WO2019055990A1 - Method and system for synthesis of long molecules using photonic device - Google Patents

Abstract

An apparatus includes a nanophotonic cavity, a waveguide, a microfluidic channel, and a photon detector. The cavity and the waveguide are fabricated out of a wide band gap semiconductor with an index of refraction that provides index contrast with the synthesis medium. One arrangement includes a photonic crystal having walls defining a channel for directing fluid flow. A functionalized spot is disposed in the channel for being tethered to a seed molecule to which a plurality of mers will be added. The walls extend downstream of the functionalized spot by a length that is at least as long as said molecule when assembled. The molecule can be linearized by a suitable fluid flow.

Description

METHOD AND SYSTEM FOR SYNTHESIS OF LONG MOLECULES USING

PHOTONIC DEVICE

RELATED APPLICATIONS

E o o o i ] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 62/559,790, filed on September 18, 2017, which is incorporated herein by reference in its entirety.

: 0002 ] It is known in the art to take a strand of DNA and identify the sequence of base pairs. This process (known as "sequencing") has been helpful in promoting the

understanding of genetics. Various techniques for biomolecule identification are described by J. Bernstein in U.S. Patent Application Publication No. US 2006/0011862, published on January 19, 2006 and U.S. Patent No. 7,511,285, issued on March 31, 2009.

[ 0003 ] In many cases, however, it is desirable to not only know what base pairs are in naturally-occurring DNA but to be able to synthesize new strands of DNA with one's own choices for base pairs. The ability to do so could give rise to many commercial and medical applications. Known methods of attaching nucleotides include standard phosphoramidite solid phase synthesis. Also relying on phosphoramidite chemistry are approaches that use printed DNA microarrays for enabling chip-based chemical DNA synthesis with error correction.

[ 0004 ] Some recent techniques for synthesizing molecular chains such as single- stranded DNA are described by I.W. Frank et al. in International Publication No. WO 2017/222710 Al, published on December 28, 2017. This document is incorporated herein by this reference in its entirety. r 0005 ] In one approach, WO 2017/222710 describes an apparatus for optically-verified de novo DNA synthesis. The apparatus includes a microfluidic system that has channels leading in and out of a synthesis chamber having a functionalized region on a fl oor thereof on which a single-strand of DNA, to which nucleotides are to be attached, can be fixed. The chamber is in optical communication with both an illumination system, which excites an electron in a fluorophore that is attached to the DNA strand, and a detection system, which detects a signature photon emitted as the excited electron decays into its ground state,

SUMMARY OF TH E I NVENTION

[ 0006 ] A need continues to exist, however, for methods and devices suitable in synthesizing molecular chains.

[ 0007 ] In one aspect, invention concerns a system for the synthesis of long molecules monitored using a photonic device. This photonic device typically includes a photonic crystal cavity, an input waveguide, and an output waveguide. The system further includes an excitation source, a microfluidic channel assembly providing fluid flow transversely across the photonic cavity, and a photon detector. The photonic crystal and waveguides can be fabricated out of a wide band gap semiconductor with an index of refraction that provides a sufficient index contrast with the synthesis media flowing through the microfluidic assembly channel such that the light remains guided in the cavity and waveguides.

[ 0008 ] A functionaiized spot is disposed both in the assembly channel and in the photonic crystal cavity. A backbone is tethered to the spot and a plurality of units

(nucleotides or other "mers") are then added to form a molecule. Preferably, the fluid flow- in the assembly channel stretches the backbone and all mers already assembled to the backbone during assembly of the molecule. The photonic structures on either side of the functionaiized spot are generally at least as wide (in the transverse direction) as the molecule is long, when assembled,

[ 0009 ] The input waveguide, the photonic crystal cavity, and the output waveguide have impedances selected such that excitation light, coupled into the cavity from the input waveguide, results in a spatially confined resonant optical modes inside the cavity that are strongly coupled to the input and output waveguides to facilitate light transfer into and out of the cavity. This significant coupling enables signal light generated in the photonic cavity due to the interaction between the excitation light and the molecule being assembled to first couple into the cavity and then exits on the output waveguide for detection with a high probability.

[ 0010 ] In another aspect, the invention features a method for synthesizing a molecule. The molecule is tethered in photonic crystal cavity. The cavity is typically at least as wide (in the transverse direction) as the molecule will be long, when assembled. A microfluidic assembly channel provides fluid flow transversely through the crystal . The assembly channel is configured for supporting fluid flow and includes the gap between the photonic structures on either side of a functionalized spot, and possibly other gaps formed between other photonic structures of the photonic crystal cavity. The synthesis is conducted by growing the molecule, tethered to the functionalized spot, one or more units (e.g., one or more nucleotides) at the time.

[ 0011 ] The photonic crystal is used to interrogate the molecule with excitation light and then capture one or more signal photons emitted from possibly a beacon coupled to the most recently attached unit. In specific examples, solutions containing ingredients, for instance loaded or empty carriers, enzymes, byproducts, etc., are directed into and out of the microfluidic assembly channel. In many cases, the fluid flow through the channel is such that the molecule is stretched out along the assembly channel. r 0012 j In general, according to one aspect, the invention features a system for processing one or more molecules. The system comprises a photonic cavity, a channel extending through the photonic cavity, and a spot for holding a molecule such that the molecule is in the channel and at least partially in the photonic cavity.

[ 0013 ] In embodiments, the photonic cavity includes photonic structures, in which some of the photonic structures define the channel in the photonic cavity. A width of the photonic structures preferably increases along at least part of a length of the cavity with the channel being defined by longer ones of the photonic structures. Then media for processing the one or more molecules flows continuously or intermittently through the channel in a direction that is at least partial ly transverse to an optical axis of the photonic cavity. This function can be performed by a pump for flowing media for processing the one or more molecules through the channel.

