for a total of 2 hours, and fluorescence was measured using a BioTek (Winooski, VT) Synergy 4 Microplate Reader (with filters for excitation at 475 nm and emission at 508 nm).

[0103] Sequence The types of generation were designed by combinations of template and primer DNA. In this disclosure, throughout all generation and degradation experiments, the following sequences were used (except for some control experiments, DASH-based detection, and cell-free protein expression experiments). Primer (T1c): GACCACCTTCGCGTCCAAAGC (SEQ ID NO: 1) Template (T2-Eco): CGAAGGTGGTCTTTTTTTTTATATAGAATTCTATATATTTTTTTTGCTTTGGACG (SEQ ID NO: 2)

[0104] Note: The sequences are written in the 5’->3’ direction. The 5’ and 3’ pairs represent complementary sequences. For the ligation process, the template DNA was prepared with 5’ phosphorylation. Primer (T1c-NCTRL): CAACCAAACACCCCAACCACC (SEQ ID NO: 3) Template (T2-NCTRL): GTGTTTGGTTGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGGTGGTTGGG (SEQ ID NO: 5)

[0105] The template sequence consists of two segments of the complementary sequence to the primers (blue and red), with an additional central domain linked by a linker using a poly T sequence. The sequences were designed by the DNADesign MATLAB toolbox of the in - house version run on Eclipse (developed from the original MATLAB version by the Winfree group (Caltech Centrosome DNAdesign website)). Different sets of templates / primers with specific sequences were designed for the generation of species.

[0106] Preparation of the generation of species: Gel electrophoresis By gel electrophoresis, it was confirmed that 1x and 2x sized circular templates were successfully formed after the reaction. In this case, according to the results, due to the palindromic sequence in the template, 2x circular templates were also generated, which led to hybridization with another template and ended up with double the length. T2-NCTRL and T1c-NCTRL showed only 1x sized circular template formation after ligation.

[0107] Experimental configuration of the device Simultaneous synthesis and assembly using microfluidics were carried out with a micro -... connected to tubing and syringes Concretized by a combination of fluid devices (Figure 8). The prepared generated mix solution was connected to a 1 mL BD Medical Tuberculin Syringe (Franklin Lakes, NJ) and a Cole-Parmer Microbore with a short Microgroup subcutaneous catheter (Medway, MA) as the insertion tip. It was drawn into Puri-Flex Autoanalysis Tubing (Vernon Hills, IL). Immediately after preparing the solution, the syringe was then attached to a Harvard Apparatus PHD-2000 syringe pump (Holliston, MA) and injected. Before the experiment, the DASH device was pre-filled with nuclease-free water; both the inlet and outlet were also covered with water. Once the generated mix appears at the tip, the tip is then immediately inserted into the DASH device. Note that both the device and the tip were covered with solution to ensure that no air bubbles entered the device during the process. Typically, the generated mix was injected into the DASH device at 0.1 μL / min.

[0108] Additional records regarding the configuration of the generation-decomposition / movement / competition experiment Generation-decomposition, movement, and competition experiments For the generation-decomposition experiment, a design with three inlets (Figure 36, #14-2) was designed. The central inlet (1: red in the #14-2 catalog) was connected to the generation solution (final species concentration 0.1 nM). The lateral inlets (2: blue, split into two inlets) were connected to the decomposition solution (DNase I (1 unit / μL) in the final 1x Phi29 reaction buffer). Both the generation and decomposition solutions were injected at 0.1 μL / min. For the creative movement experiment, tracks with two and three inlets having a gradient vorticity region (Figures 40, 41, 42, #22-3, 23-3, 23-4) were used. Both solutions were injected at 0.15 μL / min. For the creative competition experiment, a track with three inlets having a gradient vorticity region (Figure 41, #23-3) was used. Both solutions were injected at 0.15 μL / min.

[0109] Repeated generation-decomposition experiments To ensure a certain synthesis reaction time, an apparatus (ID#20-2) with three inlets having additional T-junction modules was designed. An external pipe with a length corresponding to a 2-hour synthesis reaction time (under a flow rate of 0.1 μL / min) was connected between the outlet of the T-junction and inlet 1 of the apparatus with two inlets. A reaction mixture solution containing Phi29 enzyme but no dNTP was used at inlet 1-1; a solution containing no Phi29 but the final 2 mM dNTP was used at inlet 1-2. Both 1-1 and 1-2 were injected at 0.05 μL / min. The solutions were mixed in the central reservoir of the T-junction; synthesis occurred while flowing through the external pipe for 2 hours. For inlet 2, the same DNase I solution used in the generation-decomposition experiment was used (injected at 0.1 μL / min).

[0110] Transfer method for SEM observation For SEM observation of samples containing DASH-AuNP patterns, a transferable DASH device was prepared by a "peelable" PDMS device configuration using adhesive tape as the substrate. It should be noted that the overall process can be used as a general transfer technique for future applications of this DASH platform in addition to SEM sample preparation. By placing the 3M Scotch tape (Maplewood, MN) upside down on the slide glass (i.e., with the adhesive side up) as the substrate, and then gently placing the PDMS chamber device on top and sealing it. After DASH pattern generation and gold nanoparticle attachment according to the typical method described above, the device was immediately frozen in a -80 °C freezer overnight. Then, the PDMS device was peeled off from the substrate at room temperature before the solution inside the chamber started to melt. As a result, the DASH pattern was transferred to the substrate side because the structure remained inside the ice during the removal process.

