Devices and methods involving metasurface oligonucleotide synthesis

Abstract

In exemplary aspects, oligonucleotides are synthesized via a microfluidic reaction chamber integrating nanostructure / high-Q dielectric nanoantenna arrays of a metasurface device, the arrays providing synthesis sites. The method comprises; the chamber processing input reagents for producing oligonucleotides at outlets of the chamber; and optically exciting the synthesis sites by altering optical parameter(s) to cause at least one enzymatic synthesis cycle. In more specific examples, the method further includes effecting, after the cycle(s) occurs, PCR amplification of certain of the synthesized products by selecting and optically exciting one or more of the synthesis sites, and / or microfluidically segregating the one or more synthesized products, in response to the amplification step, into homogenous oligo pools.

Description

[0001]STFD.463PCT (S24-133) 1      DEVICES AND INVOLVING METASURFACE OLIGONUCLEOTIDE SYNTHESIS FEDERALLY-SPONSORED RESEARCH AND DEVELOPMENT This invention was made with Government support under contract AI152072 awarded by the National Institutes of Health. The Government has certain rights in the invention. BACKGROUND In exemplary contexts, aspects of the present disclosure are directed to methods and apparatuses for the synthesis of oligonucleotides. Recent developments have realized a number of different platform types configured for the synthesis of oligonucleotides (oligos). For example, one such platform type is a microarray-based solid-phase synthesis platform which is capable of producing a diversity of high density oligonucleotides. However, known microarray platform types are limited to producing oligos that are not more than 300 nucleotides long due to accumulating reactions errors. Consequently, approaches using such microarray platforms can be generally error-prone and can yield low-quality synthesis products that give rise to subsequent purification challenges. Moreover, such approaches can generate excess wastes (which in certain instances may be toxic), and drive up the cost per base of the error free synthesis products. Recent cost estimates for such microarray synthesis are approximately $0.0005 per nucleotide. Therefore, synthesis of anywhere from one hundred or more unique sequences, and in many instances, hundreds to thousands of unique sequences (one sequence per site) with roughly around one attomole (“aM”, representing approximately 600k units) of a sequence per site having 300 nucleotides per sequence can be economically intractable. While there have been efforts to mitigate these errors, which arise primarily due to the misalignment of optical beams and reagent droplets with the synthesis sites, the efforts have resulted in limitations in terms of the density and diversity of synthesis. These factors confine the solid-phase synthesis of oligonucleotides to a limited number of vendors who are capable of providing the product as a service. Consequently, this has led to inadequate access, long lead times and high cost of synthetic oligos, and to ongoing research efforts seeking ways to harness the potential of synthetic biology for the potential of synthesizing and screening over large genetic design spaces for optimal biological functions relevant to specifically-targeted applications. STFD.463PCT (S24-133) 2      These and other matters have challenges to efficiencies in production of genetically engineered biology including but not limited to apparatuses and methods involving synthesis of oligos (e.g., nucleotide long oligonucleotide strands) for a variety of applications and markets. SUMMARY OF VARIOUS ASPECTS AND EXAMPLES Various examples / embodiments as described in the present disclosure are directed to issues such as those addressed above and / or others which may become apparent from the following disclosure, which discusses various aspects that may be implemented alone as a separate feature and / or in combination with one or more of the other aspects. In certain examples, aspects of the present disclosure involve the synthesis of oligonucleotides via a microfluidic reaction chamber integrating nanostructure using high-Q dielectric nanoantenna arrays integrated as part of a metasurface device, wherein the arrays provide synthesis sites which are optically excited by altering one or more optical parameters to cause an enzymatic synthesis cycle. In other more-particular examples, aspects of the present disclosure are directed to overcoming the inadequate access to a diverse set of high-quality strands of long oligonucleotides that limit the ability of synthetic biology to engineer complex genetic functions. In specific example implementations consistent with the present disclosure, synthesis of oligonucleotides uses an iterative design-build-test-learn cycle (e.g., with site selective thermolytic deblocking step and coupling step). In certain exemplary aspects, the present disclosure is directed to use of a microfluidic reaction chamber and optical responses of one or more metasurface-based structures that form part of a chip. The chip, while integrated within the microfluidic-reaction chamber, includes synthesis sites that are activated to prompt the optical responses and cause one or more enzymatic-synthesis cycles for the synthesis of oligonucleotides. Among a variety of more-specific aspects according to the present disclosure, it has been discovered that the above technique is particularly effective for amplifying synthesized products, in response to the enzymatic-synthesis cycle(s), while the products are at individual sites of the activated sites. In certain exemplary aspects involving a method of the present disclosure, oligonucleotides are synthesized via a microfluidic reaction chamber that is integrated with a metasurface device having nanostructure and associated high-Q dielectric nanoantenna arrays, and wherein the arrays provide synthesis sites. The method comprises: the chamber STFD.463PCT (S24-133) 3      processing input reagents for producing at outlets of the chamber; and optically exciting selected ones of the synthesis sites by altering optical parameter(s) to cause at least one enzymatic synthesis cycle (e.g., including thermolytic deblocking and coupling via catalyzation by terminal deoxynucleotidyl transferase (TdT)). In more specific examples, the method further includes effecting, after the cycle(s) occurs, (e.g., PCR) amplification of certain of the synthesized products by selecting and optically exciting one or more of the synthesis sites, and / or microfluidically segregating the one or more synthesized products, in response to the amplification step, into homogenous oligo pools. In related more specific example, the nanostructures are configured to be act independently as heaters to amplify synthesized products associated with the one or more output reagents at individual ones of the synthesis site, and the method further comprises: generating heat, via the at least one of the synthesis sites, by the step of optically exciting, to amplify one or more synthesized products associated with the one or more output reagents at individual one of the synthesis sites, and microfluidically segregating the one or more synthesized products, in response to the one or more synthesized products being amplified, into homogenous oligo pools. Other exemplary aspects of the present disclosure involve an oligonucleotide- synthesis apparatus, comprising a microfluidic reaction chamber and a metasurface-chip device. The microfluidic reaction chamber includes inlets to receive one or more input reagents and includes outlets to yield one or more output reagents as synthesized oligonucleotides. The metasurface-chip device, as integrated within the microfluidic reaction chamber, includes each of one or more arrays, with each such array including at least one