[ 0014 ] A controller is preferably provided that controls the flow of media for processing the one or more molecules at a rate sufficient to prevent the folding of the molecules. r o o 15 j In operati on, the photonic cavity form s a standing wave of light for

photoiuminescing the one or more molecules and couples signal light generated by the molecules photoiuminescing out of the photonic cavity, A portion of the photonic device is reflective to excitation light and transmissive to signal light. [ 0016 ] In general, according to another aspect, the invention features a method for processing one or more molecules. The method comprises holding a molecule such that the molecule is in channel and at least partially in a photonic cavity, determining a status of the molecule by detecting signal light generated by exciting the molecule with excitation light forming a standing wave in the photonic cavity, and flowing media for processing the molecule through the channel.

[ 0017 ] Practicing aspects of the invention can provide a fast, reliable, high throughput and possibly inexpensive approach for preparing molecular chains, and, in particular long molecular chains. In some cases, embodiments described herein can be used to prepare DNA chains having more than 200 bases, preferably more than 10,000 bases. Equipment and procedures according to the invention can be incorporated in larger systems, taking advantage of microfluidic technologies, for example. Many of the approaches described herein can be applied or adapted to sequencing processes. r o o 18 j The above and other features of the invention including various novel details of construction and combinations of parts, and other advantages, will now be more particularly described with reference to the accompanying drawings and pointed out in the claims. It will be understood that the particular method and device embodying the invention are shown by way of illustration and not as a limitation of the invention. The principles and features of this invention may be employed in various and numerous embodiments without departing from the scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[ 0019 ] In the accompanying drawings, reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale; emphasis has instead been placed upon illustrating the principles of the invention. Of the drawings:

[ 0020 ] FIG. 1 is a top plan view of a system for synthesis of long molecules according to the invention;

[ 0021 ] FIG. 2 is a side cross-section of the photonic crystal device shown in FIG. 1 ;

[ 0022 ] FIG. 3 is a partial side view of photonic crystal cavity with a molecule under construction in the assembly channel, with only a few mers attached to the molecule; [ 0023 ] FIG. 4 is a partial si de view of assembly channel looking in the direction of the optical axis of the cavity showing the molecule s extending along the substrate as a result of fluid flow;

[ o 024 ] FIG. 5 A i s a plot of electric field magnitude as a function of transverse position (along x-axis) in the assembly channel as the channel width is varied from 700 nm to 2489 nm;

[ 0025 ] FIG. 5B is a plot of electric field magnitude as a function of position along the length (y-axis) of the cavity showing changes in the magnitude the electric field as the channel length is varied from 700 nm to 2489 nm; and

[ 0026 ] FIGS. 6A and 6B are top views illustrating an input waveguide transition region and an output waveguide transition region.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[ 0027 ] The invention now will be described more fully hereinafter with reference to the accompanying drawings, in which illustrative embodiments of the invention are shown. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[ 0028 ] As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items. Further, the singular forms and the articles "a", "an" and "the" are intended to include the plural forms as well, unless expressly stated otherwise. It wil l be further understood that the terms; includes, comprises, including and/or comprising, when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements,

components, and/or groups thereof. Further, it will be understood that when an element, including component or subsystem, is referred to and/or shown as being connected or coupled to another element, it can be directly connected or coupled to the other element or intervening elements may be present.

[ 0029 ] It will be understood that although terms such as "first" and "second^'" are used herein to describe various elements, these elements should not be limited by these terms. These terms are only used to distinguish one element from another element. Thus, an element discussed below could be termed a second element, and similarly, a second element may be termed a first element without departing from the teachings of the present invention.

[ 0030 ] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

[ 0031 ] The invention generally relates to the synthesis of molecules, and, in particular, to the synthesis of long molecules. In many cases, the molecule to be assembled is a chain type molecule such as found, for instance, in DNA strands. Examples of molecules that can be assembled or grown include polymeric biomolecules, e.g., nucleic acid sequences such as DNA, single stranded for instance, RNA, proteins, polypeptides, enzymes, antibodies, aptamers and so forth.

[ 0032 ] Many long molecules have a backbone that is made up of units, often called "mers," placed along the backbone. For example, in an oligomer involving genetic material, the units are nucleic acids. In a protein, the units are the "R" groups that distinguish one amino acid from another.

[ 0033 ] Various synthetic approaches can be employed, as currently known in the art or as developed in the future. Possible chemistries that can be used are described in Griswold et al., International Publication No. WO 2018/102554 Al, Sprachman et ai., International Publication No. WO 2017/22271 1 Al, and Magyar et al ., in International Application No. PCT/US2018/033798 with the title Modified Template -Independent DNA Polymerase, filed on May 22, 2018. An alternative compatible chemistry for non-DNA polymer synthesis is described in Magyar et al, International Publication No. WO 2018/1293281 A.

[ 0034 ] In many embodiments of the invention, the synthesis is conducted by the iterative addition of molecular units on to the end of the immobilized seed molecule.

Reagent addition is controlled microfluidicaily and photoluminescence, such as fluorescence or phosphorescence, is monitored through the photonic device at different steps to verify successful incorporation. [ 0035 ] The apparatus and methods described herein are configured to assemble a molecule by building it step by step, possibly one unit at a time. Specific embodiments also include approaches for confirming, at each step, that the desired unit has indeed been added to the chain.

[ 0036 ] In the case of growing a DNA chain, for instance, the assembly involves adding one nucleotide (naturally occurring, a synthetic analog, etc.) at the time. In further embodiments, the synthetic process employed prevents subsequent undesired additions of the same nucleotide. In many cases, the synthetic route uses an enzyme, such as, for instance, terminal deoxynucleotidyl transferase (also commonly known by the abbreviation TdT or TDT) for attaching the nucleotide at the growing end. The nucleotide can be added to the to the backbone of the strand, typically at the 3' end. For a tethered arrangement, the addition can occur at the free end or at the attached end. As a result, the molecule (DNA chain) grows progressively longer. r 0037 ] A typical nucleotide sequence can have thousands of nucleotides. Since the synthesis procedure involves adding one nucleotide at a time, being able to add nucleotides quickly may present significant advantages. Also, the functionality of a DNA strand depends a great deal on the absence of any errors in the nucleotide sequence. Even a small error is enough to impair, if not destroy, a DNA molecule's functionality. Thus, a practical approach must be both fast, reliable, and able to fix errors as they occur. A suitable synthesis procedure is described below.