[0111] Sequences used for pathogen detection CMV target: CTGAGTGTGACCTAGGCCGGCATCATTGGATGC (SEQ ID NO: 5). Non-target: CTGAGTGTGACCTAGGAAGGCATCATTGGATGC (SEQ ID NO: 6). Template: GCCTAGGTCACACTCAGTTTTTTTTCGGTGCGAGTTTACGCTCTACTTTTTTTTGCATCCAATGATGCCG (SEQ ID NO: 7). DASH generation primer: GTAGAGCGTAAACTCGCACCG (SEQ ID NO: 8).

[0112] Sequences used for generating DASH-AuNP hybrid materials

Chemical formula

[0113] The linker DNA was designed using a segment (shown in italics) having poly T followed by the complementary sequence of the synthesized DNA derived from the T2-Eco template. For the above conjugation process, a thiol group modification was added to the 5'-side.

[0114] DASH-enzyme functionalization method DASH pattern generation was performed by following a standard protocol with a final seed concentration of 500 pM. The generation process was monitored for up to 4 hours until pattern generation was completed using a fluorescence microscope. Following pattern generation, an avidin-HRP solution from Bio-rad (Hercules, CA) was prepared at a concentration of 10 μg / ml or 100 μg / ml in a final 1x RepliPHI buffer with a final 1x SYBR Green I and injected into the device at 0.1 μL / min for 1 hour. Next, excess avidin-HRP was washed by flushing a solution of the final 1x RepliPHI buffer with a final 1x SYBR Green I at 0.1 μL / min for 1 hour. For the HRP activity assay bound to the DASH pattern, a 1-Step Ultra TMB-ELISA substrate solution from Thermo Fisher Scientific (Waltham, MA) was used. The TMB substrate solution was injected into the device at 0.1 μL / min for 1 hour. The in situ HRP reaction was monitored by using a QuantaRed Enhanced Chemifluorescent HRP Substrate Kit from Thermo Fisher Scientific (Waltham, MA) for confirmation of the localization of HRP activity from the DASH pattern. A QuantaRed solution at 1 ng / mL or 1 μg / mL was injected into the device at a flow rate of 0.1 μL / min and continuously monitored for up to 2 hours using a fluorescence microscope.

[0115] Additional methods and sequences for use in cell-free protein expression Preparation of seeds for generation A circular DNA template for DASH generation seeds was prepared by using a plasmid containing the sfGFP (super-folded Green Fluorescent Protein) sequence. First, a final 0.25 unit / μL of New England 100 ng of plasmid was prepared in a solution of the final 1x NEBuffer from New England Biolabs (Ipswich, MA) mixed with the Nb.BsmI nicking endonuclease from Biolabs (Ipswich, MA). The solution was incubated at 65 °C for 5 hours, then at 80 °C for 20 minutes, and then cooled to room temperature at -1 °C / min to perform the enzyme inactivation step. Next, exonuclease I at a final 0.2 unit / μL and exonuclease III at a final 1 unit / μL were then added and the reaction was incubated at 37 °C for 5 hours. The exonuclease was then inactivated at 80 °C for 20 minutes, and then the annealing process was carried out at -1 °C / min to room temperature. Gel bands showed the success of the formation of circular template DNA from the original double-stranded plasmid DNA. Next, use the 30k Amicon Ultra centrifugal filter from EMD Millipore (Billerica, MA) together with 8 μL of nuclease-free water per 1 μL of the reaction solution, and buffer exchange the solution containing the single-stranded circular template by centrifugation at 10,000 xg. The addition of water and subsequent centrifugation were repeated twice before collecting the template. Then, the DASH generating primer was hybridized to the template at a 1:1 molar ratio by annealing the solution from 95 °C to room temperature at -1 °C / min. Sequence DASH generating primer: CAAAAAACCCCTCAAGACCC (SEQ ID NO: 10) Protein expression primer: TAATACGACTCACTATAGGG (SEQ ID NO: 11) Green fluorescent protein expression template (plasmid) (SEQ ID NO: 12).

Example

[0116] Control experiment of the DASH pattern formation process Redistribution after pre-formation of large (gel-like) networks Instead of simultaneous synthesis and formation, for controls, a preformed DNA network was redistributed inside the DASH device. The generated mix (final 0.5 nM) in 0.6 mL tubes was incubated at room temperature for 1, 2, 4 hours, then heated to 90 °C for 20 minutes, immediately quenched with ice, and flowed through the device for 2 hours (0.1 μL / min). Note that the samples were heated to inactivate the enzymatic reaction during the assembly (device-flow) process to stop further synthesis and then immediately quenched to promote network formation. Under equivalent conditions (device type, flow rate, seed concentration), the DASH structure typically begins to form a fibrous network structure approximately 2.5 hours after the start of the reaction. However, all samples produced random gel-like aggregates throughout the device with these conditions; no DASH pattern was observed. This result suggests that the redistribution of small networks (and in situ network formation) may be one of the important mechanisms behind DASH formation (i.e., preformed large aggregates cannot readily change their morphology to a fibrous pattern during the redistribution process once they are formed).