nanostructure integrated into at least one high-Q dielectric nanoantenna. Each array is associated with one or more responses (spectral- / wavelength-specific and polarization- specific) and is to provide at least one of a plethora of synthesis sites, with each such site being optically excitable by switching or varying at least one of a wavelength parameter and a polarization parameter. The optical excitation is to cause at least one enzymatic synthesis cycle, acting on the one or more input reagents, such as thermolytic deblocking and coupling via catalyzation by TdT. In a more specific example, the at least one enzymatic synthesis cycle is implemented as a two-step enzymatic synthesis cycle for oligonucleotides including the site selective thermolytic deblocking and coupling steps. In more specific examples, the above-characterized method and / or apparatus includes or is associated with additional aspects. As examples: each of the arrays is associated with at least one of geometry and spatial orientation to characterize the one or STFD.463PCT (S24-133) 4      more wavelength (or specific spectral) and polarization responses; and / or the metasurface-chip device and the microfluidic reaction chamber are cooperatively configured with each of the synthesis sites being independently and optically excitable by switching or varying said at least one of a wavelength parameter and a polarization parameter to cause one or more multi-step enzymatic synthesis cycles. In related examples, the apparatus further includes: an optically-transparent synthesis substrate, as part of the metasurface-chip device, on or over which the one or more arrays are patterned; an optical source, including controls for affecting wavelength and polarization parameters, to direct light at and selectively excite the synthesis sites between respective on and off states by switching or varying the wavelength and polarization parameters; aqueous enzymatic reagents as input reagents to provide the oligonucleotides without generation of toxic wastes (e.g., eliminating potential toxic wastes generated by traditional phosphoramidite reaction cycle that employs organic reagents); a synthesis-site density, as part of the metasurface chip, of more than one million of the synthesis sites per square centimeter of surface area on the metasurface chip; and / or one or more metasurfaces as part of the metasurface chip, each of the one or more metasurfaces being associated with a respective optical response that depends on dimension and orientation of the one or more arrays and / or being configured to include activatable metasurfaces based on an associated optical response (e.g., each being independently activatable by tuning wavelength and polarization of light). Yet further aspects, also according to the present disclosure, may be used with the above-characterized method and / or apparatus. Such further aspects includes: the arrangement being configured to provide the oligonucleotides at least in part due to the thermolytic deblocking step, wherein each of the high-Q nanoantennas are associated with a Q factor being in a range from several hundred an order of one thousand; the synthesis sites being configured to be excited selectively without causing synthesis errors (e.g., as experienced in solid-phase synthesis); the nanoantennas being spectrally and spatially isolated to facilitate generation of an electric field and heat confinement to facilitate synthesis of the oligonucleotides; the metasurface chip being configured to facilitate using fabrication steps common to MOS-based fabrication that includes patterning of the arrays for integration or creation of the synthesis sites; each of the high-Q nanoantennas being associated with a Q factor that is sufficiently high (e.g., in a range from multiple hundreds to 1000 spectral responses) to minimize spatial and spectral overlap between the synthesis sites, and is to facilitate generation of an electric field; and / or the nanostructures (e.g., precisely-aligned gold STFD.463PCT (S24-133) 5      nanostructures) being integrated into the arrays and configured to act independently as site-selective resonant heaters to amplify synthesized products associated with the one or more output reagents at individual ones of the synthesis sites. Various aspects and examples according to the present disclosure (including the Appendices of the underlying U.S. provisional application) are directed to issues such as those above and / or others which may become apparent from the following disclosure. In one particular exemplary context, one aspect of the present disclosure is directed to addressing the inadequate access to a diverse set of high-quality strands of long oligonucleotides that limit the ability of synthetic biology to engineer complex genetic functions (e.g., through the above iterative design-build-test-learn cycle). In certain example embodiments, aspects of the present disclosure involve one or more exemplary solid-phase synthesis platforms that enable the high fidelity, density and diversity synthesis of long oligonucleotide strands at low costs and high purity to decentralize the synthesis process. Such platforms can also be used to accelerate research in synthetic biology by making synthetic oligos more accessible to a wider body of researchers. In particular examples, aspects of the present disclosure are directed to apparatuses (e.g., system, platform, circuitry, components, etc.) and methods involving any one, or a combination, of the following: provide and / or increase access to diverse set of high- quality strands of long oligonucleotides (synthetic DNA); overcoming problems with current technology which attempts to align optical beams and reagent droplets, which are limited to oligos that are not more than 300 nucleotides long due to accumulating reactions errors, and which produce low quality oligonucleotides and excess waste. In more specific examples, the present disclosure is directed to exemplary methods or apparatuses (or aspects of such methods or apparatuses) such as: arrays of dielectric nanoantennas patterned on an optically transparent synthesis substrate; implementation and use of external controls to selectively excite desired synthesis sites based on the geometry and spatial orientation of these nanoantennas (which dictates their unique spectral and polarization response), for example, without needing to resort to (or relying on) moving mechanical component-based alignments; and controlling synthesis sites via manipulating the wavelength and polarization of the optical excitation (e.g., switching them on and off by simply switching the wavelength and polarization of the optical excitation). In yet other specific examples, aspects of the present disclosure are directed to apparatuses and methods involving nanoantenna arrays as resonant heaters. To drive site selective thermolytic deblocking reactions, gold (e.g., precisely aligned) nanostructures may STFD.463PCT (S24-133) 6      be integrated into the nanoantenna arrays to as resonant heaters. The dimension and position of the gold nanostructures are chosen to achieve the combined and mostly contradictory goals of high heat and high-Q (e.g., approximately 500). The above discussion is not intended to describe each aspect, embodiment or every implementation of the present disclosure. The figures and detailed description that follow also exemplify various embodiments. BRIEF DESCRIPTION OF FIGURES Various example embodiments, including experimental examples, may be more completely understood in consideration of the following detailed description in connection with the accompanying drawings, each in accordance with the present disclosure, in which: FIG.1 is a diagram illustrating a multi-step enzymatic synthesis cycle, in accordance with exemplary aspects of the present disclosure; FIG.2A is a diagram of a synthesis platform, and