[ 0038 ] To begin the assembly process, one end of an initial or seed molecule (an initial DNA strand, for example) is exposed to a solution, provided, for instance, via

microfluidics. The solution contains many molecules of a loaded carrier and many- molecules of an enzyme such as TDT.

[ 0039 ] The carrier is designed to include a blocking group appended to a signaling group. In many cases, the signaling group carries one fluorophore. The carrier exists in two states: a loaded state, and an empty state. In the loaded state, the carrier covalently bonds to its payload, e.g., the nucleotide being added. In the empty state, the carrier is no longer bonded to its payload. Or, stated in a different manner, in the empty state the carrier can accept a payload.

[ o o 4 o ] To transition from the loaded state to an empty state, the carrier undergoes a cleaving of a covalent bond between itself and its payload. This covalent bond is configured such that the cleavage mechanism will cleave this bond while leaving other bonds undisturbed. The cleaving can be carried out in a variety of ways. For example, it is possible to illuminate the carrier with photons of appropriate energy, thus promoting optical cleavage. In other approaches, the bond is cleaved chemically, electro-chemical ly or by another suitable mechanism.

[ o o 41 ] In one illustration, if the nucleotide to be added is guanine, the environment around the DNA strand is flooded with many loaded carriers that contain guanine. Then, to attach, for example, cytosine on top of the guanine on the DNA strand, one would rinse away any loaded carriers carrying guanine, and then flood the environment with a whole new set of loaded carriers, this time carrying cytosine instead. This permits the serial attachment of different kinds of nucleotide to the growing DNA strand,

[ 0042 ] Typically, attachment to the DNA strand does not happen instantly and a waiting period may be required. The waiting time interval can be selected based on routine experimentation, prior experience and/or other considerations. Preferably, the waiting period is long enough so that it is very likely that one of the enzymes and one of the loaded carriers will encounter each other at the appropriate end of the growing DNA strand.

When this happens, the enzyme causes the loaded carrier to attach to the DNA end. In some implementations, the waiting period is further optimized to reduce or minimize unnecessary delays in the overall synthesis.

[ 0043 ] In many embodiments, the loaded carrier is provided with a blocking group and once one loaded carrier has been attached to the DNA strand, its associated blocking group prevents any further loaded carriers from attaching themselves.

[ 0044 ] After having waited for the full attachment interval, there is still a possibility that nothing was able to attach. It is therefore important to confirm that the loaded carrier did in fact attach to the appropriate DNA end. This is performed by taking advantage of the signaling group provided on the loaded carrier. Under illumination by interrogatory photons, at the interrogation wavelength, the fluorophore provided by the signaling group emits signature photons in response. As used herein the term "interrogation" refers to the process of illuminating the fluorophore with interrogatory photons, while the term

"response" refers to the resulting emission of signal photons at one or more signal wavelengths. [ 0045 ] Since in solution each carrier has its own signaling group with its own fluorophore, it is useful to rinse away the carriers in the solution before the interrogation step, thus avoiding detection of spurious signature photons. As a result, if attachment was successful, there will be one signaling group remaining, namely the one belonging to the signaling group of the one carrier that ultimately attached to the DNA strand, bringing with it the newly-added nucleotide.

[ 0046 ] Since only one signature photon can be emitted at a time, collection efficiency becomes important. Even with high collection efficiency, it is often necessary to repeatedly interrogate. If, after repeated interrogation, no signature or signal photon is detected, one can infer that nothing was able to attach. Therefore, another attempt is made to attach the carrier. For example, a new solution containing the same loaded carrier can be provided, essentially as described above.

[ 0047 ] On the other hand, if a signal photon is detected, one can infer that the carrier is now attached to the appropriate end of the DNA strand. At this point, both the signaling group and the blocking group have performed their role and are removed for one or more reasons. For example, their presence in the finished product may interfere with its function. Also, if the blocking group remains, no further attachments can occur. And, if the signaling group is retained on the molecule being assembled, its fluorophore may emit signature photons during subsequent interrogation phases, causing confusion since a detector would have no way of knowing where a photon was coming from.

[ o o 48 ] In one impleme tation, the carrier i s detached from the molecule being assembled electrochemically, using electrodes placed adjacent to the nanophotonic device. The electrodes are biased appropriately to either oxidize or reduce the bond that attaches the carrier to the molecule being assembled. A possible chemistry to achieve this is described in Sprachman et al, WO2017/222711 Al. The reduction potential of the bond between the signaling group and the blocking group differs from that of the bond between the earner and its payload, a nucleotide in this case. This ensures that the signaling group and the blocking group can be removed together, as a unit. Removing the carrier thus results in only the nucleotide remaining attached to the DNA strand. Other suitable techniques for detaching the carrier include mechanisms that are purely chemical by exposure to a cleavage medium, purely optical or other suitable approaches including hybrid procedures. [ 0049 ] After the carrier removal step, it may be useful to confirm that the signaling group and the blocking group are no longer present. This can be accomplished using the same interrogation procedure described above. If the signaling group is no longer attached, there will be no response. Hence, one can infer, from the absence of any response, that the DNA strand is now ready for the next desired nucleotide. On the other hand, if a signature photon is detected, one simply repeats the detaching step. Generally, since the signaling group is appended covalently to the blocking group, if the signaling group is not present, one can reasonably infer that the blocking group is also no longer present. r o o 5 o j In many aspects of the inventi on, the synthesi s i s conducted using techniques and equipment that promote assembly of long molecules. In one example, DNA chains being assembled by adding one nucleotide at a time can be a hundred nucleotides long or more, e.g., within the range of from about 50 to about 200 to 300 or more nucleotides long.