[0117] Redistribution without preformation of large gel-like networks To clarify the formation process during DASH generation, another control experiment was performed. At the optimal timing (2.5 hours), but this time without promoting the preformation of gel-like large aggregates by quenching (i.e., all processes were carried out at room temperature), the redistribution of pre-synthesized DNA (T2-NCTRL template, final 5 nM) was tested. Here, after 2.5 hours of growth in 0.6 mL tubes, instead of using heat-based inactivation of the enzymatic reaction, Proteinase K from New England Biolabs (final 0.02 units / μL) was mixed. The solution was then flowed through at 0.1 μL / min for 4 hours.

[0118] The results show that in this way (non-autonomously, based on manual operations), the pre-synthesized long DNA can be redistributed and a DASH pattern can be formed. However, in this case, simply, probably in the form of a smaller pre-formed network, formation occurs due to the redistribution of the synthesized long DNA, so a non-uniform pattern is observed inside the device (i.e., only the upstream side (the right half of the image) contained a fibrous pattern). As mentioned in the text, continuous and autonomous synthesis and flow, especially vorticity, are important for inducing local formation of the network on the side of the column, and thus lead to the uniform generation of the DASH pattern inside the device.

[0119] Contribution of DNA hybridization during the formation process Finally, using redistribution as in the experiments shown above, the mechanism behind network formation is further investigated. The final 5 nM concentration of the T2-NCTRL template was used in the production mix. Here, after 2.5 hours of synthesis in the tube, proteinase K was mixed as in the previous tests, and then formamide (final 50%) was also mixed into the solution. Using formamide is a well-known method for in situ hybridization of DNA, and basically reduces the melting temperature of double-stranded DNA in a linear fashion by approximately 0.65 °C for each percent of formamide.

[0120] From the triple tests, it was shown that no DASH pattern was observed using the samples after formamide treatment. Comparing with the successful redistribution results shown in the previous section, the results suggest that in addition to the physical entanglement by long DNA polymers, hybridization plays at least a partial role in this production process.

Example

[0121] SEM images of DASH patterns and spherical structures in the fibrous network Measurement Using a total of 30 points selected from the SEM images of the samples, the diameters of the spherical structures seen in the DASH pattern were measured (Supplementary Figure S12). An average of 0.26 ± 0.10 μm was obtained.

[0122] Estimated critical molecular weight of ssDNA for DASH formation By following the technical specifications provided by the manufacturer, the average ssDNA length synthesized by the reaction can be roughly estimated. According to the manufacturer, 1 unit of RepliPHI Phi29 can process 25 pmol of dNTP in 30 minutes, i.e., incorporate 50 pmol of dNTP into ssDNA in 1 hour. A typical reaction contains 5.7 units / μL of enzyme at a final seed concentration of 5 nM. The average length of ssDNA after 1 hour of synthesis based on this parameter is:

Number

[0123] For example, the typical minimum generation time required for apparatus #9-1 (Figure 30) using a 5 nM seed concentration was approximately 2 hours. Thus, in this case, the average length of N (2時間) = 1.1×10 5 nt, i.e., a molecular weight of 3.3×10 7 (molecular mass over 10 million Da) was calculated as a rough estimate of the critical molecular weight for DASH formation under these conditions.

Example

[0124] Control experiments for sensitivity analysis of turbidity and flow rate To roughly understand the relationship between DASH generation and flow (rate, velocity, turbidity) on the side of the column, several additional measurements and comparisons were made using actual experiments and CFD simulations with an apparatus having the same column geometry.

[0125] Experiments using an apparatus with three chambers An apparatus having three chambers (Figure 38, #12-1) was used to measure the relationship between flow rate and DASH generation start time. All three chambers shared the same column dimensions (#3-1 the same); only the width of the main chamber differed (narrow: 175 μm, medium: 385 μm, wide: 805 μm). The width was determined based on the maximum image capture size of the microscope. This design enabled simultaneous DASH generation tests at three different flow rates with the same column design in one test. In both simulations and actual experiments, three flow rates were selected (slow: 0.1155 μL / min, medium: 0.231 μL / min, fast: 0.462 μL / min). The middle flow rate was set to have the same flow rate as a standard experiment (with a 1-inlet device) in the middle chamber (e.g., device #3-1 (500 μm width) at a flow rate of 0.1 μL / min (approximately between 0.2 and 0.5 mm / s)). From the CFD simulation results, as intended, differences in the flow rate and vorticity inside the device corresponding to the chamber width and flow rate were successfully shown (Figures 10A - 10C and Figures 11A - 11C).

[0126] Relationship between vorticity / velocity and DASH generation start time ​The average vorticity on the side of the column was compared with respect to the start time of DASH generation in the actual experiment. Using the #12-1 device at a seed concentration of 5 nM and setting the above three different flow rates, the actual experiment was tested; each generation process was measured by a fluorescence microscope (150 seconds / frame). The signal-to-noise ratio (SNR) calculation of the generated DASH pattern based on one-dimensional Fourier transform was performed row by row, chamber by chamber, and frame by frame. Then, six consecutive rows of samples (row numbers 213-218) in each chamber were selected, and the average value was used as the sample data representing the DASH generation process for each condition (Figure 12). A SNR value of 2.0 was set as an arbitrary threshold for quantitatively determining the start time of generation; the time point (frame number) exceeding the threshold in each sample was recorded as the start frame of DASH generation. Note that the "characteristic" flow rate (μm / second) inside the device was used as a legend in the graph; the value was simply calculated based on the cross-sectional size of the chamber (W×H (μm 2 ); W = chamber width, H = 17.4 μm and flow rate (μL / min). Then, each characteristic flow rate under each condition was converted to vorticity by CFD simulation. A clear correlation between the two values was shown from the plot between the characteristic flow rate and vorticity.