FIG.2B is a diagram illustrating how a metasurface structure functions as part of the synthesis platform of FIG. 2A, both diagrams being in accordance with certain exemplary aspects of the present disclosure; FIGs.3A-3D are respectively, each in accordance with certain exemplary aspects of the present disclosure: optical and SEM images of arrays of silicon nanostructures (FIG. 3A), a set of graphs depicting wavelength dependent temperature rise of resonators with different dimensions (FIG.3B and FIG.3C), and image capture of an exemplary microfluidic reactor (FIG.3D); FIGs.4A-4B are a set of diagrams showing an example of how coupling reaction, in accordance with certain exemplary aspects of the present disclosure, functions, with FIG. 4A showing cycling (with deblocking and coupling steps) for a particular reaction affecting an initiator strand, and FIG.4B showing aspects of temperature dependent enzymatic coupling; FIG.4C is a graph showing aspects of previously-known decoupling efforts, relative to aspects of the present disclosure; and FIGs.5A-5B are a set of system-level diagrams, with FIG.5A showing an example experimental setup, and FIG.5B showing an expanded view of components used in the example experimental setup of FIG.5A. While various embodiments discussed herein are amenable to modifications and alternative forms, aspects thereof have been shown by way of example in the drawings and STFD.463PCT (S24-133) 7      will be described in detail. It should be however, that the intention is not to limit the disclosure to the particular embodiments described. On the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the scope of the disclosure including aspects defined in the claims. In addition, the term “example” as used throughout this application is only by way of illustration, and not limitation. DETAILED DESCRIPTION Aspects of the present disclosure are believed to be applicable to a variety of different types of methods and apparatuses (e.g., systems, components and devices) characterized at least in part by use of a microfluidic reaction chamber and optical properties associated with metasurface-based structures. While the present disclosure is not necessarily limited to any particular type of apparatus or type of method, an understanding of specific examples in the following description may be understood from discussion in such specific contexts. Accordingly, in the following description various specific details are set forth to describe specific examples presented herein. It should be apparent to one skilled in the art, however, that one or more other examples and / or variations of these examples may be practiced without all the specific details given below. In other instances, well known features have not been described in detail so as not to obscure the description of the examples herein. For ease of illustration, the same connotation and / or reference numerals may be used in different diagrams to refer to the same elements or additional instances of the same element. Also, although aspects and features may in some cases be described in individual figures, it will be appreciated that features from one figure or embodiment can be combined with features of another figure or embodiment even though the combination is not explicitly shown or explicitly described as a combination. Exemplary aspects of the present disclosure are related to use of a microfluidic reaction chamber and optical responses of one or more metasurface-based structures that form part of a chip. For an example method consistent with such aspects of the present disclosure, one or more metasurfaces (e.g., planarly-configured nanostructures) are part of a metasurface chip, which is integrated within a microfluidic-reaction chamber. Via excitation from a light source and (while the metasurface chip is within the microfluidic-reaction chamber, one or more enzymatic-synthesis cycles are used with optical responses of the metasurface chip to synthesize oligonucleotides. Specific uses of this type of method are directed to the manufacture of synthetic genes to engineer biological functions for various STFD.463PCT (S24-133) 8      industries. Such industries include, without cancer therapy, nitrogen-fixing super bacteria, bio-manufactured products such as hyaline for use in transparent flexible electronics (e.g., as a more sustainable alternative to traditional petroleum-based films), genetically engineered foods (e.g., impossible burger), and certain pharmaceutical products (e.g., sitagliptin for managing blood sugar levels in adults with type 2 diabetes). In other specific examples, aspects of the present disclosure are directed to apparatuses and methods involving nanoantenna arrays as resonant heaters. To drive site selective thermal (e.g., thermolytic deblocking) reactions, precisely-configured (e.g., aligned gold) nanostructures are integrated into the nanoantenna arrays and, when selectively activated, function as resonant heaters. The dimension and position of such nanostructures are chosen to achieve the combined and mostly contradictory goals of high heat and high-Q (e.g., a few to several hundred). In further specific examples, aspects of the present disclosure are directed to apparatuses and methods involving a metasurface chip integrated into a microfluidic reaction chamber with the required inlets and outlets to implement an iterative enzymatic synthesis cycle for oligonucleotides including the site-selective reaction activity such as a thermolytic deblocking and TdT coupling steps. In other example embodiments, the iterative enzymatic synthesis cycle is implemented using one or more alternatives to thermolytic deblocking and TdT coupling. Also, in certain specific examples, aqueous enzymatic reagents may be used to eliminate potential toxic wastes generated by the traditional phosphoramidite reaction cycle that employs organic reagents. One such method, consistent with such above-noted aspects, includes using the chamber to process reagents input to the chamber for producing oligonucleotides at outlets of the chamber, and optically exciting the synthesis sites by altering optical parameter(s) to cause at least one enzymatic synthesis cycle (e.g., including thermolytic deblocking and coupling via catalyzation by terminal deoxynucleotidyl transferase (TdT)). Among a variety of more-specific aspects also according to the present disclosure, it has been discovered that the above technique is particularly effective for amplifying synthesized products, in response to the enzymatic-synthesis cycle(s), while the products are at individual ones of the activated sites. In more specific examples, this approach is used as part of the well-known molecular biology technique PCR (Polymerase Chain Reaction) amplification for replicating copies of a specific DNA sequence manifesting by way of the synthesis. STFD.463PCT (S24-133) 9      In a specific example PCR is catalyzed by the thermophilic enzyme Taq polymerase. The process involves repetitive heat cycles, with each cycle including multiple thermal steps including for example: denaturation, annealing and extension. This includes metasurface-based photothermal heaters, via nanostructures (e.g., including gold) integrated with the array-based sites which are configured to be site selective for activation. In this manner, by activating one or more sites selectively, the products are selectively amplified for synthesis at individual sites associated with the nanoantennas. At each site, because the synthesized products may be in relatively small quantities (compared to what is typically used for gene assembly), the synthesized products may be amplified. Advantageously, the amplification occurs before synthesis products are detached from the substrate, which facilitates realizing a desired assembling order. Another exemplary aspects of the present disclosure involves an