[ 0051 ] A suitable system for assembling a desired molecule, a DNA strand, for example, by adding one or more units at a time, includes a photonic device, which typically includes a photonic crystal cavity, an input waveguide, and an output waveguide, a microfluidic channel assembly providing fluid flow transversely through the crystal cavity, and a signal photon detector.

[ 0052 ] The photonic crystal cavity is formed in a photonic (also referred to herein as a "nanophotonic") crystal structure. These typically periodic optical nanostructures affect the propagation of photons in a manner similar to that in which ionic lattices affect electrons in solids. Some suitable materials that can be used include, silicon, silicon nitride, silicon carbide, and diamond.

[ 0053 ] Specific implementations of the invention utilize stoichiometric silicon nitride (Si₃N₄ or, sometimes, SiN, non-stoichiometric silicon nitride SixNy - where x is not equal to 3 and/or y is not equal to 4 - may also be appropriate in some situations) formed

(patterned) in a layer having a suitable thickness, 200 nanometers (nm), for example.

Generally, however the thickness is related to the operational wavelengths of the light used in the system. Thus, the thickness is often in a range of about 50-2000 nm, or more.

Advantages associated with this material involve the ease with which one can obtain a high-quality film and the well-known technology for processing silicon nitride. On the other hand, any convenient material could be used so long that it is adequately transmissive to light at the interrogation and signal wavelengths and has an appropriate index of refraction at those same wavelengths.

[ 0054 ] In addition, silicon nitride is relatively easy to functionalize, has a refractive index greater than that of water, and is transparent at many potential operational wavelengths. This makes it a good choice for guiding light through a waveguide that contacts an aqueous medium. On the other hand, its index of refraction, while adequate, is not impressive. Moreover, silicon nitride can have a tendency to fluoresce, giving rise to a background emission that may interfere with detection of the signature or fluorescent photons. As a result, other options are silicon carbide, diamond, or titanium dioxide (all of which are currently more difficult to process for photonic integrated circuits). Despite its drawbacks silicon nitride has been shown to enable photonic designs that provide high enough quality factors and Purceil factors to overcome background fluorescence.

[ 0055 ] Specific embodiments utilize a photonic cry stai cavity in which the

electromagnetic radiation at the interrogation wavelength, the fluorescent signal(s) emitted from a fluorophore used during the growth of a molecular chain, for example, is well behaved, e.g., less likely to result in photobleaching (the destruction of the

photoluminescence (fluorescence, for instance) from the fluorophore). The cavity can have properties that promote one dominant longitudinal cavity mode, with a reduced or minimized number of harmonics.

[ 0056 ] Suitable photonic crystals can be fabricated by currently known methods or by techniques developed in the future. anophotonic structures can be made, for example, from a submicron layer (a couple of hundred nanometers thick, e.g., within the range of from about 150 ran to about 300 nm, for instance 200 nm, for signal wavelengths around 700 nm) of a material such as silicon nitride. The nanophotonic structures are disposed on possibly a silicon oxide (SiOi) layer (commonly referred to as a buried oxide layer (BOX)) should be at least 500 nm thick but can be a few microns (μηι) thick (within a range of from 2 to about 10, e.g., 5 to 7 um). From an optical perspective there is no maximum thickness for the BOX. Limitations in the BOX are related to difficulty in growing extremely thick thermal oxides including time of processing and the build-up of stress which can lead to wafer bowing and cracking in the layers. Support for the SiO:? layer can be provided by a thicker e.g., a few hundred micrometers (500 um thick, for instance) silicon handle wafer. [ 0057 ] Photonic structures can be defined through electron-beam (e-beam) lithography exposure of 500 nm ZEP-520A e-beam resist (Many other e-beam resists are possible, hydrogen siisesquioxane based resists or others). The e-beam resist is developed in xylenes and the pattern is transferred in to the silicon nitride device layer through an anisotropic reactive ion etch (inductively-coupled reactive-ion-etch (ICP RIE)) using carbon tetrafluoride chemistry. Other approaches rely on any lithographic process that provides sufficient resolution, such as extreme ultraviolet lithography, or direct ion milling with a focused ion beam to define the device structures. r 0058 j Coupling light into and out of the photonic device and specifically, the device's input and output waveguides, can be achieved by fabricating grating structures at one or either end of the waveguides that enable far field coupling of fluorophore excitation light into the device to excite fluorescence and collection of signal photons for detection. In other approaches, fluorophore excitation light is coupled into the device, and fluorescence emission collected via feeder waveguides which are exposed at the chip edges. Optical coupling can then be performed using, for example, a fiber v-groove array, a commercially available chip with precisely spaced grooves designed so that a standard fiber will rest in them at a predictable position and height and then these fibers are butt-coupled to the waveguides.

[ 0059 ] Photon detection can be performed using a single-photon-sensitive detector, such as an avalanche photodiode. The signal from the photodetector is monitored with a controller such as a computer, which determines if the signal is sufficient to indicate the incorporation of a monomer into the strand that is being synthesized.

[ 0060 ] Fig. 1 shows a system 100 for synthesis of long molecules, which has been constructed according to the principles of the present invention.

[ 0061 ] In general terms, the system 100 comprises a micro fiuidic channel subsystem 60 and a photonic crystal cavity 14, The micro fiuidic channel subsystem 60 provides and circulates the chemicals required to grow one or more molecules. The photonic crystal cavity 14 interrogates that molecule or molecules during its assembly to confirm whether the steps in the assembly have been successfully completed.

[ 0062 ] In more detail, an excitation source 15, such as a laser, generates light at the interrogation wavelength. This light is coupled into an input waveguide 12 that couples the light into the photonic crystal cavity 14. The efficiency with which the excitation light is coupled into the cavity 14 is partially dictated by the input waveguide transition region 1 10.