[0127] Based on these data, a comparison between vorticity and the start time of DASH generation was plotted. From the graph, a correlation between vorticity and the inverse function of the start time of generation (1 / frame) was roughly shown, suggesting that vorticity and the start time of DASH generation have an inverse correlation (i.e., higher vorticity results in an earlier start time of generation).

Example

[0128] Vorticity comparison between different column shapes Simulation Based on the protocol mentioned in Materials and Methods (Example 2), CFD simulations of the geometric shapes of various devices were tested. Specifically, the devices with a square pillar (Figure 28, #3-1) and a rhombic pillar (Figure 39, #3-2) share the same overall geometric shape of the device (including the same periodicity between the pillars, the number of pillars, and the same width of the pillars), but the shapes of the pillars are different. As a result, the overall flow velocity became almost the same (Figures 13A - 13B), but the magnitude of vorticity on the side of the pillar was significantly reduced in the rhombic pillar compared to the square pillar due to its streamlined shape (Figures 14A - 14B). This difference between the two geometric shapes can be used as a model case for comparing whether the degree and size of the high vorticity region on the side of the pillar affect the DASH generation process.

[0129] Generation Time Comparison Experiment To examine the effect of the difference in vorticity, the signal-to-noise ratio (SNR) of the DASH patterns generated from both the square and rhombic pillar devices was experimentally measured by time-lapse observation during the process. The values quantitatively represent the "intensity" of the generated DASH pattern (one-dimensional line pattern) observed in each frame (a higher S / N represents stronger pattern generation). The tests were performed in triplicates (red: square pillar, blue: rhombic pillar) under the same experimental conditions (0.5 nM generation seed concentration, 0.1 μL / min flow rate).

[0130] The results (Figure 15) showed a clear trend between the differences in the shapes of the two types of pillars; the device with a square pillar generated the DASH pattern faster than the device with a rhombic pillar even when other parameters including the flow velocity and seed concentration were the same. The results suggest that the difference in vorticity on the side of the pillar affected the generation process (higher vorticity leads to faster generation).

Example

[0131] Generation - Decomposition of DASH Pattern FSA Display As a finite state automaton (FSA), a mathematical model commonly used in robotics, system engineering, and computer science, describes continuous generation and decomposition behavior (Figure 3B). M:Q = {start, growth, decay} Σ = {initiated generation, changed flow, completed digestion} F = {start}

[0132] Here, Q represents a set of states (behaviors), Σ represents a set of input stimuli (triggers for each behavior), and F is the final state. The model enables an approximation of the overall behavior individually using three different states such as start, growth, and decay. Start represents the starting state without any DASH formation; all three solutions flowing into the device remained layered. The generation of the DASH pattern induces a state transition to the growth state. During this state, due to the layered flow, the decomposition mix remains separated from the generation mix, so the assimilation process occurs. When the generated DASH pattern begins to fill the gap between the columns, the flow changes due to this physical feedback, and thus a state transition occurs and changes to the decay state. The mixing of the generation and decomposition solutions predominates the catabolism process inside the device, so the pattern decomposes. When digestion is complete, the state returns to the original starting state and, if the DNA synthesis time is kept constant, the loop can be repeated.

[0133] Detailed description of experimental results Time-lapse videos were recorded using standard protocols. The average fluorescence intensity plot (Figure 3C) in the text was plotted by measuring the average intensity of line 837 (which intersects multiple segments of the DASH pattern) between columns 120 - 190. To further show that generation / decomposition occurred synchronously at multiple positions, for example, the fluorescence intensities from three sample points were plotted. At all three sample points, a single intensity peak was observed at approximately 200 - 250 minutes, which is in good agreement with the average fluorescence intensity described above. Points were selected from three different segments of the DASH pattern. Also, the average intensity of a rectangular region (including two segments of the DASH pattern) was also calculated to further show that the behavior is not due to the irregular behavior of a specific sample point but rather due to the overall generation / decomposition behavior. Triplicate recordings from three different DASH devices successfully repeated the same trend. Also, a negative control test (degradation mix without DNase I) did not show disintegration behavior. From the results, it is shown that the DASH pattern was generated (until approximately 250 minutes) and then completely decomposed synchronously due to DNase I activity.

[0134] CFD simulation Here, we simulated the flow behavior inside the DASH device following the development of the DASH pattern with controlled accumulation (Supplementary Figure S24); to simplify the configuration, we tracked the experimentally accumulated region recognized as the solid region and performed CFD simulations. A particle trace model (particle density: 1.34 g / cm ; particle radius: 13.8 μm, coefficient of restitution: 0.5) was applied. Particles from the central inlet (inlet 1; generation mix) were colored black; particles from the side were colored red. 3 ; particle radius: 13.8μm, coefficient of restitution: 0.5) was applied. Particles from the central inlet (inlet 1; generation mix) were colored black; particles from the side were colored red.

