DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Formal Matters
Applicants’ claim amendments and arguments in the reply filed on 12 May 2026 are acknowledged and have been fully considered. Claims 65, 67-104, and 106-121 are pending. Claims 65, 67-81, 83, 85-86, 88-90, 93, 95-104, 106-112, and 114 are under consideration in the instant office action. Claims 82, 84, 87, 91-92, 94, 113, and 115-121 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention and/or species, there being no allowable generic or linking claims. Claims 1-64, 66, and 105 are canceled. Applicant amended claim 65.
Withdrawn Objections/Rejections
Rejections and/or objections not reiterated from the previous office actions are hereby withdrawn as are those rejections and/or objections expressly stated to be withdrawn.
Rejections-Maintained
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Note: The claims are examined with respect to the elected species of a PFPE-PEG di-block copolymer as the surfactant type, a first water based fluid and a second oil based fluid as the fluid types, cholesterol-tagged DNA as the specific and defined first compound, an amine tagged DNA as the specific and defined second compound, water as the specific and defined first fluid, oil as the specific and defined second fluid, and a DNA-based link as spacer.
Claims 665, 67-81, 83, 85-86, 88-90, 93, 95-104, 106-112, and 114 remain rejected under 35 U.S.C. 103 as being unpatentable over Holtze et al. (US20150217246, IDS reference) and Ishikawa et al. (ChemRxiv, 05 September 2018, Version 1, pages 1-27).
Applicant claims
Applicant claims a surfactant-stabilized fluid interface.
Determination of the Scope and Content of the Prior Art
(MPEP 2141.01)
Holtze et al. teach surfactants (e.g., fluorosurfactants) for stabilizing aqueous or hydrocarbon droplets in a fluorophilic continuous phase are presented. In some embodiments, fluorosurfactants include a fluorophilic tail soluble in a fluorophilic (e.g., fluorocarbon) continuous phase, and a headgroup soluble in either an aqueous phase or a lipophilic (e.g., hydrocarbon) phase. The combination of a fluorophilic tail and a headgroup may be chosen so as to create a surfactant with a suitable geometry for forming stabilized reverse emulsion droplets having a disperse aqueous or lipophilic phase in a continuous, fluorophilic phase. In some embodiments, the headgroup is preferably non-ionic and can prevent or limit the adsorption of molecules at the interface between the surfactant and the discontinuous phase. This configuration can allow the droplet to serve, for example, as a reaction site for certain chemical and/or biological reactions. In another embodiment, aqueous droplets are stabilized in a fluorocarbon phase at least in part by the electrostatic attraction of two oppositely charged or polar components, one of which is at least partially soluble in the dispersed phase, the other at least partially soluble in the continuous phase. One component may provide colloidal stability of the emulsion, and the other may prevent the adsorption of biomolecules at the interface between a component and the discontinuous phase. Advantageously, surfactants and surfactant combinations of the invention may provide sufficient stabilization against coalescence of droplets, without interfering with processes that can be carried out inside the droplets (see abstract). As used herein, “nonaqueous” is meant to define material such as a fluid that is immiscible with water. That is, a liquid that when mixed with water will form a stable two-phase mixture. The non-aqueous phase need not be liquid, but can be a solid or semi-solid lipid or other nonpolar substance that is not soluble in water. In some instances, the nonaqueous phase can include a lipophilic component (e.g., a hydrocarbon) or a fluorinated component (e.g., a fluorocarbon). The aqueous phase can be any liquid miscible with water; that is, any liquid that, when admixed with water, can form a room-temperature, single-phase solution that is stable. In some cases, the aqueous phase can comprise one or more physiologically acceptable reagents and/or solvents, etc. Non-limiting examples of aqueous phase materials include (besides water itself) methanol, ethanol, DMF (dimethylformamide), or DMSO (dimethyl sulfoxide) (paragraph 0049). The choice of size and geometry of a surfactant (including outer and headgroup components) as applied to the stabilization of emulsions including an alcohol as a discontinuous phase is one example of tailoring droplets using description contained herein. Without wishing to be bound by any theory, the inventors have discovered the following trends and observations. In order of decreasing polarity, methanol (MeOH), ethanol (EtOH), and i-propanol (i-PrOH) are similar in their chemical properties and each does not dissolve in certain fluorocarbon oils. In some cases, each of methanol, ethanol, and i-propanol dissolve in a substance that can be used as an headgroup component of a fluorosurfactant. In one embodiment, the substance is PEG or a derivative thereof, which suggests that PEG-fluorophilic (e.g., PFPE)-block copolymers can stabilize emulsions comprising the alcohols in a fluorophilic continuous phase. However, surprisingly, it was discovered that the fairly polar methanol group may be stabilized with any of the applied surfactants, Table 1 shows that surfactants of certain block lengths provided long-term stabilizing i-propanol emulsions. These observations may be applied to other emulsions including low polarity solvents as the discontinuous phase (paragraph 0088). Surfactants with small PEG- and small PFPE-blocks may decrease the surface tension; however, they may not provide colloidal stabilization of the emulsion. Increasing the length of both the PEG- and the PFPE-blocks may improve the long-term stability of the emulsion. In some cases, the influence of the outer-facing portion of the surfactant (e.g., PFPE) may be more important for long term stabilization than that of the headgroup portion (e.g., PEG). This suggests that the failure of emulsion stabilization may be dominated by the formation of a bare patch on the interface of two adjacent droplets, giving rise to neck formation and subsequent coalescence. This may also suggest that an inappropriate surfactant geometry or too short of an headgroup portion of a surfactant (e.g., PEG) that will facilitate surfactant desorption may be counterbalanced by a thicker stabilizing (e.g., outer-facing portion) layer, such as longer fluorophilic components of a surfactant (paragraph 0089). As the difference in polarity of the disperse and continuous phases becomes smaller, larger PEG-blocks and/or larger PFPE-blocks may be required for stabilizing emulsions. Larger PEG-blocks may provide a better anchoring strength to the interface compared to smaller PEG-blocks. Larger PFPE-blocks may shield a greater interfacial area more efficiently against coalescence compared to smaller PFPE-blocks. In some cases, the effect of the PFPE-block variation is more pronounced than that of the PEG-block variation. This may be due to the capability of a greater stabilizing moiety to cover a nearby bare patch or prevent or inhibit the bare patch from forming. In some embodiments, an increase in the size of one or both of the blocks could decrease the surfactant mobility on the interface, making the formation of bare patches less likely (paragraph 0091). In another embodiment, an emulsion of the present invention comprises THF as a discontinuous phase and a fluorophilic continuous phase stabilized by fluorosurfactants described herein. In one embodiment, the surfactant comprises PEG and PFPE. In some cases, both longer PEG and longer PFPE blocks may afford an improved stabilization; the effect of the PFPE blocks may be more pronounced. In other cases, however, there are exceptions to this trend. For instance, surfactants including a PEG portion that has a higher molecular weight than the PFPE portion may stabilize droplets better than surfactants including a PFPE portion that has a higher molecular weight that the PEG portion. This result may be associated with the pronounced geometry of the surfactant molecules that cause the formation of thermodynamically stable, swollen micelles that cannot coalesce (paragraph 0092). As mentioned, in some embodiments, the emulsions of the invention include discontinuous aqueous and/or lipophilic (e.g., hydrocarbon) droplets in a continuous, fluorophilic phase. This means that separate, isolated regions of droplets of an aqueous and/or lipophilic component are contained within a continuous fluorophilic phase, which may be defined by a fluorocarbon component. The discontinuous aqueous and/or lipophilic droplets in the nonaqueous phase typically have an average cross-sectional dimension of greater than 25 nm. In some embodiments, the average cross-sectional dimension of the droplets is greater than 50 nm, greater than 100 nm, greater than 250 nm, greater than 500 nm, greater than 1 micron, greater than 5 microns, greater than 10 microns, greater than 50 microns, greater than 100 microns, greater than 200 microns, or greater than 500 microns, etc. As used herein, the average cross-sectional dimension of a droplet is the diameter of a perfect sphere having the same volume as