oligonucleotide- synthesis apparatus that includes a microfluidic reaction chamber and a metasurface-chip device. The microfluidic reaction chamber includes inlets to receive one or more input reagents and includes outlets to yield one or more output reagents as synthesized oligonucleotides. The metasurface-chip device, as integrated within the microfluidic reaction chamber, includes each of one or more arrays, with each such array including at least one nanostructure integrated into at least one high-Q dielectric nanoantenna. Each array is associated with one or more responses (spectral- / wavelength-specific and polarization- specific) and is to provide at least one of a plethora of synthesis sites, with each such site being optically excitable by switching or varying at least one of a wavelength parameter and a polarization parameter. The optical excitation is to cause at least one enzymatic synthesis cycle, acting on the one or more input reagents (e.g., including thermolytic deblocking and TdT coupling). In a more specific example, the at least one enzymatic synthesis cycle is implemented as a two-step enzymatic synthesis cycle for oligonucleotides including the site selective thermolytic deblocking step and coupling step. As exemplified in the specific example shown in FIG.1, the multi-step enzymatic synthesis cycle may be implemented as an iterative design-build-test-learn cycle, such as a two-step enzymatic DNA synthesis cycle involving site-selective thermolytic deblocking step and coupling step in each cycle. In the deblocking step depicted on the right side of FIG.1, the blocking group is removed to open up the 3’ end of the oligo chain, thereby providing a free hydroxyl (-OH) group at the 3' end for the coupling of an incoming nucleotide as part of the synthesis. STFD.463PCT (S24-133) 10      On the left side, FIG.1 shows second step as TdT coupling, which involves the ability of the enzyme to add nucleotides to the 3' end of a DNA molecule without the need for a template. This approach is advantageous in that it can occur without the need for a template and can be used to add homopolymer tails or label DNA with modified nucleotides. In this particular example as illustrated, the starting point of the cycled synthesis starts with a (DNA or RNA) sequence or strand, 3' T G C A 5' (3’ and 5’ referring to the ends of the strand and T G C representing the sequence of nucleotides in the strand, read from the 3' end to the 5' end). This sequence or strand may be received as an input reagent at an inlet of a thermal reaction chamber. In the deblocking step, the 3’ end of the oligo chain is opened up for the coupling of an incoming nucleotide as part of the synthesis. As depicted in the cycle(s) of FIG.1 and with the 3' end having a freed up hydroxyl (-OH) group due to the deblocking, the second step effects the TdT coupling of the incoming nucleotide (C) at the 3' end. Advantageously and as discussed hereinabove, this type of synthesis approach is not limited in terms of the density and diversity of synthesis, with the generation of synthetic oligos in relatively short lead times, at a low cost and with broad synthetic biology applications including synthesizing and screening over large genetic design spaces for optimal biological functions relevant to specifically-targeted applications. The above-described method, using a multi-step enzymatic synthesis cycle as exemplified in FIG.1, can be implemented in a variety of specific metasurface oligonucleotide synthesizers according to the present disclosure. The certain embodiments are directed to a metasurface-based oligonucleotide synthesizer (i.e., an oligonucleotide synthesizer integrating one or more metasurfaces) which has one or more of the following salient technical features. In some specific example embodiments, an oligonucleotide synthesizer uses one or a combination of the following aspects: very high-Q nanoantennas, multi-step enzymatic synthesis cycle, CMOS-type fabrication compatibility, and reduction of unwanted byproducts. These listed aspects are further discussed below. Nanoantennas arrays: the synthesizer includes arrays of dielectric nanoantennas patterned on an optically transparent synthesis substrate. The geometry and spatial orientation of these nanoantennas dictate their unique spectral and polarization response which are used as external controls to selectively excite desired synthesis sites without resorting to moving mechanical component-based alignments used in current state-of-the-art photolithographic and / or inkjet printer-based microarrays. Synthesis sites can be switched on and off by simply STFD.463PCT (S24-133) 11      switching the wavelength and polarization optical excitation, thereby mitigating (or in many implementations, eliminating) synthesis errors in solid-phase synthesis processes. Very high-Q nanoantennas. The above-discussed nanoantennas can have extremely high-Q factor (e.g., a few hundred to several hundred) and in certain implementation many (e.g., on the order of hundreds, or approximately 1000) spectral responses due to the lossless dielectrics. To drive site selective thermolytic deblocking reactions, certain embodiments integrate precisely aligned gold nanostructures into the nanoantenna arrays, and the gold nanostructures act as resonant heaters. The dimension and position of the gold nanostructures are chosen to achieve the combined and mostly contradictory goals of high heat and high-Q (e.g., approximately 500). In certain implementations, this is another important feature in operation of platform, according to the present disclosure, for the oligo synthesis. While high heat is required to drive thermolytic deblocking at the reaction sites, the spectral and spatial isolation of the nanoantennas due to the high-Q lends the much-needed electric field and heat confinement necessary for synthesis selectivity, density and diversity. The large resonant field enhancement due to the high-Q also leads to the generation of more heat at a particular power of optical excitation thus improving energy efficiency of the system. Multi-step enzymatic synthesis cycle. The metasurface chip is integrated into a microfluidic reaction chamber with the required inlets and outlets to implement the multi-step (e.g., two-step) enzymatic synthesis cycle for oligonucleotides such as a cycle including the site selective thermolytic deblocking and TdT coupling steps (the coupling step is catalyzed by the enzyme TdT). Fabrication compatibility. Fabrication of the metasurface oligonucleotide synthesizer chip can be implemented via CMOS compatible steps and related equipment, and allowing the patterning and integrating of millions of synthesis sites on a chip. Consistent with such approaches as exemplified by the present disclosure, one specific example has a current density of five million (5 M) synthesis sites per square centimeter. Reduction of unwanted byproducts. Aqueous enzymatic reagents may be used in the synthesis, according to the present disclosure, to eliminate potential toxic wastes generated by the traditional phosphoramidite reaction cycle that employs organic reagents. Further, the improved synthesis efficiency reduces unwanted byproducts which also cuts down on waste generation. As previously indicated, synthetic DNA drives the field of synthetic biology which can address some of the most urgent global challenges. Therefore, the possible STFD.463PCT (S24-133) 12      products and services that can be developed this invention are plentiful. Some of them are: Oligonucleotide synthesis as a service; Chip-scale synthesizer for research labs; DNA based sensors (aptamer sensors); Disease diagnostics; Vaccine and drugs; Platform for DNA data storage; Biofuels; Biofertilizers; and Nanomaterials. Based on successful proof-of-concept experimental / more-detailed implementations in accordance with the present disclosure, numerous