[0135] First, a laminar flow creates two distinct regions within the flow channel (Figure 16A) such that the effect of the decomposition solution from the side is minimized during the generation process in the region with black particles (generation mix). Thus, in the actual experiment, a DASH pattern was generated in the central region of the device. Once controlled accumulation occurs at the center of the device (Figure 16B), during deployment, this structural change begins to alter the solution, resulting in a mixture of black and red particles (i.e., a mixture of the generation and decomposition solutions) (Figures 16C and 16D). As a result, the decomposition mix exceeds generation in the mixed region, and thus the DASH pattern is digested. CFD simulations clearly showed that the state switch from growth to decay was due to the spatio-temporal feedback induced by the controlled accumulation of the DASH pattern.

[0136] Repeated generation - decomposition Similar to the generation / decomposition experiment, the repeated generation / decomposition of the DASH pattern at a static position was tested using device #20 - 2. Here, it should be noted that we controlled the synthesis time to a constant 2 - hour reaction to minimize irreversible accumulation inside the device and enable a repeatable redistribution process throughout the 12 - hour observation. As with other experiments, this experiment was also conducted without human operation / intervention; all generation / decomposition reactions were executed autonomously.

[0137] Tests were tried using the final 0.1 nM generation mix solution. The graph (Figure 17) shows the overall behavior by taking the average at row 822 (which intersects multiple segments of the DASH pattern) between columns 135 - 165. Also, to show the synchronized behavior at different positions, three sample points were selected from the time - lapse video and the intensities were plotted. Additionally, to show that the phenomenon is not due to the irregular behavior of a particular sample point, the average from a rectangular region containing the DASH pattern (one fiber segment) was also plotted. By showing two peaks during the 12 - hour test, all results showed overall consistency with two cycles of generation and decomposition. Similar results were also repeated by using different concentrations of the generation mix (0.5 nM).

Example

[0138] Emergent movement behavior driven by DASH Analysis The inventors quantitatively analyzed body movement using two parameters such as the center of mass plot (CoM) and the boundary line detection. CoM represents the overall movement of the mass in the image; the boundary line indicates the continuous movement of the entity.

[0139] CoM plot The center of mass (CoM) of the DASH pattern during movement using two types of straight tracks (wide width: Figure 40, #22 - 3 and narrow width: Figure 41, #23 - 3) was plotted (Figure 3D). The x - axis distance between the CoM and the left end of the time - lapse image was plotted during movement.

[0140] The initial decrease in value (i.e., CoM movement towards the downstream side) is due to the initial deployment of the pattern at the extreme left end (downstream) of the device. (Since pattern generation does not occur inside the device, the initial CoM position is at the center of the image; finally, the CoM moved due to the first pattern generation in the most downstream region.) Once the pattern moves upstream against the direction of the flow, the CoM corresponded to the position of the pattern and represented the movement accurately. Once the pattern reached the right end (the most upstream region) of the device, the CoM almost "stopped" at the final position. The average speed of movement was calculated as 1.2 mm / hour (#22 - 3) and 2.3 mm / hour (#23 - 3) from the initial and final positions of the CoM.

[0141] Boundary line analysis The DASH pattern that occupies the widest continuous region in the flow path was defined as the body. Based on this definition, boundary line analysis was calculated based on the following algorithm using MATLAB. First, software within the tissue was used to read in the time-lapse images from the fluorescence microscope frame by frame, and then they were converted into binary (black and white) images using an arbitrary threshold (0.015). Next, the "holes" in the binary image were filled and the region of the body was determined. Finally, the widest occupied region in the image was selected frame by frame, and then the boundary line of the region was displayed as shown in the video.

Example

[0142] Details of Detection Based on DASH Detection based on DASH was designed using a combination of recognition based on hybridization / ligation, amplification based on DASH pattern generation, and readout based on DASH pattern recognition (Figure 18). The recognition process uses a template with a complementary sequence to the target DNA or RNA; to simplify the example, in this demonstration, the negative control uses an "incorrect" target sequence with a 2-base pair mismatch at the ligation site of the template. Only the correct combination of target and template results in successful ligation (circularization) of the template, which enables the enzymatic synthesis process for the next amplification step. Note that both amplification and readout were performed simultaneously and autonomously by taking advantage of DASH's mesoscale pattern generation ability.

[0143] Results Time-lapse recordings were made using (150 seconds / frame) for a total of 78 frames, observing triplet samples from both positive and non-target (5 pM to 500 pM). To quantitatively represent the presence of patterns inside the device, the generated results were analyzed and plotted using FFT software within the tissue (Figures 19A - 19B and Figures 20A - 20B). A comparison plot (Figure 21) was generated by using the maximum value of the signal-to-noise ratio (S / N or SNR) of the generated DASH pattern as one axis and using another axis by plotting the maximum value from the average fluorescence intensity inside the chamber (after subtracting the background intensity). Here, the SNR display was used to quantitatively show the presence / absence of the pattern (i.e., quantitatively simulate a naked-eye readout); when we set an arbitrary threshold of SNR = 15, as shown in Figure 4A, the results corresponded well with our qualitative observations. The plot also showed a clear comparison that our pattern recognition-based detection could improve the detection sensitivity by more than 10 times (detectable at 500 pM and 50 pM) using the target concentration compared to the average intensity values (undetectable at both concentrations), and could maintain specificity compared to negative control samples (target sequences with 2-base pair mismatches).