[ 0063 ] The photonic crystal cavity 14 is characterized by a typically linear array of photonic structures 20 that extend along the length and optical axis AO of the cavity. In the preferred embodiment, these photonic structures 20 gradually grow in their transverse width (lengt along the x-axis) from being sized to match the input waveguide 12 at the cavity input and to having a maximum transverse width W at the center of the cavity 14. After the center of the cavity, the photonic structures 20 then progressively decrease in their transverse width until they match the transverse width of output waveguide 16, at the cavity output. This accomplishes a modulation of the effective index experienced by- photons travelling along the waveguide. Other modulation techniques include keeping the width fixed but varying the size or period of the "holes" that comprise the photonic crystal (lower index areas). r 0064 j Signal photons are coupled out of the cavity into the output waveguide 16. The design of the output waveguide transition region 1 16 ensures efficient coupling of those photons into the output waveguide 16.

[ 0065 ] In general, the length of each structure in the y-axis direction is between 100 nm and 200 nm. The size of the gaps 22 between the structures 20 in the y-axis directions are generally between about 50 and 100. Moreover, while only 18 photonic structures 22 are shown in the cavity 14, typically the number of these structures is much larger, number in the range of 50 to 100 or more structures where the number is chosen to ensure a strong band-gap effect. For structures with larger refractive indices and therefore stronger photonic bandgaps the number can be smaller. r 0066 ] In specific implementations, the width of the structure 22 of the cavity 14 in the x-axis direction can curve quadraticaily between two values. In one example the width at the center of the cavity W, is 700 nm and the width of the waveguide at the end of the photonic crystal, WW, is 500 nm. The width of the photonic crystal along the y axis, w(y) is given by: w(y) =^:: W - (W -WW)*(2 *y/L)^A2, where L is the length of the photonic crystal of 20 micrometers. This design can be modified for a larger transverse width where W can range from 700 nm (W 0) to 3000 nm (W MAX). In order to maintain strong optical properties WW must be modified to match W using the formula: WW = (W- W_0)/(W_MAX-W_0)*500 nm+500. [ 0067 ] The photonic cavity 14 is designed such that the excitation light is efficiently coupled into the cavity 14 from the input waveguide 12. Light in the cavity 14 then forms a standing wave between the junctions with the input waveguide 12 and the output waveguide 16 due to the impedance differences between the waveguides and the photonic cavity 14. Such an impedance mismatch can be caused by introducing structural inhomogeneity. In one embodiment, the structural in homogeneity is introduced by the periodic nature of the photonic structures 20 separated by the gaps 22 and the quadratic growth of the width of these structures. r 0068 j In the preferred embodiment, the spacing and the y-axis length of the photonic structures 22 of the cavity are controlled to make the cavity One-way' for the interrogation wavelengths and the signal photons. In more detail, at the input end 1 12 of the cavity 14, the photonic structures 22 (spacing and length) are designed to maximize the reflectivity at the wavelengths of the signal photons. For example, the input end 1 12 has a reflectivity of greater than 99% and preferably greater than 99.9% at the wavelengths of the signal photons. Further, the input end 112 of the cavity is also at least somewhat reflective (reflectivity of greater than 80% and preferably greater than 99%) at the interrogation wavelengths generated by the excitation source 15 in order to create the resonant cavity to support the confined mode. On the other hand, at the output end 114 of the cavity 14, the photonic structures 22 (spacing and length) are designed to maximize the reflectivity at the wavelengths of the excitation light. Preferably, region 1 16 is a second photonic crystal structure that is designed to function as a dichroic filter that provides a reflectively of greater than 99.5% at the wavelengths of the excitation light. At the same time, the region 116 is transmissive at the wavelengths of the signal photons. For example, the region 116 has a transmissivity of greater than 80% at the wavelengths of the signal photons. Fig. 6B shows a "fishbone design" crystal structure that performs these functions.

[ 0069 ] Among the gaps 22 is an assembly channel gap 22-C between the two center and largest photonic structures 20-L and 20-R. The channel 22-C extends in a direction transverse to the optical axis AO. The one or more molecules 30, such as a single DNA strand, is attached to a fundi onalized spot 26 and is synthesized in the assembly channel gap. In other implementations, however, multiple molecules might be located in the channel and/or in adjacent channels and tethered to the same or their respective spots. [ 0070 ] The signal light is coupled out of the cavity 14 into the output waveguide 16 and detected by the signal photon detector 74. Often, dichroic filters or other filter modalities are used to improve the extinction ratio and ensure that the photon detector 74 is only sensitive to signal photons of the desired wavelength and not photons from the excitation source 60. The signal photon detector 74 is monitored by the controller 75 which determines whether the new unit (e.g., a nucleotide) was successfully attached.

[ 0071 ] Photon detection can be performed using a single-photon-sensitive detector, such as an avalanche photodiode. The signal from the photodetector is monitored with controller 75, which determines if the signal is sufficient to indicate the incorporation of a monomer into the strand that is being synthesized.

[ 0072 ] The signal photon detector 74 is coupled to the output waveguide 16 such as by epoxy bonding the detector 74 to an end facet of the waveguide 16 or by coupling light from the waveguide to the detector using a grating, for example. r 0073 ] Further, electrodes 80 A, SOB are located on either side of the cavity 14 and at either end of the assembly channel 22-C. These electrodes are electrically biased by the controller 75 to electrochemically detach the carrier at the appropriate time by either oxidizing or reducing the bond that attaches the carrier to the molecule being assembled. Preferably, the electrodes are attached to or formed in the oxide layer 18 and can be fabricated from a transparent conducting oxide such as Indium tin oxide (ITO) in order to avoid spoiling the optical cavity.

[ 0074 ] FIG. 2 is a side view. It shows the input waveguide 12 and the output waveguide 16 along with the photonic structures 20, all structures 20 having a consistent height in the Z axis direction. This allows them to be formed by the same layer of silicon nitride for example. In the preferred embodiment, the silicon nitride is formed on a silicon dioxide layer 18, which is, in turn, supported by a silicon wafer substrate 8. On the other hand, at least some of the gaps are sealed with a top cover 7, such a microscope side cover slip.