advantages and improvements have been recognized. For example, such implementations have been shown to minimize reaction errors stemming from misalignment of optical beams and reagent droplets with respect to the synthesis sites as in traditional photolithographic and inkjet printing based synthesizers (competing technologies). Moreover, in the absence of reaction errors, longer oligo strands (e.g., at least 300 nucleotides) can be synthesized to encode complex genetic functions, and the high-Q of the nanoantennas can be used to lessen or minimize (e.g., optimize for a particular application) spatial and spectral overlap of synthesis sites, thereby enabling high-density packing of synthesis sites (approximately 5M per centimeter square) as well as many unique spectral addresses for the generation of an extremely diverse set of oligo sequences. This is advantageous compared to electrochemical array-based synthesizers (competing technology) where it can be difficult to have a large number of independently controllable electrodes. Further, such approaches of this type can aid in the high-throughput screening of a large sequence space for the optimal genetic function. Ready access to diverse sets of complex oligo sequences will reduce the cost of synthesis per base. This will help democratize oligonucleotide synthesis which is currently provided as a service by a handful of vendors. Ready access to oligo sequences will accelerate the engineering of biological systems for applications across sustainable fuel and energy, food security, healthcare and climate change. Yet further aspects of the present disclosure are directed to a synthesizer which manifests error free solid-phase synthesis of at least 100 unique sequences using the B-MOS technology as described in the present disclosure. One such metasurface oligonucleotide synthesizer manifests: transduction of the wavelength and polarization dependent switching of optical resonance into switching of photothermal heating; wavelength and polarization dependent switching in photothermal heating for site selective synthesis; uses photothermal switching to demonstrate the synthesis of at least 100 unique sequences; and uses sequencing, HPLC and gel electrophoresis to characterize the error rate of synthesized strands as a function of the strand length. STFD.463PCT (S24-133) 13      FIGs.2A and 2B illustrate an type of oligonucleotide-synthesis apparatus, according to the present disclosure. FIG.2A is a general schematic diagram of the apparatus and its components. The components include an on-chip microfluidic-reaction- chamber platform with inlets for receiving reagents, a (transparent dielectric) substrate, a metasurface chip integrated onto the substrate as part of the on-chip microfluidic-reaction- chamber platform, and light from a light source as indicated by the illumination (or optical beam). Integrated (and sealed) within the metasurface chip are fluidic channels and a culture chamber. Other aspects of the apparatus, as shown in FIG.2A, are relevant to core operations of the synthesis. The microfluidic reaction chamber includes sufficient inlets to receive the input reagents and sufficient outlets to yield one or more output reagents as synthesized oligonucleotides The outlets are also for the extraction of wastes and products. In the middle, the metasurface-chip device is integrated within the microfluidic reaction chamber and is uses an integration of the fluidic channels and the culture chamber, to enable the enzymatic synthesis cycle to act on the input reagents. The metasurface-chip device also includes one or more arrays, with each such array including at least one nanostructure integrated into at least one high-Q dielectric nanoantenna. Each array is associated with one or more relatively-unique responses (spectral- / wavelength-specific and polarization-specific) and is to provide at least one of a plethora of synthesis sites, with each such site being optically excitable by switching or varying at least one of a wavelength parameter and / or a polarization parameter. With each of the nanoantennas being associated with a respectively unique optical response, by manipulation of one or both such illumination parameters, each site is independently excitable. As such, the external alignment of the optical beam is not required. Avoiding the need to provide external alignment of the optical beam can be highly advantageous. While previous approaches have provided site-selective synthesis in the context of microarray based synthesizers, alignment steps are needed to precisely direct the light to targeted regions of the arrays. For example, inkjet and photolithographic approaches use external mechanical motion of the printhead or deflection of the optical beam using digital micromirror devices to selectively activate intended synthesis sites for deblocking. Consequently, these types of devices suffer from errors due to misalignment of the optical beam or droplet with respect to the synthesis sites, and these errors accumulate to limit the synthesizable strand length (N). Previous approaches have also used on-chip activation as in electrochemical synthesis or thermal synthesis, and this involves use of electrodes in physical STFD.463PCT (S24-133) 14      contact with the synthesis sites, which difficult to connect uniquely addressable electrodes to each site. This limits the number of unique oligo sequences that can be synthesized. Moreover, these electrodes take up valuable device footprint and this limits site density on the array. Accordingly, the apparatus of FIG.2A uses one or more arrays, which is associated with one or more relatively-unique responses, to enable each array-based synthesis site to be independently excited by adjusting light beam parameter(s), thereby overcoming the deficiencies of systems needing external alignment of the optical beam. The optical excitation causes the enzymatic synthesis cycle (which may be iterative) to act on the input reagent(s). In a more specific example, the enzymatic synthesis cycle is implemented as a two-step enzymatic synthesis cycle for oligonucleotides with the two steps being and / or including the site selective thermolytic deblocking step and the coupling step. The synthesis product is shown in FIG.2A at the independently-excited regions of the arrays, with synthesis efficiencies at least 99.9%. The upper portion of FIG.2A shows a replication of the arrays to illustrate that the exemplary platform can not only drive site selective synthesis of many unique sequences, but can also drive the site-selective PCR amplification of the products using photothermal heating -- also at the independently-excited regions of the arrays. This may be realized as part of a post-amplification effort and can be used to microfluidically segregate the products into homogenous oligo pools (“PCR Amplification on-chip” as in FIG.2A), thereby significantly simplifying subsequent assembly efforts. In specific example implementations using PCR amplification, PCR is catalyzed by the thermophilic enzyme Taq polymerase by using repetitive heat cycles, with each cycle including multiple thermal steps including for example: denaturation (e.g., at 94-98C), annealing (e.g., at 50-65C) and extension (e.g., at 75-80C). By using gold as (or included in) nanostructures integrated with nanoantennas at the array-based sites, each of which is enabled to be activated selectively as discussed above, the platform can also be used as metasurface based photothermal heaters. In this manner, by activating one or more sites selectively, the products are selectively amplified for synthesis at individual sites associated with the nanoantennas. At each site, because the synthesized products may be in relatively small quantities (compared to what is typically used for gene assembly), the synthesized products may be amplified. The amplification