Example

[0144] DASH-Avidin Hybrid Material From the results, it is shown that avidin successfully bound to DASH (Figures 4B and 22A - 22B), but streptavidin as a control experiment did not bind (Figures 23A - 23B). Both avidin and streptavidin bind to the coenzyme biotin The abilities of these are well-known. However, there are distinct biochemical differences between these related proteins that explain the observed differences in their affinity for DNA (including the DASH pattern). First, avidin has an approximately strongly basic isoelectric point of 10 and has a net positive charge itself in the RepliPHI reaction buffer (pH 7.5). Thus, avidin is electrostatically attracted to DNA with a high negative charge. On the other hand, streptavidin has a pI of about 5 or 6 and thus has a slightly negative net charge in the RepliPHI reaction buffer. Beyond electrostatic attraction, avidin is glycosylated, while streptavidin does not contribute to the increase in non-specific binding between avidin and various substrates. In previous studies, the non-specific interaction between avidin and DNA has been characterized in detail, and it has been shown to have high affinity due to both the overall positive charge and the unique structural motif of the protein.

[0145] This avidin-based binding can be utilized as a standard functionalization method for the DASH pattern via an avidin-protein conjugate or a biotin-binding molecule using the avidin-biotin interaction. The inventors further demonstrated the functionalization of the DASH pattern with quantum dots and HRP based on this method.

Example

[0146] DASH-Quantum Dot Hybrid Material Based on the successful results of the binding of a fluorescent-avidin conjugate to the DASH pattern, this technique was extended as a versatile functionalization method for the DASH pattern. The attachment of quantum dots (Qdot) to the DASH pattern (DASH-Qdot) was tested by using the "sandwich" binding method. First, avidin was attached to the DASH pattern, and then biotin-binding quantum dots were attached to the structure by using the avidin-biotin interaction.

[0147] Positive and Control Experiments Both the positive and control samples were tested in triplicate to confirm the consistency of the results. Representative results from the positive and control samples are shown (Figures 4C and 24A - 24D). From the results, it was clearly shown that only the positive samples (having avidin and then Qdot) were successful in the binding of Qdot to the DASH pattern even after an additional 1-hour wash with 1x RepliPHI reaction buffer. On the other hand, no binding was observed in the control samples (without avidin binding). The results indicate the success of the functionalization of Qdot and also suggest that the binding mechanism of Qdot is actually based on the avidin-biotin interaction. This result, together with the results with HRP binding, suggests that the functionalization of DASH by this "sandwich" method (using avidin and then biotinylated target molecules) can be extended to a wide range of molecules from organic (proteins) to inorganic (nanoparticles).

Example

[0148] DASH-AuNP Hybrid Material Dark-Field Microscopy Dark-field optical microscopy enables clear characterization of gold nanoparticles by strong light scattering. For the observation of the DASH-AuNP pattern, an Olympus BH-2 microscope (Japan) in dark-field configuration was used (40 nm AuNP was used for the following results) (Figures 4D and 25).

[0149] SEM The success of the attachment of AuNP-DNA to the DASH pattern was confirmed by SEM. For sample preparation, a transferable DASH device configuration was used. The gold nanoparticles were clearly attached along the fibrous form of the DASH structure; some showed "wire-like" regular gold nanoparticles. 。

Example

[0150] Functionalization of the DASH Pattern with Avidin-HRP Based on the success of the binding experiment of the fluorescence-avidin conjugate and the biotin-binding molecule (via the "sandwich" binding method) to the DASH pattern, the technology was further extended by attaching other avidin-binding functional molecules (in this case enzymes), and it was tested whether the enzyme maintained its biochemical activity on the DASH pattern. Here, avidin-binding horseradish peroxidase (avidin-HRP) was selected as the model enzyme and attached to the DASH pattern; the enzyme activity was measured to show the success of the functionalization of the DASH pattern.

[0151] The activity of HRP bound to the DASH pattern was first confirmed by using the 1-Step Ultra TMB-ELISA substrate solution from Thermo Fisher Scientific (Waltham, MA). The kit detects HRP activity by converting the TMB (3,3’,5,5’-tetramethylbenzidine) substrate into a blue (intermediate) complex (Amax = 370 nm and 652 nm). The blue product of TMB was found to directly stain the DASH pattern, probably due to the electrostatic interaction between the negatively charged DNA and the positively charged TMB product. The results can also be observed by the naked eye.

[0152] Next, to further confirm that the HRP activity is localized to the DASH pattern, in The situ HRP reaction was monitored by using the QuantaRed Enhanced Chemifluorescent HRP Substrate Kit from Thermo Fisher Scientific (Waltham, MA). The QuantaRed substrate uses the ADHP (acetyl-3,7-dihydroxyphenoxazine) chemifluorescent reaction and, by reacting with HRP, converts from a non-fluorescent compound to the fluorescent compound resorufin with an excitation / emission of 570 / 585 nm. The QuantaRed solution was injected into the device and continuously monitored by a fluorescence microscope. During the process, it was observed that the product actually began to develop (and downstream) corresponding to the position of the DASH pattern until the overall fluorescence saturated throughout the device, probably due to the high sensitivity of the reaction.