the droplet (paragraph 0047). In one set of embodiments, a headgroup of a fluorosurfactant is connected to a linking moiety. In some cases, the linking moiety is a relatively small entity. The linking entity may comprise, for example, a morpholino group (e.g., dimorpholino and monomoropholino groups). The linking entity also may comprises a phosphate group in some instances. In certain embodiments, the linking entity comprises both a morpholino group and a phosphate group (e.g., a dimoporpholino phosphate) (paragraph 0064). In some embodiments, a linking moiety (e.g., positioned between A and B components of a fluorosurfactant) may be chosen to assist the self assembly and the packing of the fluorosurfactant at the interface. Additionally, a linking moiety may have a good impact on the CMC (critical micelle concentration), and therefore on the diffusion to a newly formed interface from the fluorophilic phase, which may be important for emulsification (paragraph 0065). In some embodiments, fluorosurfactants of the invention comprise two oligomeric (or polymeric) components including a fluorophilic component (e.g., component “A”) and a hydrophilic component (e.g., component “B”). These components may form a diblock-copolymer (e.g., a “A-B” structure), or other structures including those described herein (paragraph 0085). In certain embodiments, fluorosurfactants of the invention include triblock-copolymers (e.g., A-B-A structures), whose mid-block is soluble in the discontinuous phase. This “double-tail” morphology is known to have advantages in the colloidal stabilization properties over certain “single-tail” (e.g., A-B) surfactants. In some embodiments, the mid-block can include a poly(ethylene glycol) moiety. Many poly(ethylene glycol)s are available with two reactive headgroups on either end of the polymer-chain, which can facilitate the synthesis of double-tail morphologies. However, the synthetic routes described herein may be used for the synthesis of other surface active morphologies, such as diblock-copolymers, multi-block-copolymers, polymer brushes, etc. In some cases, the triblock copolymer may also contain one or more linking moieties, for example, as in the structure (A-X1—B—X2)n, where each “X” independently represents a covalent bond or a linking moiety, and the each X may be the same or different (paragraph 0093). In one embodiment, emulsions of the invention are prepared using microfluidic systems. For instance, the formation of droplets at intersection 92 of device 90 is shown in FIG. 6. As shown in illustrative embodiment, fluid 94 flows in channel 96 in the direction of arrow 98. Fluid 94 may be, for example, an aqueous or lipophilic solution that forms the discontinuous phase of a droplet. Fluid 104 flows in channel 106 in the direction of arrow 107, and fluid 108 flows in channel 110 in the direction of arrow 112. In this particular embodiment, fluids 104 and 108 have the same chemical composition and serve as a carrier fluid 116, which is immiscible with fluid 94. In other embodiments, however, fluids 104 and 108 can have different chemical compositions and/or miscibilities relative to each other and to fluid 94. At intersection 92, droplet 120 is formed by hydrodynamic focusing after passing through nozzle 122. These droplets are carried (or flowed) in channel 124 in the direction of arrow 126 (paragraph 0115). In certain embodiments, the discontinuous aqueous and/or lipophilic phase of a droplet/emulsion may include one or more physiologically acceptable reagents. The reagents may be dissolved or suspended in the discontinuous phase. In another set of embodiments, the discontinuous aqueous and/or lipophilic phase of a droplet/emulsion may include one or more reagents that can participate in a chemical and/or in a biological reaction of interest. Non-limiting examples of reagents that can be involved in a chemical and/or biological reaction, or other chemical and/or biological process, include: buffers, salts, nutrients, therapeutic agents, drugs, hormones, antibodies, analgesics, anticoagulants, anti-inflammatory compounds, antimicrobial compositions, cytokines, growth factors, interferons, lipids, oligonucleotides polymers, polysaccharides, polypeptides, protease inhibitors, cells, nucleic acids, RNA, DNA, vasoconstrictors or vasodilators, vitamins, minerals, stabilizers and the like. In other embodiments, the discontinuous aqueous and/or lipophilic phase can contain toxins and/or other substances to be tested, assayed, or reacted within the droplet. Accordingly, chemical and/or biological reactions may be performed within droplets of the invention. Because conditions of pH, temperature, reactant concentration, and the like will be adjusted for a particular reaction that is to take place within the disperse phase of the emulsion, in some cases, the surfactant system may be tailored so as to preserve the emulsion under these conditions (paragraph 0124). FIGS. 2A-2C show various non-limiting embodiments of fluorosurfactants of the invention. As shown in the illustrative embodiment of FIG. 2A, fluorosurfactant 80 includes headgroup 82 and fluorophilic component 84. As used herein, a fluorophilic component such as component 84 is referred to as an “A”-block and a non-fluorophilic component of a surfactant, e.g., headgroup 82, is referred to as a “B”-block. The combination of a headgroup with a single fluorophilic component forms an “A-B” structure. The A-B structure is referred to as a diblock structure (paragraph 0052). One aspect of the invention involves the formation of stabilized emulsions using fluorosurfactants including those described herein. Surprisingly, in order to obtain long-term stabilized emulsions, certain geometries of the fluorosurfactants are needed in some cases. For instance, certain ratios of molecular weights of the fluorophilic component to the headgroup component may be required for steric stabilization of the droplets. In addition, fluorophilic components having large molecular weights can contribute to long term colloidal stabilization, according to certain embodiments. These and other considerations for choosing appropriate components of fluorosurfactants and suitable mixtures of fluorsurfactants may be suitable for forming certain emulsions, for instance, emulsions comprising droplets having an average diameter in the micron or micrometer range. These and other criteria are described in more detail below (paragraph 0054). Accordingly, the droplets and emulsions produced in accordance with various embodiments of the present invention have a variety of uses. For example, in one embodiment, the droplets are used as reaction vessels for carrying out chemical and/or biological reactions within the droplet. Increasing effort is being put into investigating biological systems on very small scales. This involves the observation of cells and their interaction with the environment as well as the investigation of strands of DNA, even of single genes. There are certain advantages to the encapsulation of cells and DNA into aqueous droplets (e.g., a dispersed phase of an emulsion) that are separated from one another with oil (e.g., a continuous phase), as is discussed herein. This is called compartmentalization and it generally allows the screening of much larger numbers of cells or genes at greater rates using much less chemicals than in classical experimental setups, such as Petri-dishes or microtiter plates (paragraph 0118). In certain embodiments, colloidal stabilization of droplets can be achieved while preventing the adsorption of biological materials. For instance, using passivating agents such as PEGs as headgroups of surfactants, droplets containing solutions of biomolecules may be stabilized in fluorocarbon oils while preventing the adsorption of DNA, RNA, proteins or other materials to the interfaces. In some embodiments, cells may be encapsulated in aqueous droplets without adsorbing to the droplet interfaces. They may therefore be investigated as if they were floating in an aqueous bulk medium (paragraph 0142). The emulsions of the present invention may be formed using any suitable emulsification procedure known to those of ordinary skill in the art. In this regard, it will be appreciated that the emulsions can be formed using microfluidic systems, ultrasound, high pressure homogenization, shaking, stirring, spray processes, membrane techniques, or any other appropriate method. In one particular embodiment, a micro-capillary or a microfluidic device is used to form an emulsion. The size and stability of the droplets produced by this method may vary depending on, for example, capillary tip diameter, fluid velocity, viscosity ratio of the continuous and discontinuous phases, and interfacial tension of the two phases (paragraph 0112).
Ascertainment of the Difference Between Scope of the Prior Art and the Claims
(MPEP 2141.02)
Holtze et al. do not specifically teach the incorporation of cholesterol tagged-DNA and the fact that wherein the hydrophobic part of the first compound is interacting with the layer of
surfactant by secondary non-covalent interactions and the molecular recognition site of the first compound extends from the layer of surfactant into the center of the emulsion droplet. These deficiencies are cured by the teachings of Ishikawa et al.