[ 0075 ] Shown in FIG. 3 is the assembly channel 22-C, located between the two center photonic structures 20-L, 20-R that also function as assembly walls, and including a functionalized spot 26. The backbone 24 of the molecule being assembled or grown, (molecule 30 in FIG. 3) attaches, at tethered end 32, to functionalized spot 26. [ 0076 ] Methods for immobilization of the seed DNA molecule are described in Frank et al. in WO 2017/222710 Al. In one example, functionalized binding spot 26 is a carboxysiiane-activated binding spot, for example. Other materials that can be employed in making a functionalized spot include but are not limited to orthogonal chemistries such as biotin/streptavidin or alkyne/azide click chemistries.

[ 0077 ] Returning to Fig. 1, a micro fluidic channel assembly 60 is also provided. It includes an input channel 62 that provides media via a manifold 64 to the assembly channel 22-C. Often, the media might additionally flow through other gaps in parallel. The input channel 62 receives media from connected media sources. Each source supplies a carrier loaded with a corresponding one of a plurality of naturally-occurring nucleotides. These include loaded-carrier sources 56A, 56G, 56C 56T that would be loaded with adenine (A), thymine (T), guanine (G) and cytosine (C) in the case of assembling DNA. Also included are one or more flushing-medium sources 58 that contain one or more types of flushing media, as well as one or more engineered-enzyme sources 59. Each media source has a corresponding valve 50 for selectively connecting that source to the first channel 62 under control of the controller 75.

[ 0078 ] On the other side of the photonic crystal cavity 14, a second manifold 66 collects the media after it has passed through the cavity 14 and provides it to a pump 68 such as a peristaltic pump. In the illustrated example, the media is returned via a return line 72 so that it can be continuously flowed through the assembly channel 22-C, in the illustrated example. A waste reservoir 70 is also provided for removing media from the microfluidic channel system 60.

[ o o 79 ] To maximize the length of the linear chain that can be monitored during synthesis, the seed molecule can be attached upstream of the center line of the crystal cavity 64, and close to edge of the structures in the transverse direction,

[ 0080 ] This preferred placement in assembly channel 24 is shown in the cross-sectional view of FIG. 4.

[ 0081 ] In a typical embodiment, the width W of the assembly channel 22-C is between 30 nm and 80 nm wide. In specific examples, the two center photonic structures 22-L, 22- R form an assembly channel 24 having a width that is comparable to the length of the molecule 30 once synthesis has been is completed. For instance, the length of assembly channel 24 can be the same or greater than the final length of molecule 30 that is being assembled. In one example, the channel 24 has a length that is at least as large as the unfolded length of molecule 30 when completely assembled. In another example, assembly channel 24 is located midpoint of the cavity 14. In a further example, assembly channel 24 is the longest channel formed by structures 20. In yet another example, assembly channel 24 is the longest among channels (gaps) 22.

[ 0082 ] For preparing short chains, or at the early stages of the assembly process, while not too many units (mers) 34 have been attached to the backbone 35, molecule 30 is likely to remain essentially linear. As a result, there is little to interfere with attaching additional mers 34. Thus, in one mode of operation, the controller 75, which controls the pump 68 may only operate the pump 68 intermittently, as necessary to flush new media through the assembly channel 22-C. However, as additional mers 34 are being added, it becomes increasingly likely that the minimal-energy configuration of the growing molecule 30 will involve folding. Such folding may interfere with its further assembly. Thus, this propensity to fold can render the process of making a longer molecule 30 less reliable.

[ 0083 ] Thus, during assembly of the molecule 30, especially as the molecule gets longer, fluid flows through the gaps 22, including the assembly channel 22-C,

continuously, by operation of the pump 68, under control of the controller 75, for example. Moreover, as the molecule get longer, the flow rate can be increased by the controller 75 to further prevent folding.

[ 0084 ] For a seed molecule immobilized on the left-hand side of the crystal, microfluidic flow from left to right perpendicular to the crystal axis can be used to linearize the molecule during synthesis, as illustrated in FIG. 4. In specific embodiments, the fluid velocity is selected to be high enough to exert a force that tends to straighten out the molecule 30 and/or inhibit the folding of the molecule 30 as it is being assembled. This makes assembly of even very long molecules 30 more reliable.

[ o o 85 ] The fluid velocity can be further adjusted by the control ler 75 to be low enough to avoid tearing the molecule 30 away from the functionalized spot 26. In practice, a fluid flow that is high enough to prevent or minimize folding, yet not high enough to rip the molecule away from functionalized spot 26 can be determined by routine experimentation, modeling calculations, prior experience or other suitable approaches.

[ 0086 ] In one example, the flow is within the range of from about 10 nL/min to about 10 uL/min. [ o o 87 ] Although the process is being described in terms of a photonic crystal 14, the benefits of suppressing the tangling of a molecule can be useful even when no photonic structure is being employed and can also be implemented by chemical means such as through the use of an denaturing agent such as urea .

[ 0088 ] The synthesis described above with respect to growing a DNA strand can be applied, adapted or expanded to assembling molecule 30, whether the molecule is a nucleic acid chain, a protein, polypeptides, enzyme, antibody, aptamer, another oligomer, and so forth.

[ o 089 ] Briefly, mer 34 can be attached to a beacon that emits a signaling photon in response to illumination by an interrogation photon. An example of such a beacon is a fluorophore that emits a signature photon in response to illumination by an interrogator}' photon. Some fluorophores that can be used are shown in Table 1 below:

Fluorescent Dye Excitation Max (nm) Emission

Texas Red X 599 617

Carb oxy-X- Rliodamine 585 612

Carb oxyFlu oresce in 521

Carb oxyTetraMethyl- 561 591

Rhodamine

Carboxycyanine 5.0 650 667

[ 0090 ] To add a particular mer using techniques that involve a beacon, the assembly channel 24 is first flooded with a solution that contains a complex formed from a mer bonded to a beacon. One of these complexes will attach to the backbone 27 before any others. In so doing, it prevents others from attaching. In one example, undesired subsequent attachments are prevented by providing the complex with a blocking group.