[0153] Cell-free protein expression from the DASH pattern In addition to the quantitative measurement of the expressed protein (Figure 4E), direct observation of CFPE from the DASH pattern was performed (Figure 4E and Figures 26A - 26B). The observations were made according to a standard protocol by using a fluorescence microscope. The results show that successful sfGFP expression occurred only from devices with the DASH pattern.

Claims

1. A system for generating materials with ordered structure and artificial metabolism, comprising: Includes equipment and generated mix, wherein the product mix is ​​a reagent containing components for forming a polymer, wherein the apparatus comprises a main chamber designed to allow a directed flow of a solution therethrough, the main chamber comprising an obstacle that induces vorticity in the directed flow of the solution comprising the product mix to initiate and promote assembly of the polymers synthesized in the apparatus to form the material, said system.

2. 2. The system of claim 1, wherein the main chamber comprises at least one inlet port and at least one outlet port that allow for the introduction of a solution comprising the product mix into the main chamber through the at least one inlet port and for flow from the at least one inlet port through the main chamber to the at least one outlet port.

3. 3. The system of claim 1 or 2, further comprising a degradation mix comprising a reagent for depolymerizing the polymer.

4. 4. The system of claim 3, wherein the main chamber comprises at least two inlet ports for separately injecting a solution containing a production mix and a solution containing a degradation mix.

5. 4. The system of claim 3, wherein the main chamber comprises three inlet ports, wherein the middle inlet port is for injecting a solution containing the production mix, and wherein the two outer inlet ports are for injecting a solution containing the degradation mix.

6. The system of any one of claims 1 to 5, wherein the device comprises multiple main chambers.

7. The system of any one of claims 1 to 6, wherein the material has a static pattern.

8. The system of any one of claims 1 to 6, wherein the material has a dynamic pattern.

9. The system of claim 8 , wherein the pattern is a movement behavior or a competitive behavior between two moving objects.

10. The system according to any one of claims 1 to 9, wherein the polymer is DNA.

11. The system according to any one of claims 1 to 9, wherein the polymer is RNA.

12. The system of claim 10 , wherein the production mix comprises dNTPs, a template nucleic acid, a primer, and a DNA polymerase.

13. The system of claim 12 , wherein the primer and the template nucleic acid are annealed before being provided to the main chamber.

14. The system of claim 12 or 13, wherein the template nucleic acid is a circular DNA.

15. The system according to any one of claims 12 to 14, wherein the DNA polymerase is Phi29 DNA polymerase.

16. The system of any one of claims 10 to 15, wherein the degradation mix comprises one or more nucleases.

17. The system of any one of claims 1 to 16, wherein the product mix comprises reagents that produce a detectable signal.

18. 3. The system of claim 1 or 2, wherein the polymer is DNA and the product mix comprises (i) dNTPs, a template nucleic acid, and a DNA polymerase, (ii) dNTPs, a primer, and a DNA polymerase, or (iii) dNTPs, a template DNA, a primer, a DNA polymerase, and a ligase.

19. The system of any one of claims 1 to 18, wherein the main chamber has an at least substantially planar shape.

20. The system of any one of claims 1 to 19, wherein the main chamber has dimensions on the micron to millimeter scale along the direction of directed flow.

21. A method for producing materials with ordered structure and engineered metabolism, comprising: Providing an apparatus and a product mix, wherein the product mix is ​​a reagent containing components for forming a polymer, and wherein the apparatus includes a main chamber designed to allow a directed flow of a solution therethrough, the main chamber including an obstacle that induces vorticity in the directed flow of the solution; and providing a solution containing the product mix to the main chamber and directing the flow of the solution through the main chamber, thereby allowing synthesis of polymers and assembly of the synthesized polymers to form the material; The method comprising:

22. 22. The method of claim 21, wherein the main chamber comprises at least one inlet port and at least one outlet port, wherein a solution comprising the product mix is ​​introduced into the main chamber through the at least one inlet port and is directed to flow from the at least one inlet port through the main chamber to the at least one outlet port.

23. A method for producing materials with ordered structure and engineered metabolism, comprising: Providing an apparatus, a generated mix, and a digested mix, wherein the production mix is ​​a reagent containing components for forming a polymer, and the degradation mix contains components for depolymerizing the polymer; wherein the apparatus includes a main chamber designed to permit a directed flow of a solution therethrough, having obstacles spaced in a predetermined pattern and shaped and sized to permit the generation of vorticity in the directed flow of the solution; and providing a solution containing a production mix and a solution containing a degradation mix to a main chamber of the device and directing the flow of the solutions through the main chamber to allow the processes of polymer synthesis and assembly and polymer degradation to occur autonomously and combinatorially, thereby forming the material; The method comprising:

24. 24. The method of claim 23, wherein the main chamber comprises at least two inlet ports for separately injecting a solution containing the production mix and a solution containing the degradation mix.

25. The main chamber contains three inlet ports, where the middle inlet port is the generation mix.

25. The method of claim 24, wherein the two outer inlet ports are for injecting a solution containing the digestion mix, and wherein the two outer inlet ports are for injecting a solution containing the digestion mix.