Ishikawa et al. teach Bio-inspired functional microcapsules stabilised with surfactants, copolymers, and nano/microparticles have attracted much attention in many fields from physical/chemical science to artificial cell engineering. Although the particle-stabilized microcapsules have advantages for their stability and rich ways for functionalisation such as surface chemical modifications and shape control of particles, versatile methods for their designable functionalisation are desired to expand their possibilities. Here, we report a DNA-based microcapsule composed of a water-in-oil microdroplet stabilised with amphiphilised DNA origami nanoplates. By utilising function programmability achieved by DNA nanotechnology, the DNA nanoplates were designed as a nanopore device for ion transportation as well as the interface stabiliser. Microscopic observations showed that the microcapsule formed by amphiphilic DNA nanoplates accumulated at the oil-water interface. Ion current measurements demonstrated that pores in the nanoplates functioned as ion channels. These findings provide a general strategy for programmable designing of microcapsules for engineering artificial cells and molecular robots (see abstract). Ishikawa et al. teach as follows:
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Our approach for microcapsules stabilised with the DNA nanoplates is illustrated in Fig. 1; the nanoplates self-assemble at the oil-water interface and then produce a microcapsule based on a W/O microemulsion. We created two types of nanoplates without and with a pore based on the previous report37. The non-pored DNA nanoplate had a hexagonal shape (44 nm each side); the pored DNA nanoplate had a hexagonal shape (52 nm each side) with a centred hexagonal pore (30 nm each side) (Supplementary Fig. 1). Atomic force microscopy (AFM) imaging showed that the designed shapes were accurately formed (Supplementary Fig. 2) (see page 6). An aqueous solution of ~7.5 nM amphiphilic non-pored or pored DNA nanoplates with 1× SYBR Gold nucleic acid stain was added to mineral oil, and then W/O microemulsions were produced by hand tapping. Confocal laser-scanning microscopy (CLSM) images of the W/O microemulsions clearly showed that the amphiphilic non-pored and pored DNA nanoplates localised at the oil-water interface, although DNA nanoplates without the Chol-TEGs were homogeneously dispersed in W/O droplets (Figs. 2c and d, Supplementary Fig. 5). Figs. 2e and f show cross-sectional fluorescence intensity profiles of the droplets stabilised with non-pored and pored nanoplates with 24 Chol-TEG, respectively, which show that the amphiphilic DNA nanoplates localised at the interfacial area with a thickness of 6.2% ± 1.5% (mean ± standard deviation) of the radius of the droplets. These results indicated that the amphiphilic nanoplates allowed the formation of W/O microemulsions. The non-pored DNA nanoplates with 12 or more Chol-TEGs sufficiently localised on the oil-water interface (Fig. 2c), whereas pored DNA nanoplates with even 48 Chol-TEGs less localised (Fig. 2d). To quantitatively evaluate the localisation degree of the nanoplates onto the oil-water interface, we calculated the fluorescence intensity ratio of the droplet interface to its inside (Figs. 2e and f; histograms of Supplementary Figs. 5c and d). The higher the ratio, the more the nanoplates localised on the interface. When the value was lower than 1, the nanoplates did not localise at the interface but dispersed inside. These results showed that the amphiphilic non-pored DNA nanoplates had higher ratios than the pored DNA nanoplates, indicating that the amphiphilic non-pored DNA nanoplates were easier to localise on the interface than the amphiphilic pored DNA nanoplates. This difference may be explained by the difference in rigidity and wettability of the amphiphilic non-pored and pored DNA nanoplates. More specifically, the center large pore may result in less rigidity of the pored DNA nanoplate because of its lower density structure, and also may result in its less wettability because of the lower surface density of the Chol-TEGs; these likely caused the pored DNA nanoplates to form more aggregates dispersed in the water phase (see page 7). Ishikawa et al. teach as follows:
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Figure 2. Water-in-oil droplets stabilised with the amphiphilic DNA nanoplates. (a) and (b) AFM images of Chol-TEG-modified non-pored nanoplates (a) and pored nanoplates (b). The cross-sectional profiles were taken along the A–B and C–D lines in the AFM images, respectively. Scale bars: 100 nm. (c) and (d) CLSM images of W/O droplets containing nonpored (c) and pored (d) nanoplates with 0–48 Chol-TEGs. Green fluorescence areas show the location of the DNA nanoplates. Scale bars: 100 µm. (e) and (f) Cross-sectional fluorescence intensity profiles of the droplets stabilised with amphiphilic non-pored (e) and pored (f) nanoplates with 24 Chol-TEGs. The profiles were measured along the yellow lines. (g) and (h) Localisation degree of (g) non-pored and (h) pored nanoplates modified with 12–48 Chol-TEG groups. The localisation degree was defined as the interfacial fluorescence intensity normalised by the internal fluorescence intensity. The interfacial fluorescence intensity was defined as the average intensity of the interfacial brighter annular area with a thickness of ~6.2% of the droplet radius [shown in (e) and (f)]. The internal fluorescence intensity was defined as the average intensity of the droplet internal area except for the interfacial annular brighter area (see pages 9-10). To investigate the state of localisation of the nanoplates, we performed fluorescence recovery after photobleaching (FRAP) of the oil-water interface stabilised with non-pored DNA nanoplates with 24 Chol-TEGs. Here, a fluorophore, 6-carboxyfluorescein group (6-FAM), was conjugated to the nanoplates using the same procedure for Chol-TEG conjugation (Fig. 3a). Fluorescence of the interface of W/O droplets constructed with the fluorescent amphiphilic nonpored DNA nanoplates was recovered to only ~5% after photobleaching (Figs. 3b and c). In contrast, freely diffusing lipids on liposomes or cell membranes are known to generally recover to ~80%41. Therefore, lateral diffusion of the amphiphilic nanoplates on the interface was very slow unlike lipids. This result suggests that the nanoplates not only localise on the oil-water interface by amphiphilic adsorption but also partly accumulate by hydrophobic interaction with each other based on partial overlap of the amphiphilic nanoplates To evaluate how the accumulated amphiphilic nanoplates affected droplet stabilisation, interfacial tensiometry of the W/O droplets was performed. The interfacial tensions of W/O droplets based on the non-pored DNA nanoplates with 0, 12, and 48 CholTEGs were 28.8 ± 0.3, 28.2 ± 0.3, and 26.1 ± 0.5 mN m−1, respectively; those based on the pored DNA nanoplates were 28.1 ± 0.1, 27.1 ± 0.1, and 26.1 ± 0.2 mN m−1, respectively. The presence or absence of the centre pore of the DNA nanoplate did not significantly affect the interfacial tension, but the interfacial tensions tended to slightly decrease with the number of Chol-TEGs for both nanoplates. These interfacial tensions were much higher than those of wellknown lipids, such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or 1,2- dioleoyl-sn-glycero-3-phosphocholine (DOPC)42 (about 5.3 and 0.5 mN m−1, respectively). These results suggest that the accumulation of the nanoplates at the oil-water interface did not so strongly contribute to the reduction of interfacial tension but the accumulation involving hydrophobic interaction between nanoplates contributed to preventing the unintended droplet coalescence. These results are reasonable if the DNA-nanoplate-stabilised droplet was a Pickering-like emulsion as expected because stabilising particles of Pickering emulsions generally interact with each other to form a weakly flocculated state (see pages 11-12).