can occur before the synthesis products are detached from the substrate (e.g., processed out as depicted via channel and outlet to the right of FIG. 2A. If the synthesis products were to be detached from the substrate before amplification, all STFD.463PCT (S24-133) 15      the different sequences would form a mixed and upon amplification there would be a mixed pool of amplified oligos that would result in subsequent segregation efforts effectively impossible for realizing a desired assembling order. Where known segregated-amplification approaches require either the additional synthesis of barcoded primers for segregation or alignment efforts as used in error-prone inkjet printing, aspects in accordance with the present disclosure enable selective activation of the metasurface based heaters to amplify the synthetic products while they are at the activated site. Post-amplification efforts can be used to microfluidically segregate the products into homogenous oligo pools, thereby significantly simplifying subsequent assembly efforts. FIG.2B is a diagram illustrating how a metasurface structure functions as part of the synthesis platform of FIG.2A. In this particular illustrated example, the metasurface includes arrays of silicon nanostructures which have different nanostructure dimensions and orientations and heat up when illuminated by light of specific wavelength and polarization. A thin layer of gold may be deposited on the nanostructures for more efficient absorption of light and transduction to heat. As discussed in connection with FIG.1 and elsewhere, the two-step enzymatic synthesis cycle is used to grow the oligonucleotide chain one nucleotide at a time by enzymatically adding it to the accessible 3’ end after the deblocking step. Both the enzymatic coupling and deblocking step are temperature sensitive, in part due to the heat transduction. For example, the deblocking step (from which the strand has a solid support) may occur at more than approximately 50° C, and coupling step may occur at approximately 37° C. FIGs.3A-3D respectively depict further aspects, in accordance with certain exemplary aspects of the present disclosure. FIG.3A illustrates in more detail exemplary aspects of the apparatus shown in FIG.2A. In particular, FIG.3A shows optical and SEM images of fabricated biperiodic arrays of silicon nanostructures. FIG.3B is a graph showing wavelength dependent temperature rise of resonators with different dimensions. FIG.3C is a graph showing polarization dependent switching between resonators of different orientations. FIG.3D is a set of pictures showing incorporation of the metasurface chip into a 3D printed plastic microfluidic device. FIGs.4A-4B are a set of diagrams useful in showing an example of how coupling reaction is enabled to function, in accordance with certain exemplary aspects of the present disclosure. FIG.4A shows deblocking and coupling steps, according to the present disclosure, for a particular reaction affecting an initiator strand. This particular example shows temperature dependent enzymatic coupling of fluorescently labelled nucleotide STFD.463PCT (S24-133) 16      (dCTP-AF647) to the initiator strand. In the high temperature drives the thermolytic removal of the blocking group (TBE-tert butoxy ethoxy) from the 3’ end of the growing oligonucleotide chain. Removal of the blocking group facilitates incorporation of an incoming fluorescently labelled nucleotide FIG.4B shows temperature dependent enzymatic coupling. The coupling is shown to manifest varying degrees, with the images in the three columns at temperatures of 35° C, 4° C and -20° C, respectively from left to right. The degree of coupling is directly proportional to the brightness in red in the bottom row, thereby indicating that the left column (35°) shows the highest coupling. FIG.4C is graph showing aspects associated with the deblocking and coupling of FIG.4A, and in particular, showing that activation occurs with a temperature increase or pH decrease. FIGs.5A-5B are a set of system-level diagrams, consistent with the above aspects (e.g., as disclosed in connection with FIGs.1, 2A-2B, etc.) and also in accordance with the present disclosure. FIG.5A shows an example experimental setup including a tunable laser light source (e.g., available from Toptica) that is tunable to provide high power tunable laser light source for wavelength control. The high power, for example, with power being adjustable to exceed 100mW, is useful to generate sufficient heat for certain of the functions including the site-selective excitation at array-based regions. As denoted by P1, a linear polarizer is used to facilitate (or ensure) that the output of the laser has a single well- defined polarization state. The light path passes from the linear polarizer (P1) to a half wave plate (HWP1) and a convex lens (L1). The half wave plate is used to rotate the polarization passing out of P1, for controlling the polarization of light incident on the sample. The sample, as shown near the top of FIG.5A, is contained in the platform (or metasurface chip) for DNA synthesis, as previously discussed. The convex lens is used to focus the incident beam on the back focal plane of the lens so that the light coming out of the objective that is incident on the sample is collimated. The light path passes from the convex lens to a beamsplitter, which splits the light into an incident beam path for reaction processing and collection beam path (including lens L2 passing scattered light in response to the incident light directed towards the sample) for observation at a camera (e.g., available from NIRvana), which is used to capture IR images of the sample during and as an effect of cyclic processing as indicated by the scattered light. The lens L2 is configured to focus scattered light (scattered by the sample), which is STFD.463PCT (S24-133) 17      collected by the depicted “Objective Lens”, the camera so that every point on the sample is mapped onto a single point on the camera. A temperature-controlled microscope stage is used, in conjunction with metasurface chip, to mount the sample. This stage includes control for adjusting the x-y position to ensure the intended section of the sample is illuminated. The temperature control allows to maintain the different temperature ranges required to ensure selective sited activation (e.g., the two-step cycle including coupling and deblocking). Another camera, which is a thermal camera as shown above a low-pass filter (F1), is used to capture thermal images of the sample due to photothermal heating (as controlled via the microscope stage). The low-pass filter (F1), is used to block optical and NIR wavelengths and pass long wavelength thermal radiation to facilitate the thermal imaging. FIG.5B shows an expanded view of certain components used in the example experimental setup of FIG.5A, and in particular with a first-level expanded view focusing on the upper portion of FIG.5A and a second-level expanded view showing an example of the metasurface-based structure including nanoantenna arrays, each with an integrated gold- based nanostructure. The components shown in the first-level expanded view include the thermal camera, the metasurface-based structure and the light beam after the light beam would pass through the objective lens of FIG.5A. The thermal camera is shown to have picked up three thermal images, respectively indicating three selectively activated (or excited) and include array regions, and / or entire array(s) (depending on the particular implementation). In this particular example, the metasurface-based structure of FIG.5B shows a bottom layer, which is a transparent dielectric substrate (to pass the incident and scattered light), a quartz-based layer on or over the transparent dielectric substrate, and gold (e.g., as a coating or layer) on or over the quartz-based layer. The gold is deposited over