26. 26. The method according to claim 24 or 25, wherein the solution containing the production mix and the solution containing the degradation mix are injected into the main chamber simultaneously, sequentially or in a predetermined order.

27. A method according to any one of claims 21 to 26, further comprising the step of visualising the pattern of material produced.

28. 28. The method of claim 27, wherein visualization is achieved by the naked eye, a camera, a fluorescent microscope, a light microscope, or an electron microscope.

29. The method of any one of claims 21 to 28, wherein the material has a static pattern.

30. The method of any one of claims 21 to 28, wherein the material has a dynamic pattern.

31. The method of claim 30, wherein the pattern is a movement behavior or a competitive behavior between two moving entities.

32. The method of any one of claims 21 to 31, wherein the polymer is DNA.

33. The method of any one of claims 21 to 31, wherein the polymer is RNA.

34. 33. The method of claim 32, wherein the product mix comprises dNTPs, a template nucleic acid, a primer, and a DNA polymerase.

35. 35. The method of claim 34, wherein the primer and the template nucleic acid are annealed prior to being provided to the main chamber, and optionally the template nucleic acid is a circular DNA.

36. 36. The method of claim 34 or 35, wherein the DNA polymerase is Phi29 DNA polymerase.

37. The method of any one of claims 32 to 36, wherein the degradation mix comprises one or more nucleases.

38. The method of any one of claims 21 to 37, wherein the product mix comprises reagents that produce a detectable signal.

39. The method according to any one of claims 21 to 38, wherein the main chamber has a planar shape.

40. The method of any one of claims 21 to 39, wherein the main chamber has dimensions on the micron to millimeter scale.

41. A material produced according to the method of any one of claims 21 to 40.

42. 1. A method for detecting nucleic acid of a pathogen, comprising: Providing an apparatus, a product mix, and a sample, wherein the product mix comprises (i) dNTPs, template nucleic acid, and DNA without primers; (ii) a polymerase, without a template nucleic acid, dNTPs, a primer, and a DNA polymerase; or (iii) a reagent comprising dNTPs, a template DNA, a primer, and a ligase, wherein the template DNA is circularized in the presence of the pathogen nucleic acid and the ligase, wherein the apparatus includes a main chamber designed to allow a directed flow of a solution therethrough, the main chamber including an obstacle that induces vorticity in the directed flow of the solution; and Providing in the main chamber (i) a solution comprising a product mix and a sample or (ii) a solution comprising a product mix, wherein the template DNA is treated with the sample and a ligase to allow circularization of the template DNA if nucleic acid of the pathogen is present in the sample; and directing the flow of the solution through the main chamber, thereby allowing synthesis of polymers and assembly of the synthesized polymers to form a DASH material with ordered structure and engineered metabolism in the presence of pathogen nucleic acid in the sample. The method comprising:

43. 43. The method of claim 42, wherein the main chamber comprises at least one inlet port and at least one outlet port, wherein a solution comprising the product mix is ​​introduced into the main chamber through the at least one inlet port and is directed to flow from the at least one inlet port through the main chamber to the at least one outlet port.

44. 44. The method of claim 42 or 43, wherein the main chamber has dimensions on the micron to millimeter scale and has a planar shape.

45. The method of any one of claims 42 to 44, wherein the pathogen nucleic acid is DNA.

46. The method of any one of claims 42 to 44, wherein the pathogen nucleic acid is RNA.

47. The method of any one of claims 42 to 46, wherein the pathogen is a bacterium, a fungus or a virus.

48. The method of any one of claims 42 to 47, wherein the DNA polymerase is Phi29 DNA polymerase.

49. The method of any one of claims 42 to 48, wherein the product mix comprises reagents that produce a detectable signal.

50. 50. The method of claim 49, wherein the reagent is a fluorescent compound that binds to DNA.

51. 1. A method for producing a hybrid material, comprising: Producing a material with ordered structure and artificial metabolism according to the method of any one of claims 21 to 40, wherein the polymer is DNA; and injecting a solution containing a reagent that binds to the material formed from the assembled DNA into the main chamber, thereby forming a hybrid material, wherein the material having an ordered structure and an engineered metabolism formed from the assembled DNA binds to the reagent; The method comprising:

52. 52. The method of claim 51, wherein the reagent comprises avidin.

53. 53. The method of claim 52, further comprising injecting into the main chamber a solution containing an enzyme (such as HRP) or biotin that binds to the quantum dots.

54. 52. The method of claim 51, wherein the reagent comprises gold nanoparticles.

55. 1. A method for cell-free protein expression comprising: Producing a material with ordered structure and artificial metabolism according to the method of any one of claims 21 to 40, wherein the polymer is DNA; and injecting the cell-free protein expression solution into the main chamber and allowing production of proteins encoded by the DNA in the material; The method comprising:

56. 1. A method for designing obstacles for a main chamber of an apparatus for producing materials with ordered structure and artificial metabolism, comprising: defining a main chamber for producing a material having an ordered structure; defining a pattern of material to be produced therein; and determining the size, shape and location of a plurality of obstacles in the main chamber of the device necessary to direct the flow of the solution along the shortest path within the main chamber and between adjacent obstacles; The method comprising:

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