Finding of Prima Facie Obviousness Rational and Motivation
(MPEP 2142-2143)
It would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the instant invention to modify the teachings of Holtze et al. by incorporation of a cholesterol tagged-DNA Ishikawa et al. teach Bio-inspired functional microcapsules stabilised with surfactants, copolymers, and nano/microparticles have attracted much attention in many fields from physical/chemical science to artificial cell engineering. Although the particle-stabilized microcapsules have advantages for their stability and rich ways for functionalisation such as surface chemical modifications and shape control of particles, versatile methods for their designable functionalisation are desired to expand their possibilities. Here, we report a DNA-based microcapsule composed of a water-in-oil microdroplet stabilised with amphiphilised DNA origami nanoplates. By utilising function programmability achieved by DNA nanotechnology, the DNA nanoplates were designed as a nanopore device for ion transportation as well as the interface stabiliser. Microscopic observations showed that the microcapsule formed by amphiphilic DNA nanoplates accumulated at the oil-water interface. Ion current measurements demonstrated that pores in the nanoplates functioned as ion channels. These findings provide a general strategy for programmable designing of microcapsules for engineering artificial cells and molecular robots (see abstract). Ishikawa et al. teach as follows:
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One of ordinary skill in the art would have been motivated to include Chol-Teg tagged DNA and wherein the hydrophobic part of the first compound is interacting with the layer of
surfactant by secondary non-covalent interactions and the molecular recognition site of the first compound extends from the layer of surfactant into the center of the emulsion droplet because Ishikawa et al. teach that our approach for microcapsules stabilised with the DNA nanoplates is illustrated in Fig. 1; the nanoplates self-assemble at the oil-water interface and then produce a microcapsule based on a W/O microemulsion. We created two types of nanoplates without and with a pore based on the previous report37. The non-pored DNA nanoplate had a hexagonal shape (44 nm each side); the pored DNA nanoplate had a hexagonal shape (52 nm each side) with a centred hexagonal pore (30 nm each side) (Supplementary Fig. 1). Atomic force microscopy (AFM) imaging showed that the designed shapes were accurately formed (Supplementary Fig. 2) (see page 6). An aqueous solution of ~7.5 nM amphiphilic non-pored or pored DNA nanoplates with 1× SYBR Gold nucleic acid stain was added to mineral oil, and then W/O microemulsions were produced by hand tapping. Confocal laser-scanning microscopy (CLSM) images of the W/O microemulsions clearly showed that the amphiphilic non-pored and pored DNA nanoplates localised at the oil-water interface, although DNA nanoplates without the Chol-TEGs were homogeneously dispersed in W/O droplets (Figs. 2c and d, Supplementary Fig. 5). Figs. 2e and f show cross-sectional fluorescence intensity profiles of the droplets stabilised with non-pored and pored nanoplates with 24 Chol-TEG, respectively, which show that the amphiphilic DNA nanoplates localised at the interfacial area with a thickness of 6.2% ± 1.5% (mean ± standard deviation) of the radius of the droplets. These results indicated that the amphiphilic nanoplates allowed the formation of W/O microemulsions. The non-pored DNA nanoplates with 12 or more Chol-TEGs sufficiently localised on the oil-water interface (Fig. 2c), whereas pored DNA nanoplates with even 48 Chol-TEGs less localised (Fig. 2d). To quantitatively evaluate the localisation degree of the nanoplates onto the oil-water interface, we calculated the fluorescence intensity ratio of the droplet interface to its inside (Figs. 2e and f; histograms of Supplementary Figs. 5c and d). The higher the ratio, the more the nanoplates localised on the interface. Ishikawa et al. teach that when the value was lower than 1, the nanoplates did not localise at the interface but dispersed inside. These results showed that the amphiphilic non-pored DNA nanoplates had higher ratios than the pored DNA nanoplates, indicating that the amphiphilic non-pored DNA nanoplates were easier to localise on the interface than the amphiphilic pored DNA nanoplates. This difference may be explained by the difference in rigidity and wettability of the amphiphilic non-pored and pored DNA nanoplates. More specifically, the center large pore may result in less rigidity of the pored DNA nanoplate because of its lower density structure, and also may result in its less wettability because of the lower surface density of the Chol-TEGs; these likely caused the pored DNA nanoplates to form more aggregates dispersed in the water phase (see page 7). Ishikawa et al. teach as follows:
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Figure 2. Water-in-oil droplets stabilised with the amphiphilic DNA nanoplates. (a) and (b) AFM images of Chol-TEG-modified non-pored nanoplates (a) and pored nanoplates (b). The cross-sectional profiles were taken along the A–B and C–D lines in the AFM images, respectively. Scale bars: 100 nm. (c) and (d) CLSM images of W/O droplets containing nonpored (c) and pored (d) nanoplates with 0–48 Chol-TEGs. Green fluorescence areas show the location of the DNA nanoplates. Scale bars: 100 µm. (e) and (f) Cross-sectional fluorescence intensity profiles of the droplets stabilised with amphiphilic non-pored (e) and pored (f) nanoplates with 24 Chol-TEGs. The profiles were measured along the yellow lines. (g) and (h) Localisation degree of (g) non-pored and (h) pored nanoplates modified with 12–48 Chol-TEG groups. The localisation degree was defined as the interfacial fluorescence intensity normalised by the internal fluorescence intensity. The interfacial fluorescence intensity was defined as the average intensity of the interfacial brighter annular area with a thickness of ~6.2% of the droplet radius [shown in (e) and (f)]. The internal fluorescence intensity was defined as the average intensity of the droplet internal area except for the interfacial annular brighter area (see pages 9-10). To investigate the state of localisation of the nanoplates, we performed fluorescence recovery after photobleaching (FRAP) of the oil-water interface stabilised with non-pored DNA nanoplates with 24 Chol-TEGs. Here, a fluorophore, 6-carboxyfluorescein group (6-FAM), was conjugated to the nanoplates using the same procedure for Chol-TEG conjugation (Fig. 3a). Fluorescence of the interface of W/O droplets constructed with the fluorescent amphiphilic nonpored DNA nanoplates was recovered to only ~5% after photobleaching (Figs. 3b and c). In contrast, freely diffusing lipids on liposomes or cell membranes are known to generally recover to ~80%41. Therefore, lateral diffusion of the amphiphilic nanoplates on the interface was very slow unlike lipids. This result suggests that the nanoplates not only localise on the oil-water interface by amphiphilic adsorption but also partly accumulate by hydrophobic interaction with each other based on partial overlap of the amphiphilic nanoplates To evaluate how the accumulated amphiphilic nanoplates affected droplet stabilisation, interfacial tensiometry of the W/O droplets was performed. The interfacial tensions of W/O droplets based on the non-pored DNA nanoplates with 0, 12, and 48 CholTEGs were 28.8 ± 0.3, 28.2 ± 0.3, and 26.1 ± 0.5 mN m−1, respectively; those based on the pored DNA nanoplates were 28.1 ± 0.1, 27.1 ± 0.1, and 26.1 ± 0.2 mN m−1, respectively. The presence or absence of the centre pore of the DNA nanoplate did not significantly affect the interfacial tension, but the interfacial tensions tended to slightly decrease with the number of Chol-TEGs for both nanoplates. These interfacial tensions were much higher than those of wellknown lipids, such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or 1,2- dioleoyl-sn-glycero-3-phosphocholine (DOPC)42 (about 5.3 and 0.5 mN m−1, respectively). These results suggest that the accumulation of the nanoplates at the oil-water interface did not so strongly contribute to the reduction of interfacial tension but the accumulation involving hydrophobic interaction between nanoplates contributed to preventing the unintended droplet coalescence. These results are reasonable if the DNA-nanoplate-stabilised droplet was a Pickering-like emulsion as expected because stabilising particles of Pickering emulsions generally interact with each other to form a weakly flocculated state (see pages 11-12). If applicants resort to argue the reference does not provide any motivation to wherein the hydrophobic part of the first compound is interacting with the layer of surfactant by secondary non-covalent interactions and the molecular recognition site of the first compound extends from the layer of surfactant into the center of the emulsion droplet, it must be remembered that “[w]hen a patent simply arranges old elements with each performing the same function it had been known to perform and yields no more than one would expect from such an arrangement, the combination is obvious.” KSR v. Teleflex, 127 S.Ct. 1727, 1740 (2007) (quoting Sakraida v. A.G. Pro, 425 U.S. 273, 282 (1976)). “[W]hen the question is whether a patent claiming the combination of elements of prior art is obvious,” the relevant question is “whether the improvement is more than the predictable use of prior art elements according to their established functions.” (Id.). Addressing the issue of obviousness, the Supreme Court noted that the analysis under 35 USC 103 “need not seek out precise teachings directed to the specific subject matter of the challenged claim, for a court can take account of the inferences and creative steps that a person of ordinary skill in the art would employ.” KSR at 1741. The Court emphasized that “[a] person of ordinary skill is… a person of ordinary creativity, not an automaton.” Id. at 1742. Ishikawa et al. clearly teach that when the value was lower than 1, the nanoplates did not localise at the interface but dispersed inside. These results showed that the amphiphilic non-pored DNA nanoplates had higher ratios than the pored DNA nanoplates, indicating that the amphiphilic non-pored DNA nanoplates were easier to localise on the interface than the amphiphilic pored DNA nanoplates. Furthermore, in the case where the amount of ingredients, particle sizes, etc., "overlap or lie inside ranges disclosed by the prior art" a prima facie case of obviousness exists. In re Wertheim, 541 F.2d 257, 191 USPQ 90 (CCPA 1976); In re Woodruff, 919 F.2d 1575, 16 USPQ2d 1934 (Fed. Cir. 1990). Similarly, a prima facie case of obviousness exists where the claimed ranges or amounts do not overlap with the prior art but are merely close. Titanium Metals Corp. of America v. Banner, 778 F.2d 775, 783, 227 USPQ 773, 779 (Fed. Cir. 1985) Furthermore, differences in concentration or size will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration is critical. "[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233,235 (CCPA 1955). One of ordinary skill in the art would have had a reasonable chance of success in combining the teachings of Holtze et al. and Ishikawa et al. because both references teach emulsion compositions containing DNA.