nanostructures (e.g., amorphous silicon) which are integrated with the previously-disclosed nanoantennas. Among each of the illuminated arrays illustrated in FIG.5B, the array that is tuned to the laser wavelength and power heats up to have a sufficiently high temperature in order to activate the previously-described cycle, as exemplified by driving a deblocking reaction (e.g., >50°C while the other arrays are at room temperature) and driving a coupling reaction (e.g., 37°C while other arrays are at -20°C). The reagent supply to the metasurface array can be controlled by the microfluidics as disclosed hereinabove. Consistent with the above aspects disclosed in connection with the example systems of FIGs.2A- 2B and FIGs., 5A-5B, these and others aspects may be used with, and STFD.463PCT (S24-133) 18      better understood based on, optical devices components (e.g., including but not limited to examples of metasurfaces, materials and nanostructures) as disclosed in one or more of the following patent applications as published by WIPO and / or the USPTO (each having one or more common inventors and a common Applicant / Assignee): WO2022036314 (US20230299551A1, especially regarding FIGs.3C and 5A), PCT Publication Nos.: WO2023097050 (US 2025 / 0020596A1 (STFD.438PCT), especially regarding FIGs.3A, 3B, 5 and 6); WO2024112939 (STFD.447PCT, especially regarding FIGs.1, 2E, 2F, 2G and 4A); and WO2022076832 (U.S. Appln. Ser. No.18 / 028,873 (STFD.453PCT / S20-284), especially regarding FIGs.1A, 1B, 1C, 2A and 2C), wherein to the extent permitted, such identified subject matter is incorporated by reference in its entirety generally, specifically for the above- identified subject matter and also to the extent that further aspects and examples may be useful to supplement and / or clarify aspects associated with functions and structures of the optical aspects of the above-referenced example embodiments and aspects of the present disclosure. As research continues, alternative types of deblocking and coupling (other than thermolytic deblocking and TdT coupling) may be used with other aspects of the claimed invention including the more generally-characterized example method and apparatus embodiments. Such alternative approaches are characterized below (see also Pichon, M., Hollenstein, M. Controlled enzymatic synthesis of oligonucleotides. Commun Chem 7, 138 (2024), and online at https: / / doi.org / 10.1038 / s42004-024-01216-0). For thermolytic deblocking such alternatives include: (a) enzymatic deblocking (instead of heat, certain enzymatic methods use enzymes like phosphatases to remove blocking groups from the 3'- hydroxyl of the growing oligonucleotide; (b) photocleavable blocking groups (using photocleavable blocking groups to be removed by light exposure); and (c) phosphate blocking group: (a phosphate group as a blocking group that can be efficiently hydrolyzed by commercially available phosphatases under conditions similar to the TdT-coupling. For TdT coupling such alternatives include: (a) polymerase-nucleotide conjugates by tethering a single, unblocked nucleotide to the TdT enzyme via a linker, creating a polymerase- nucleotide conjugate, thereby facilitating each enzyme only adding one nucleotide at a time and preventing unwanted extensions, and then another enzyme is used to cleave the linker, preparing the DNA for the next synthesis cycle; (b) instead of relying solely on a blocked nucleotide, TdT activity is kinetically controlled by adding a competing enzyme, like apyrase, that degrades the nucleotide substrates and by tuning the ratio of TdT to apyrase, the addition of nucleotides can be limited to a single base; (c) use of template-dependent STFD.463PCT (S24-133) 19      polymerases for enzymatic synthesis, the enzymes catalyze the co-polymerization of nucleoside triphosphates on DNA primers, resulting in sequences of variable lengths; (d) DNA ligases for use to join fragments of DNA, including those containing modified nucleotides, which approach can be used in combination with polymerases to synthesize DNA strands containing unnatural nucleotides at specific positions or even to ligate pre- synthesized short artificial oligonucleotides; and (e) new enzymes and synthesis strategies, for example, currently being developed (e.g., Camena Bioscience which is developing "TdT- free" DNA synthesis technologies, such as their gSynth platform, that utilize a proprietary enzyme mix and process to produce DNA). Such alternatives were suggested at para.0032 without naming any in particular. Consistent with the above aspects, such a manufactured device or method of such manufacture may involve aspects presented and claimed in U.S. Provisional Application Serial No.63 / 662,912 filed on June 21, 2024 (STFD.463P1 / S24-133) with Appendices A-B, to which priority is claimed. To the extent permitted, such subject matter is incorporated by reference in its entirety generally and to the extent that further aspects and examples (such as experimental and / more-detailed embodiments) may be useful to supplement and / or clarify. As specific examples, the above-characterized figures and discussion are provided to help illustrate certain aspects (and advantages in some instances) which may be used in the manufacture of such structures and devices. These structures and devices include the exemplary structures and devices described in connection with each of the figures as well as other devices, as each such described embodiment has one or more related aspects which may be modified and / or combined with the other such devices and examples as described hereinabove and as may also be found in the Appendices A-B of the above-identified U.S. Provisional Application. The skilled artisan would recognize various terminology as used in the present disclosure. As examples, the Specification may use forms of the words “comprise” and “include” as being synonymous open-ended inclusive terms, reference to a noun in the singular in various contexts may refer to one by itself or at least one being from among a plurality, and reference to “example” is not intended to be limiting (e.g., “example” and “non-limiting example” are synonymous). The present disclosure may describe and / or illustrates aspects useful for implementing the examples by way of various semiconductor materials / circuits which may be illustrated as or using terms such as layers, blocks, modules, device, system, unit, controller, and / or other circuit-type depictions. Such optical elements and / or semiconductive materials, synthesizer elements and / or related parts and circuitry may STFD.463PCT (S24-133) 20      be used together with other elements to how certain examples may be carried out in the form or structures, steps, functions, operations, activities, etc. It would also be appreciated that terms to exemplify orientation, such as upper / lower, left / right, top / bottom and above / below, may be used herein to refer to relative positions of elements as shown in the figures. It should be understood that the terminology is used for notational convenience only and that in actual use the disclosed structures may be oriented different from the orientation shown in the figures. Thus, the terms should not be construed in a limiting manner. Based upon the above discussion and illustrations, those skilled in the art will readily recognize that various modifications and changes may be made to the various embodiments without strictly following the exemplary embodiments and applications illustrated and described herein. For example, methods as exemplified in the Figures may involve steps carried out in various orders, with one or more aspects of the embodiments herein retained, or may involve fewer or more steps. Such modifications do not necessarily depart from the true spirit and scope of various aspects of the disclosure, including aspects set forth in the claims.