[ 0091 ] The complex-containing solution is then flushed from the assembly channel 24, leaving behind only the complex that is attached to the molecule being assembled. An interrogation photon then illuminates the complex, and in particular, the beacon. This triggers emission of a signaling photon. Upon detecting the photon, attachment is verified, and the beacon can be detached and rinsed away, thus preparing the backbone 27 for adding the next mer.

[ 0092 ] Since only one signaling photon is emitted per interrogation photon, it is important to maximize the chance of detecting that photon. To maximize the chance of spontaneously emitting from an interrogation photon at a particular wavelength, it is useful to design the cavity 14 to support only a single mode of the standing wave for the excitation wavelength. In one embodiment, the length of the assembly channel 24 is on the order of 700 nanometers and the width of the first section is about 500 nanometers.

Assuming an effective index of refraction of about 1.8 for a silicon nitride photonic crystal bathed in aqueous solution and a wavelength on the order of 720 ran, this results in a confined mode whose lowest order mode has a mode volume of about 8- 10° nnr.

[ 0093 ] In situations in which the mer 34 is added to the tethered end 32, the response photon will always be emitted from the same location, namely the functionalized spot 26, However, in those cases in which the mer 34 is added to the free end 36, the location from which the response photon is emitted will move as the molecule 30 grows,

[ 0094 ] To ensure that the response photon is always emitted into the photonic crystal cavity 14, it is useful to elongate the first and second assembly wails/photonic structures 22-C, 22-R, 40. In one implementation, the first and second assembly walls 38, 40, and hence the assembly channel 24, are extended to 3000 ran in the x-axis direction.

[ 0095 ] In such an apparatus, the photonic crystal cavity 14 is likely to support standing waves of higher order modes, possibly resulting in a somewhat lower Purcell factor (the emission rate enhancement of a spontaneous emitter inside a cavity or resonator) and reduced collection efficiency. On the other hand, many of the benefits of nanophotonic structures can be maintained. These include the ability to place many such structures in a chip of a given area, the ease with which it is possible to build complex routing between such structures, and selective light delivery and collection.

[ 0096 ] For preparing long chains, it is desirable to provide as long a channel as possible. Simply increasing this channel dimension, however, can lead to a smearing out of the electric field magnitude. Further aspects of the invention relate to approaches and considerations for determining the properties of a suitable channel or cavity in which to carry out the assembly of molecule 30. Many arrangements benefit from considering trade-offs between wishing to provide an increasingly longer channel, to support the synthesis of longer and longer molecules, with selecting cavity dimensions that also take into consideration the properties of the electric field that the cavity will support. r 0097 ] Computer simulations of the absolute value of the electric field or the magnitude of the electric field for various dimensions along which molecule 30 is grown can be conducted. In many instances, such simulations show that increasing the length of the channel (from 700 rati to 2489 ran, in one example) results in a less focused, more spread out or diffuse electric field.

[ o 098] Fig. 5 A is a plot of the electric field for the stimulation light contained in the cavity 14 in the transverse direction at the location of the assembly channel 22 -C. Thus, after a certain value, increasing the channel width in the x-axis direction can be associated with significant losses in cavity effects.

[0099] Fig. 5B shows the electric field magnitude along the length of the cavity 14 in the y-axis direction. It shows that the magnitude generally peaks at the center of the cavity at the location of the assembly channel 22-C. On the other hand, it shows a fine structure of node, antinodes associated with the standing wave superimposed on the envelope of the confined mode.

[ooioo] In one embodiment, the controller 75 temperature tunes the cavity 14 or wavelength tunes the excitation source, for example, to ensure that the molecule 30 is located at a node of this fine structure of the standing wave to maximize the electric field at the molecule 30 and thus the generation of the signal photons,

[ o o l o i ] Such simulations can be employed to balance advantages associated with increased channel length with disadvantages associated with a less defined electric field. In some cases, an intermediate length can still promote the assembly of long chains and also provide a sufficiently sharp peak in the electric field absolute value along with promoting single mode standing waves. Further exploration of the parameter space may yield higher performing cavities with similar channel width for growth.

[ 00102 ] In addition to assembling molecules, techniques and devices described herein can be applied or adapted to other molecular processing such as sequencing. In one embodiment, approaches presented here for growing molecular chains are employed in DNA sequencing using sequencing by synthesis. Rather than de novo synthesis of a DNA strand using a template independent polymerase, a template dependent polymerase can be used to copy a single stranded DNA molecule. The length of the DNA molecule that can be sequenced is controlled by the width of the nanophotonic device (or the length of channel 22-C). The device can allow sequencing of molecules of up to about 10³ nucleotides.

[ 00103 ] In more detail, where the seed molecule is DNA of an unknown sequence, then the complementary DN A strand is synthesized iteratively using a DNA polymerase using an approach as described in U.S. Pat. Nos. US 8,399,188 B2 and US 9,708,358 B2, which are incorporated herein by this reference. These patents detail approaches for the industry standard sequencing by synthesis.

[ 00104 ] The nanophotonic structure described herein can be coupled with or incorporated in various methods and equipment. Further aspects of the invention relate to methods and systems for monitoring the assembly of molecule 30 and, in particular, to niicrofluidics and/or suitable arrangements and processes for illuminating attached mer 34 and detecting the fluorescence signal emitted in response.

[ 00105 ] In many cases, linearization of molecule 30 (a DNA strand that is being assembled, for example) may benefit from a specific microfluidic device geometry.

Examples of suitable techniques for preparing and/or operating microfluidic channels are described by J. Varsanik et al., Plasmonics in Biology and Medicine, Proc, Of SPIE 7577 (2010) and Bernstein et al., U.S. Patent No. 7,511,285 B2.