In light of the forgoing discussion, the Examiner concludes that the subject matter defined by the instant claims would have been obvious within the meaning of 35 USC 103. Therefore, the invention as a whole was prima facie obvious to one of ordinary skill in the art before the effective filing date of the instant invention, as evidenced by the references, especially in the absence of evidence to the contrary.
Response to Arguments
Applicant's arguments filed 12 May 2026 have been fully considered but they are not persuasive.
Applicants argue it is clear thus that the express objective of Holtze is to avoid adsorption of biomolecules at the interface so that enclosed components can be studied "as if they were floating in bulk." Holtze actively seeks to prevent biomolecules (e. g. "components 60 and 62, such as proteins, DNA, and/or cells" of Holtze) from adsorbing at the droplet interface. A person of skill in the art would have understood that DNA as mentioned above in Holtze is only present as one of the components in a droplet content, and is to be presented as being floating in a bulk medium, let alone as "a first compound containing a single hydrophobic part covalently linked to a molecular recognition site of the first compound," as recited in claim 65 which can be a building block for interface functionalization. Nothing in Holtze would have suggested DNA in Holtze is capable of programmable, DNA-based functionalization of the inner interface. In this connection, Applicant respectfully submits that any suggestion to modify Holtze, for example by application of Ishikawa to Holtze, to arrive at Applicant's claim 65 would inescapably change the principle of operation of Holtze. See MPEP 2143.01.
The above assertions are not found persuasive Holtze’s discussion of limiting nonspecific biomolecule adsorption is an optional embodiment for one use of the droplets; it is not the principle of operation of Holtze, and combining Holtze with Ishikawa would not change that principle. See MPEP 2143.01(VI). Holtze’s principle of operation is formation and steric stabilization of emulsion droplets with a block-copolymer fluorosurfactant, not a prohibition on interface functionalization. Holtze is directed to fluorocarbon-emulsion-stabilizing surfactants comprising a fluorophilic tail and a hydrophilic or lipophilic headgroup, including A-B diblock and A-B-A/multi-block architectures in which the A block is a fluorophilic polymer (e.g., Krytox/PFPE) and the B block is a hydrophilic polymer such as PEG. The surfactant adsorbs at the aqueous/fluorophilic interface, lowers interfacial tension, and sterically stabilizes droplets against coalescence so that the disperse phase can contain proteins, DNA, and/or cells and support chemical or biological reactions as clearly described above. That is the principle of operation: a block-copolymer surfactant layer at a fluid–fluid interface that keeps the droplet intact. Claim 65 still requires exactly that architecture—a center of first fluid, a second fluid on the other side of the interface, and “the layer of surfactant is a block-copolymer having at least one hydrophilic block and at least one hydrophobic block,” with the surfactant stabilizing the interface. Adding an amphiphilic first compound that inserts into that layer by secondary non-covalent interactions does not replace Holtze’s surfactant or abandon droplet stabilization. Holtze states that non-ionic headgroups “may” limit or prevent adsorption of components onto the interface “in some, but not all, cases,” and that this passivation “may” allow components to be investigated as if floating in bulk “in certain embodiments.” The same disclosure expressly teaches the opposite configuration. Figure 1B shows components 60 and 62 adsorbed at the surfactant/discontinuous-phase interface “when the headgroup is ionic and/or includes a chemical moiety that preferentially binds and/or adsorbs the components.” Holtze also teaches co-adsorption of other interfacially active species (e.g., BSA) to screen residual uncovered patches. A reference that depicts both a non-adsorbing inner interface and an adsorbing inner interface cannot be limited to a principle of “never functionalize the inner interface.” Optional passivation for bulk-like assays is not an essential operating principle that would be destroyed by a designed, amphiphilic insert. See MPEP 2143.01(VI); In re Ratti, 270 F.2d 810 (CCPA 1959) (principle of operation is the fundamental mode by which the prior-art device achieves its purpose, not every disclosed preference).
Ishikawa supplies the missing amphiphilic DNA building block without requiring Holtze’s enclosed DNA to serve as the first compound. Applicant is correct that Holtze’s DNA is described as a droplet content (components 60/62). That is not the rejection’s mapping. The rejection relies on Ishikawa for a first compound that contains a single hydrophobic part covalently linked to a molecular-recognition site (cholesterol-TEG–modified DNA origami nanoplates), accumulation of that amphiphile at the oil–water interface by hydrophobic insertion, and complementary DNA recognition used as a programmable interface function (nanopore / hybridization). Ishikawa, chemRxiv (5 Sept. 2018) v1, Abstract; pp. 1–3, 8–10; CLSM showing amphiphilic nanoplates localized at the oil–water interface while unmodified DNA remains dispersed in the aqueous interior. That is substantially similar geometry recited in claim 65: hydrophobic part interacting with the surfactant layer by secondary non-covalent interactions; molecular-recognition site extending into the first fluid; complementary sites forming a 1:1 complex. The examiner contends that combining Holtze’s PFPE–PEG (or analogous block-copolymer) interface with Ishikawa’s amphiphilized DNA does not convert Holtze’s cargo DNA into an interface building block. It adds a second, purpose-built amphiphile of the type Holtze already contemplates can sit at the inner interface (Fig. 1B; BSA co-adsorbate). The combination would not change Holtze’s principle of operation or render Holtze unsatisfactory for its intended purpose. MPEP 2143.01(VI) is not met merely because one disclosed use of Holtze prefers low nonspecific adsorption. The test is whether the modification would change the fundamental mode of operation or destroy fitness for the reference’s intended purpose. Holtze’s intended purposes include (i) stable water-in-fluorocarbon (or hydrocarbon-in-fluorocarbon) droplets, (ii) encapsulation of biomolecules and cells, and (iii) performance of reactions inside droplets. Ishikawa’s amphiphilic DNA plates stabilize W/O microdroplets and remain at the interface; they do not abolish the surfactant film or force coalescence. One of ordinary skill in the art would still use Holtze’s block-copolymer surfactant as the primary stabilizer and would use Ishikawa’s amphiphile as a minority, sequence-addressable insert. Emulsion integrity—the principle of operation—remains. Nonspecific adsorption of cargo proteins/DNA/cells is also distinct from designed insertion of an amphiphilic conjugate. Holtze’s PEG headgroups suppress unwanted binding of unmodified biomolecules. Ishikawa’s construct is not an unmodified biomolecule; it is hydrophobically tagged so that it is intended to partition to the interface. One of ordinary skill in the art would have understood that PEG passivation and programmed hydrophobic anchoring are compatible: the PEG brush can still suppress random adsorption while cholesterol (or analogous) anchors occupy the hydrophobic region of the same block-copolymer film. Holtze’s own Fig. 1B and BSA teaching confirm that additional interfacial species can be present without abandoning the surfactant-stabilized droplet.One of ordinary skill in the art would have been motivated to combine the references because Holtze already identifies DNA, proteins, and cells as droplet contents and identifies single-compartment reactions and biological assays as uses. Ishikawa expressly seeks “versatile methods for … designable functionalization” of surfactant- or particle-stabilized microcapsules and demonstrates DNA-origami plates that both stabilize the interface and present programmable recognition (complementary hybridization / nanopores) toward the aqueous interior. One of ordinary skill in the art seeking addressable inner-interface chemistry, capture, or transport in Holtze-type droplets would have looked to Ishikawa’s amphiphilized DNA and inserted it into Holtze’s PFPE–PEG film. The result is the claimed arrangement. No change in Holtze’s mode of droplet stabilization is required.
Applicants also argue Ishikawa's Chol-TEG-decorated amphiphilic DNA nanoplates self-assemble as Janus particles at the water-in-oil interface and stabilize droplets in a Pickering emulsion. These DNA nanoplates replace surfactant in the classical sense - the DNA nanoplates themselves are the "particle surfactants" of the interface. There is no PFPE-PEG layer in Ishikawa's DNA origami nanoplate-based Pickering emulsion. Applicants argue Holtze describes a PFPE-PEG layer, but explicitly for passivation of the interface against biomolecule adsorption as discussed above - the exact opposite of programmable functionalization. Ishikawa has no PFPE-PEG layer (or any other surfactant layer). Rather, the droplet is stabilized as a Pickering emulsion by DNA origami nanoplates decorated with many Chol-TEG tags forming a rigid interfacial layer. Stabilization and function are in the same component, and there is no separate surfactant and no separate DNA anchor as also discussed above.