Claims

STFD.463PCT (S24-133) 21      What is Claimed:

1. An oligonucleotide-synthesis apparatus comprising: a microfluidic reaction chamber including inlets to receive one or more input reagents and including outlets to yield one or more output reagents as synthesized oligonucleotides; and a metasurface-chip device being within the microfluidic reaction chamber and including each of one or more arrays, wherein each of the arrays includes at least one nanostructure integrated into at least one high-Q dielectric nanoantenna, each of the one or more arrays is associated with one or more wavelength or specific spectral responses and polarization responses and is to provide at least one of a plethora of synthesis sites, and each of the synthesis sites is optically excitable by switching or varying at least one of a wavelength parameter and a polarization parameter to cause at least one enzymatic synthesis cycle, acting on the one or more input reagents.

2. The apparatus of claim 1, wherein each of the one or more arrays is associated with at least one of geometry and spatial orientation to characterize the one or more wavelength (or specific spectral) responses and polarization responses.

3. The apparatus of claim 1, further including, as part of the metasurface-chip device, an optically-transparent synthesis substrate on or over which the one or more arrays are patterned.

4. The apparatus of claim 1, further including an optical source, including controls for affecting wavelength and polarization parameters, to direct light at and selectively excite the synthesis sites between respective on and off states by switching or varying the wavelength and polarization parameters.

5. The apparatus of claim 1, wherein the metasurface-chip device and the microfluidic reaction chamber are cooperatively configured with each of the synthesis sites being independently optically excitable by switching or varying said at least one of a wavelength parameter and a polarization parameter to cause at least one enzymatic synthesis cycle.STFD.463PCT (S24-133) 22      6. The apparatus of claim 1, wherein chip device has a synthesis-site density of more than one million of the synthesis sites per square centimeter of surface area on the metasurface-chip device.

7. The apparatus of claim 1, wherein each of the high-Q nanoantennas are associated with a Q factor being in a range from several hundred an order of one thousand, and the at least one enzymatic synthesis cycle includes thermolytic deblocking and terminal- deoxynucleotidyl-transferase coupling.

8. The apparatus of claim 1, wherein the synthesis sites are to be excited selectively without causing synthesis errors.

9. The apparatus of claim 1, further including aqueous enzymatic reagents as input reagents to provide the oligonucleotides without generation of toxic wastes.

10. The apparatus of claim 1, wherein the nanostructures have gold, integrated into the one or more arrays, to cause the nanostructures to act as resonant heaters.

11. The apparatus of claim 1, wherein the nanoantennas are spectrally and spatially isolated to facilitate generation of an electric field and heat confinement to facilitate synthesis of the oligonucleotides.

12. The apparatus of claim 1, wherein the metasurface-chip device is configured to facilitate using fabrication steps common to MOS-based fabrication that includes patterning of the arrays for integration or creation of the synthesis sites.

13. The apparatus of claim 1, wherein each of the high-Q nanoantennas is associated with a Q factor that is sufficiently high to minimize spatial and spectral overlap between the synthesis sites, and is to facilitate generation of an electric field.

14. The apparatus of claim 1, wherein each of the at least one high-Q dielectric nanoantenna has a high-Q factor in a range from multiple hundreds to 1000 spectral responses.STFD.463PCT (S24-133) 23      15. The apparatus of claim 1, further one or more metasurfaces as part of the metasurface-chip device, each of the one or more metasurfaces having a respective optical response that depends on dimension and orientation of the one or more arrays.

16. The apparatus of claim 1, wherein the metasurface-chip device includes a plurality of activatable metasurfaces, each being independently activatable by tuning wavelength and polarization of light.

17. The apparatus of claim 1, wherein the nanostructures are integrated into the one or more arrays and configured to act independently as site-selective resonant heaters to amplify synthesized products associated with the one or more output reagents at individual one of the synthesis sites.

18. A method involving use of a microfluidic reaction chamber for the synthesis of oligonucleotides, the method comprising: while the microfluidic reaction chamber contains and is integrated with a metasurface-chip device that includes each of one or more arrays, wherein each of the one or more arrays including at least one nanostructure integrated into at least one high-Q dielectric nanoantenna, and each of the one or more arrays is to provide at least one of a plethora of synthesis sites, receiving one or more input reagents at inlets of the microfluidic reaction chamber for yielding one or more output reagents as synthesized oligonucleotides at outlets of the microfluidic reaction chamber; and optically exciting at least one of the synthesis sites, by switching or varying at least one of a wavelength parameter and a polarization parameter, to cause at least one enzymatic synthesis cycle, to act on the one or more input reagents.

19. The method of claim 18, further including effecting, after said at least one enzymatic synthesis cycle occurs, PCR amplification of certain synthesized products by optically exciting a selected set of one or more of the synthesis sites.STFD.463PCT (S24-133) 24      20. The method of claim 18, wherein the nanostructures are configured to be act independently as heaters to amplify synthesized products associated with the one or more output reagents at individual ones of the synthesis site, and the method further comprises: generating heat, via the at least one of the synthesis sites, by the step of optically exciting, to amplify one or more synthesized products associated with the one or more output reagents at individual one of the synthesis sites, and microfluidically segregating the one or more synthesized products, in response to the one or more synthesized products being amplified, into homogenous oligo pools.

Images