[ o o 106 ] One approach for sealing the channel 22-C involves bonding the glass layer 7 to the top of the photonic structures 20 and also sealing the microfluidic channels of the channel assembly. The channels could be formed directly in a silicon nitride or silicon dioxide layer, which can be disposed on the silicon handle wafer 8.

[ 00107 ] Figs. 6A and 6B illustrate an example of input waveguide transition region 110 and output waveguide transition region 116, respectively. In one embodiment, adiabatic waveguide transitions are used.

EXAMPLE

[ 00108 ] The device in this example includes a nanophotonic cavity, a waveguide, a microfluidic channel, and a photon detector. The nanophotonic crystal and waveguide are fabricated out of a wide band gap semiconductor with an index of refraction that is at least 2, to provide sufficient index contrast with the synthesis medium (water in this case). In this example the nanophotonic cavity and waveguide are fabricated from silicon nitride.

[ 00109 ] The nanophotonic structures are fabricated from a 200 nm -thick layer of silicon nitride, which sits on a 6 μτη Si0₂ layer on a 500 μιη thick silicon handle wafer. The photonic structures are defined through electron-beam (e-beam) lithography exposure of 500 nm ZEP-520A e-beam resist. Alternatively, any lithographic process that provides sufficient resolution, such as extreme ultraviolet lithography, can be used to define the device structures. The e-beam resist is developed in xylenes and the pattern is transferred in to the silicon nitride device layer through an anisotropic reactive ion etch using carbon tetrafluoride chemistry.

[ o o 11 o ] While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

What is claimed is: . A system for processing one or more molecules, the system comprising:

a photonic cavity,

a channel extending through the photonic cavity; and

a spot for holding a molecule such that the molecule is in the channel and at least partially in the photonic cavity,

2. A system as claimed in claim 1, wherein the photonic cavity includes photonic structures, in which some of the photonic structures define the channel in the photonic cavity.

3. A system as claimed in claim 2, wherein a width of the photonic structures increases along at least part of a length of the cavity with the channel being defined by longer ones of the photonic structures.

4. A system as claimed in any of claims 1-3, wherein media for processing the one or more molecules flows through the channel in a direction that is at least partially transverse to an optical axis of the photonic cavity.

5. A system as claimed in any of claims 1-4, further comprising a pump for flowing media for processing the one or more molecules through the channel.

6. A system as claimed in any of claims 1 -5, further comprising a controller that controls the flow of media for processing the one or more molecules at a rate sufficient to prevent the folding of the molecules.

7. A system as claimed in any of claims 1-6, wherein the photonic cavity forms a standing wave of light for photoluminescing the one or more molecules and couples signal light generated by the molecules photoluminescing out of the photonic cavity.

8. A system as claimed in any of claims 1 -7, wherein a portion of the photonic cavity is reflective to excitation light and transmissive to signal light.

9. A system as claimed in any of claim 1-8, further comprising:

a detector; and

an output waveguide coupled to the photonic cavity, the output waveguide

receiving photoluminescent light generated in the cavity and coupling the light to the detector.

10. A method for processing one or more molecules, the method comprising: holding a molecule such that the molecule is in channel and at least partially in a photonic cavity;

determining a status of the molecule by detecting signal light generated by

exciting the molecule with excitation light forming a standing wave in the photonic cavity; and

flowing media for processing the molecule through the channel,

1 1 . A method as claimed in claim 10, wherein the channel extends through the photonic cavity.

12. A method as claimed in any of claims 10-1 1, wherein in the molecule is held by a spot that is in the channel.

13. A method as claimed in any of claims 10-12, wherein the photonic cavity includes photonic structures, in which some of the photonic structures define the channel in the photonic cavity.

14. A method as claimed in claim 13, wherein a width of the photonic structures increases a long at least part of a length of the cavity with the channel being defined by longer ones of the photonic structures.

15. A method as claimed in any of claims 10-14, further comprising flowing the media for processing the one or more molecules through the channel in a direction that is at least partially transverse to an optical axis of the photonic cavity.

16. A method as claimed in any of claims 10-15, further comprising controlling a flow rate of media for processing the one or more molecules to be sufficient to prevent the folding of the molecules.

17. A method as claimed in any of claims 10-16, further comprising forming a standing wave of light for photoluminescing the molecule in the photonic cavity and coupling signal light generated by the molecules photoluminescing out of the photonic cavity.

18. A method as claimed in any of claims 10-17, wherein a portion of the photonic cavity is reflective to excitation light and transmissive to signal light.

19. A system for assembling a molecule, the system comprising a photonic crystal including a first wall and a second wall, the first wall and the second wall defining an assembly channel,

wherein the assembly channel is configured for fluid flow;

wherein a functionalized spot is disposed in the assembly channel, the molecule being tethered to the spot,

wherein the first and second walls extend downstream of the functionalized spot by a length that is at least as long as the molecule when assembled, and wherein the assembly channel is longer than other channels defined in the

photonic crystal by other walls.

20. A system comprising:

photonic crystal including photonic structures, at least some of which form

multiple walls extending in a direction that is transverse to a longitudinal axis of the crystal the multiple walls defining a plurality of channels that have different lengths and are configured for fluid flow, wherein the plurality of channels includes a longer channel configured for molecular synthesis,

a sensor for detecting signal light generated in the cavity; and

a waveguide for coupling the signal light from the cavity to the sensor.

21. A method for sequencing or assembling a molecule, the method comprising; tethering a seed molecule to a functionalized spot in an assembly channel in a photonic crystal cavity, wherein the assembly channel is at least as long as the molecule when assembled; and while fluid is flowing through the assembly channel, adding iterative!}' units to an end of the immobilized seed molecule or synthesizing a complementary strand using DNA polymerase.

22. A system for synthesizing long molecules, the system comprising:

a microfluidic channel assembly, and

a photonic crystal cavity including an array of photonic structures, wherein the structures increase in a transverse width from a cavity input to a maximum width near a center of the cavity and decrease in the transverse width from the center of the cavity to a cavity output.