The above assertions are not found persuasive because That Ishikawa’s Chol-TEG DNA nanoplates can stabilize a mineral-oil W/O droplet as a Pickering (particle) emulsifier, and that Ishikawa’s working examples do not include a PFPE–PEG film, does not defeat the combination with Holtze. The rejection is under § 103, not § 102. Holtze supplies the recited block-copolymer surfactant layer; Ishikawa supplies the recited amphiphilic first compound. Ishikawa is not required to already contain Holtze’s surfactant. See MPEP 2145(IV); In re Keller, 642 F.2d 413, 425 (CCPA 1981) (one cannot show nonobviousness by attacking references individually where the rejection is based on their combination). Ishikawa is cited for the first compound and its interfacial geometry, not as a wholesale substitute for Holtze’s surfactant. Claim 65 requires two interfacial species: (i) a layer of block-copolymer surfactant that stabilizes the fluid interface, and (ii) a first compound having a single hydrophobic part covalently linked to a molecular-recognition site, the hydrophobic part interacting with that surfactant layer by secondary non-covalent interactions, with the recognition site extending into the first fluid. Holtze teaches (i): PFPE (Krytox)–PEG and related A-B / A-B-A fluorosurfactants adsorbed at an aqueous/fluorophilic interface. US 2015/0217246 at [0246]–[0253], [0304]–[0307], [0411], [0460]–[0463], Figs. 1A–1B, 2A–2C. Ishikawa teaches (ii): a DNA origami nanoplate selectively decorated on one face with Chol-TEG hydrophobic anchors covalently attached to DNA, so the hydrophobic moieties partition to the oil side of the oil–water interface and the DNA recognition architecture faces the aqueous interior. Ishikawa, chemRxiv (5 Sept. 2018) v1, Abstract; Fig. 1; pp. 2–3, 8–10 (CLSM: Chol-TEG plates localize at the interface; unmodified plates remain dispersed in the aqueous core). The absence of PFPE–PEG in Ishikawa’s examples is expected. It is not a teaching that a block-copolymer surfactant layer cannot coexist with Chol TEG DNA, and it is not a gap in the combination. Ishikawa does not teach away from a molecular surfactant layer. Ishikawa’s opening frame is the opposite of exclusivity. The paper places the work in the genus of “microcapsules stabilised with surfactants, copolymers, and nano/microparticles,” and seeks “versatile methods for their designable functionalisation.” Ishikawa Abstract (emphasis added). The experimental choice to let amphiphilic nanoplates serve as the sole stabilizer in a hand-tapped mineral-oil emulsion is one implementation. It is not a statement that molecular surfactants must be omitted, or that Chol-TEG DNA is incompatible with a pre-existing amphiphilic copolymer film. The physical mechanism Ishikawa actually uses is hydrophobic insertion of Chol-TEG into the oil side of the interface—the same class of secondary non-covalent interaction the claim recites between the hydrophobic part and the surfactant layer. One of ordinary skill in the art placing that construct at Holtze’s PFPE–PEG interface would expect the cholesterol tails to embed in the hydrophobic/fluorophilic region of the block-copolymer film and the DNA face to project into the aqueous center. That is the claimed arrangement. Nothing in Ishikawa requires the DNA plate to displace the copolymer in order for that insertion to occur. “Pickering” and “Janus” do not take Ishikawa outside the claim or outside a proper combination with Holtze. Applicant is correct that Ishikawa describes the plates as Janus particles and the emulsion as particle-stabilized. Those labels describe how Ishikawa chose to stabilize its own droplets. They do not change the molecular features mapped to the claim: one hydrophobic part (Chol-TEG) covalently linked to a molecular-recognition site (DNA), hydrophobic contacts at the interface, recognition site in the aqueous phase, and complementary DNA pairing available for a 1:1 complex. One of ordinary skill in the art is not limited to bodily incorporating Ishikawa’s entire Pickering protocol into Holtze. In re Mouttet, 686 F.3d 1322, 1332 (Fed. Cir. 2012); MPEP 2145(III). Nor does Pickering stabilization teach that particles and molecular surfactants are mutually exclusive. Mixed interfaces—block-copolymer or small-molecule surfactant plus an additional amphiphilic or particulate species—are routine. Holtze itself contemplates a second interfacially active species at the inner face (BSA co-adsorbed to cover residual patches) and an adsorbed-component embodiment (Fig. 1B). Adding Ishikawa’s amphiphilic DNA as that second species keeps Holtze’s principle of operation (steric stabilization by a block-copolymer fluorosurfactant) and adds programmable inner-face recognition. It is not a substitution that “replaces surfactant in the classical sense.” Applicant’s “replacement” theory cuts against nonobviousness, not for it.If, as Applicant contends, Ishikawa’s plates can stabilize a droplet without PFPE–PEG, one of ordinary skill in the art still had every reason to keep Holtze’s surfactant when the goal is the claimed two-component interface. Holtze’s PFPE–PEG film is the established, biocompatible stabilizer for water-in-fluorocarbon droplets used with proteins, DNA, and cells. Ishikawa’s contribution is a programmable amphiphile that sits at an oil–water interface by hydrophobic anchoring. The obvious combination is to retain Holtze’s layer (claim limitation: “the surfactant stabilizes the fluid interface”; “the layer of surfactant is a block-copolymer…”) and insert Ishikawa’s Chol-TEG DNA into that layer (claim limitation: hydrophobic part interacting with the layer by secondary non-covalent interactions; recognition site extending into the first fluid). Deleting Holtze’s surfactant would fail to meet the claim. Combining the references meets it.The examiner contends that Holtze still operates as a block-copolymer-stabilized emulsion droplet. Ishikawa’s DNA is used as an interfacial ligand, not as a mandatory replacement stabilizer. Emulsion integrity is preserved; inner-face chemistry becomes sequence-addressable. That is the predictable result of the combination, not a new principle of operation.
Applicants argue Ishikawa describes Chol-TEG-ssDNA "grafted" onto a large origami structure; typically 12-48 Chol-TEG units per plate. Applicant submits that these hydrophobic moiety containing Chol-TEG-ssDNAs in Ishikawa are an integral component of a particle stabilizer (nanoplate), and are distinct from anchors in a surfactant layer. However, the Examiner appears to view this Chol-TEG-ssDNA in isolation as a "first compound" within the meaning of our application contrary to the technical context in Ishikawa as a whole. Applicants argue that the first compound and second compound form "a 1:1 complex of the first compound and the second compound," (e.g. programmable DNA system at the inner interface, which Holtze expressly seeks to avoid to prevent or limit the adsorption of molecules at the interface between the surfactant and the discontinuous phase, and which Ishikawa does not suggest.). Applicants argue that the first compound is a monomeric unit with one recognition site that pairwise hybridizes with one recognition site of the second compound (thus they are complementary to each other). Ishikawa, in contrast, uses a multitude of such Chol-TEG units per plate; the actual interface function relies on a multivalent, rigid Janus particle – a conceptually fundamentally different system.
The above assertions are not found persuasive because none of Applicants positions are consistent with the references or the claim language because Holtze’s PFPE–PEG layer is the recited surfactant film. Passivation is one optional use of that film, not its exclusive function and not a teaching away from programmed insertion. Holtze’s surfactants are block copolymers with a fluorophilic PFPE (Krytox) block and a hydrophilic PEG (or DMP) block that adsorb at the aqueous/fluorophilic interface and stabilize the droplet. That structure is the “layer of surfactant [that] is a block-copolymer having at least one hydrophilic block and at least one hydrophobic block” in claim 65. Applicant treats “limit adsorption of biomolecules” as if it were the only disclosed purpose of that layer. It is not. Holtze states that non-ionic PEG headgroups may limit adsorption “in some, but not all, cases,” and that bulk-like behavior is a feature of “certain embodiments.” The same specification discloses an adsorbed-component inner interface when the headgroup is ionic or includes a moiety that binds or adsorbs components and co-adsorption of a second interfacially active species (BSA) at residual patches. A reference that teaches both a passivated inner face and an occupied inner face does not teach away from placing a designed amphiphile in the same film; In re Gurley, 27 F.3d 551, 553 (Fed. Cir. 1994) (a reference teaches away only when it criticizes, discredits, or otherwise discourages the path taken). Passivation of unmodified cargo protein/DNA/cells is also not the “exact opposite” of programmable functionalization. The claim does not require the PFPE–PEG layer itself to be the recognition element. It requires that layer to stabilize the interface, and a separate first compound to present the recognition site after its hydrophobic part interacts with the layer. Holtze’s PEG brush can still suppress random adsorption of cargo while an amphiphilic conjugate is deliberately anchored in the hydrophobic region of the same film. That is compatible with Holtze, not contrary to it. Additionally, Ishikawa need not contain a PFPE–PEG layer. The combination supplies both limitations. That Ishikawa’s working examples are mineral-oil W/O droplets stabilized by Chol-TEG nanoplates, without a disclosed PFPE–PEG film, is not disputed and is not material. Ishikawa is not an anticipating reference. Holtze supplies the block-copolymer surfactant layer; Ishikawa supplies an amphiphile whose hydrophobic part and DNA recognition site map onto the first (and second) compound. Attacking Ishikawa for lacking Holtze’s surfactant is an individual-reference argument against a combination rejection. In re Keller, 642 F.2d 413, 425 (CCPA 1981); MPEP 2145(IV). Ishikawa’s own framing undercuts exclusivity. The reference situates the work among microcapsules “stabilised with surfactants, copolymers, and nano/microparticles” and seeks methods for “designable functionalisation.” Using the plates as the sole stabilizer in one protocol is an experimental choice, not a prohibition on seating the same amphiphilic DNA construct in a pre-existing copolymer film. Furthermore, “Stabilization and function in the same component” does not preclude using Ishikawa’s amphiphile as the first compound in Holtze’s film. Claim 65 does not require the first compound to be the sole stabilizer, and it does not forbid the first compound from also contributing to interfacial occupancy. It requires a stabilizing block-copolymer layer and a first compound whose hydrophobic part interacts with that layer. One of ordinary skill in the art combining the references would keep Holtze’s PFPE–PEG as the primary stabilizer—the component Holtze already optimized for water-in-fluorocarbon droplets containing biomolecules—and would use Ishikawa’s Chol-TEG DNA as the programmable insert. That allocation of roles is the claimed architecture. Applicant’s observation that Ishikawa can stabilize without a separate surfactant explains why Ishikawa is a useful source of the amphiphile; it is not a reason one of ordinary skill in the art would be unable or unwilling to use that amphiphile with Holtze’s surfactant.The combination is not a bodily incorporation of Ishikawa’s entire Pickering droplet into Holtze. In re Mouttet, 686 F.3d 1322, 1332 (Fed. Cir. 2012); what is taken from Ishikawa is the structural motif the claim recites: a hydrophobic moiety covalently linked to a molecular-recognition site that partitions to an oil–water interface with the recognition site toward the aqueous phase. Even furthermore, reading Chol-TEG–ssDNA as the “first compound” is consistent with Ishikawa and with the claim. The Examiner is not required to import the entire origami plate as an indivisible particle and then declare the claim unmet. Applicant argues that each Chol-TEG–ssDNA is only an “integral component of a particle stabilizer” (typically 12–48 Chol-TEG units per plate) and therefore cannot be the claimed first compound. That is a restriction the claim does not impose and that the prior art does not require to meet. Claim 65 recites “a first compound containing a single hydrophobic part covalently linked to a molecular recognition site of the first compound.” Ishikawa’s building block is exactly that species before, and as used in, plate assembly: a Chol-TEG hydrophobic part covalently attached to an ssDNA tag that is the recognition / hybridization site, which is then hybridized onto staple tails on one face of the plate. Ishikawa at 2–3 (Chol-TEG modified onto an ssDNA tag; tags address one face). Multiplicity of such units on one plate does not change the identity of each unit. A plate displaying 12–48 copies is an array of the same first compound, not a chemically different class of “anchor-that-is-not-a-compound.” Dependent claim scope in the application itself contemplates hydrophobic parts based on cholesterol and DNA as the recognition polymer—the same pairing Ishikawa uses. The claim also does not exclude the first compound from being presented on a larger DNA assembly. The molecular-recognition site “of the first compound” may be a polymer. An origami plate is a DNA polymer assembly that presents complementary recognition sequences. Treating the Chol-TEG–DNA conjugate as the first compound, whether free or displayed on a nanoplate, is a fair reading of both the claim and Ishikawa. Isolating the amphiphilic conjugate for mapping purposes is not taking it “out of context.” It is identifying the element Ishikawa contributes to the combination. The “context” Applicant invokes—that those conjugates were used to make a Pickering plate—goes to Ishikawa’s preferred embodiment, not to what one of ordinary skill in the art would understand the conjugate to be or where it can sit.One of ordinary skill in the art would also have understood that the same hydrophobic-insertion chemistry does not depend on the plate being the sole emulsifier. Chol-TEG partitions into a hydrophobic phase or into the hydrophobic region of an interfacial film. That is why unmodified plates stay in the aqueous core and Chol-TEG plates go to the interface in Ishikawa’s CLSM. The same driving force operates at Holtze’s PFPE–PEG interface. Number of tags per plate affects coverage and rigidity of Ishikawa’s particle layer; it does not convert each Chol-TEG–DNA unit into something other than an amphiphilic first compound, and it does not teach that those units cannot insert into a copolymer surfactant film. Furthermore, the examiner notes that there is a separate surfactant and a separate DNA anchor in the combination, which is what the claim requires. Applicant’s statement that “there is no separate surfactant and no separate DNA anchor” describes Ishikawa standing alone. In the proposed combination there is both: Holtze’s PFPE–PEG (or equivalent block copolymer) as the separate surfactant layer, and Ishikawa’s Chol-TEG–linked DNA as the separate first compound whose hydrophobic part interacts with that layer by secondary non-covalent interactions. That is the mapping. Applicant’s insistence that the two functions must remain fused in one Pickering particle is an attempt to freeze Ishikawa’s example as the only permissible use of its amphiphile. Section 103 does not work that way. The “1:1 complex” limitation is complementary molecular recognition between the first and second compounds. Holtze does not teach away from that complex at a block-copolymer-stabilized interface, and Ishikawa supplies it. The claim recites a defined 1:1 recognition complex, not nonspecific adsorption of cargo. Claim 65 requires that “the molecular recognition site of the first compound is complementary to the molecular recognition site of the second compound to form a 1:1 complex of the first compound and the second compound.” That is pairwise, saturable binding between two designed partners (e.g., complementary nucleic-acid strands). It is not the uncontrolled adsorption of proteins, genomic DNA, or cells onto an unfunctionalized inner film. Holtze’s passivation language is directed at the latter. Non-ionic PEG headgroups “can prevent or limit the adsorption of molecules at the interface” so that components 60 and 62 “proteins, DNA, and/or cells” present as droplet contents—“may” be studied “as if they were floating in a bulk medium” “in certain embodiments,” “in some, but not all, cases.” That is a preference against nonspecific loss of assay cargo to the wall. It is not a ban on a second, purpose-built amphiphile that presents a complementary recognition site, and it is not a ban on 1:1 binding between two such designed partners.Holtze itself separates those concepts. Figure 1B discloses components adsorbed at the inner interface when the headgroup “includes a chemical moiety that preferentially binds and/or adsorbs the components.” Preferential binding is recognition, not the random adsorption Holtze seeks to limit in the Fig. 1A embodiment. A 1:1 complementary complex is a species of preferential binding. Holtze therefore does not “expressly seek to avoid” the claimed complex. Additionally, Holtze does not teach away from programmable DNA at the interface. A reference teaches away only when it criticizes, discredits, or discourages the claimed path. In re Gurley, 27 F.3d 551, 553 (Fed. Cir. 1994). Holtze encourages PEG passivation so that unmodified biomolecules remain available in the disperse phase for reactions and assays. It does not criticize using DNA hybridization as an interfacial tool, does not say complementary strands must not meet at the inner face, and does not say a block-copolymer film cannot host an amphiphilic DNA conjugate. DNA is identified as a legitimate droplet component. The combination does not require Holtze’s cargo DNA to become the first compound; it adds Ishikawa’s amphiphilic DNA as a separate interfacial species while Holtze’s PFPE–PEG continues to stabilize the droplet and can continue to suppress nonspecific adsorption of unmodified cargo.Those are compatible operations. Passivation of the bulk contents and programmed 1:1 binding of a designed interfacial pair are not the same process, and Holtze’s preference for the first does not forbid the second. Furthermore, the examiner notes that Ishikawa suggests the 1:1 complex. Ishikawa is DNA nanotechnology. Complementary hybridization is how the nanoplate is built and how function is programmed. Chol-TEG is first attached to an ssDNA tag; that tag is hybridized to complementary staple tails on one face of the plate—a 1:1 strand–strand complex that displays the hydrophobic part on one face and DNA recognition toward the aqueous interior (Fig. 1). The plates themselves present addressable DNA sequence. Pored plates are designed as ion channels by the same sequence-level design. Applicant’s statement that Ishikawa “does not suggest” a programmable DNA system at the inner interface is incorrect. Ishikawa’s entire result is a programmable DNA system localized at the inner oil–water interface, with recognition sites facing the first fluid. Complementary pairing is the mechanism that both assembles the amphiphile and makes the interface sequence-addressable. One of ordinary skill in the art reading Ishikawa would understand that a second strand (or second DNA construct) complementary to a displayed sequence forms a 1:1 duplex with that sequence. That is the claimed complex. The claim does not require the second compound to be a second origami plate. It requires a second compound comprising a complementary molecular-recognition site. Ishikawa’s staple/tag hybridization and the inherent 1:1 stoichiometry of Watson–Crick pairing teach that limitation.
Applicants argue that the combination proposed by the Examiner would require a skilled person in the art to: 1. Abandon Holtze's express teaching of interface passivation and instead functionalize it with biomolecules, and 2. Ignore Ishikawa's core concept (rigid DNA particle replaces surfactant) and instead extract only the Chol-TEG-ssDNA module, then use it not on a DNA plate but as a monomeric anchor in a PFPE-PEG surfactant layer.
The above assertions are not found persuasive because the proposed combination does not require one of ordinary skill in the art to abandon Holtze or to ignore Ishikawa. It requires one of ordinary skill in the art to use each reference for the element it actually teaches. First, the combination does not require abandoning Holtze’s passivation teaching. Holtze’s principle of operation is steric stabilization of aqueous (or lipophilic) droplets in a fluorophilic continuous phase by a PFPE–PEG (or analogous) block-copolymer surfactant so that the disperse phase can contain proteins, DNA, and/or cells and support reactions. Interface passivation is an optional advantage of non-ionic PEG headgroups “in some, but not all, cases” and “in certain embodiments,” so that unmodified cargo may behave as if in bulk. That preference is not discarded by the combination. The PFPE–PEG layer remains in place and continues to stabilize the droplet. PEG can continue to suppress nonspecific adsorption of unmodified cargo. What is added is a separate, designed amphiphile whose hydrophobic part is intended to occupy the film. Holtze already contemplates an occupied inner interface and a second interfacially active species such as BSA. Adding Ishikawa’s amphiphilic DNA is the same class of modification: a further species at the inner face, not deletion of the surfactant or of passivation toward bulk contents.Passivation of cargo and programmed 1:1 recognition of a designed pair are not mutually exclusive. Applicant equates them only by treating “any DNA at the interface” as the adsorption Holtze prefers to limit. Holtze limits uncontrolled fouling of assay components. It does not criticize a purpose-built Chol-TEG–DNA conjugate, and it does not say complementary strands may not form a 1:1 complex at the inner face. That is not teaching away. In re Gurley, 27 F.3d 551, 553 (Fed. Cir. 1994). One of ordinary skill in the art following the rejection would therefore keep Holtze’s express teaching: use the PFPE–PEG film, keep cargo in the droplet, limit nonspecific wall binding. One of ordinary skill in the art would add a programmed anchor. That is combination, not abandonment. Second, the combination does not require ignoring Ishikawa’s core concept. Ishikawa’s core teaching, as Ishikawa states it, is not “rigid DNA particles must replace molecular surfactant and may not be used in any other way.” It is that amphiphilized DNA origami nanoplates—Chol-TEG hydrophobic parts covalently linked to DNA recognition architecture—accumulate at an oil–water interface with DNA facing the aqueous interior, and that DNA nanotechnology makes that interface programmable (hybridization, nanopores). The paper locates that result in the genus of capsules “stabilised with surfactants, copolymers, and nano/microparticles” and seeks “designable functionalisation.” Abstract. The Pickering protocol (plates as the sole stabilizer in mineral oil; 12–48 Chol-TEG units per plate; rigid particle layer) is Ishikawa’s working example. One of ordinary skill in the art is not required to import that entire example into Holtze. In re Mouttet, 686 F.3d 1322, 1332 (Fed. Cir. 2012); In re Keller, 642 F.2d 413, 425 (CCPA 1981). What is taken is the structural motif the claim recites: a compound with a single hydrophobic part covalently linked to a molecular-recognition site that partitions to the interface by secondary non-covalent interactions and presents complementary DNA toward the first fluid, forming a 1:1 complex. That motif exists in Ishikawa whether or not the plate is the sole emulsifier. Chol-TEG–ssDNA is synthesized as a discrete conjugate and then hybridized 1:1 onto complementary staple tails. Ishikawa at 2–3. Multiplicity of copies on one plate does not change the identity of each conjugate, and it does not confine the conjugate to use only as a particle emulsifier. The same hydrophobic-insertion chemistry that drives plates to Ishikawa’s interface drives the same hydrophobic part into the hydrophobic/fluorophilic region of Holtze’s block-copolymer film. Using the plate as displayed first compound, or using the Chol-TEG–DNA module as the first compound, both map onto claim 65. Either reading uses Ishikawa; neither ignores it. Applicant’s demand that one of ordinary skill in the art retain “rigid DNA particle replaces surfactant” as an indivisible concept is the bodily-incorporation argument in another form. If that concept were treated as mandatory, the combination would omit Holtze’s surfactant and would fail the claim, which requires a separate block-copolymer surfactant layer and a first compound interacting with that layer. The obvious path is the one that meets the claim: keep Holtze’s stabilizer; add Ishikawa’s amphiphilic DNA as the recognition component. Ishikawa’s demonstration that the plates can stabilize a droplet without PFPE–PEG is evidence that the amphiphile is strongly interfacial, not evidence that it cannot sit in a PFPE–PEG film. Third, the two-step “skilled person would have to” list misstates the obviousness inquiry.Obviousness does not ask whether one of ordinary skill in the art would destroy each reference’s preferred embodiment and rebuild the claim from isolated fragments with no reason to do so. It asks whether the prior art as a whole would have suggested the claimed arrangement, with a reasonable expectation of success. Here: Holtze already is a PFPE–PEG-stabilized droplet containing DNA and supporting interfacial occupancy. Ishikawa already is amphiphilic DNA (Chol-TEG covalently linked to a recognition strand) at an inner oil–water interface, assembled and addressed by 1:1 hybridization. The art already treats surfactants, copolymers, and particles as alternative or combinable interfacial tools (Ishikawa Abstract; Holtze’s BSA co-adsorbate). The claimed droplet is that interface plus complementary 1:1 recognition extending into the first fluid. Therefore, the examiner notes that no abandonment of Holtze’s surfactant, no deletion of passivation toward unmodified cargo, and no discarding of Ishikawa’s amphiphilic DNA chemistry is required. One of ordinary skill in the art uses Holtze for the recited block-copolymer layer and Ishikawa for the recited first/second compounds and 1:1 complex.
In light of the forgoing discussion, the Examiner concludes that the subject matter defined by the instant claims would have been obvious within the meaning of 35 USC 103. Therefore, the invention as a whole was prima facie obvious to one of ordinary skill in the art before the effective filing date of the instant invention, as evidenced by the references, especially in the absence of evidence to the contrary.
Conclusion
No claims are allowed.
THIS ACTION IS MADE FINAL. Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
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/TIGABU KASSA/
Primary Examiner, Art Unit 1619
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