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 .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 15 September 2025 has been entered.
Formal Matters
Applicant’s claim amendments and arguments in the reply filed on 15 September 2025 are acknowledged and have been fully considered. Claims 65 and 67-121 are pending. Claims 65, 67-81, 83, 85-86, 88-90, 93, 95-112, 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 Claims 82, 84, 87, 91-92, 94, 113, and 115-121 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention and/or species, there being no allowable generic or linking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 and 66 are canceled. Applicant amended claim 65. Applicant’s claim amendments and arguments did not overcome the rejections set forth in the previous office action under 35 USC 103 for reasons set forth in the previous office action and herein below.
Withdrawn Objections/Rejections
Rejections and/or objections not reiterated from the previous office actions are hereby withdrawn as are those rejections and/or objections expressly stated to be withdrawn.
Moot Arguments
Applicant’s arguments with respect to claim(s) 65, 67-81, 83, 85-86, 88-90, 93, 95-112, and 114 have been considered but are moot because the new ground of rejection does not rely on any reference applied in the prior rejection of record for any teaching or matter specifically challenged in the argument.
New Rejections-Necessitated by Amendments
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 65, 67-81, 83, 85-86, 88-90, 93, 95-112, and 114 are newly 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, newly cited).
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:
PNG
media_image1.png
798
680
media_image1.png
Greyscale
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:
PNG
media_image2.png
690
642
media_image2.png
Greyscale
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:
PNG
media_image1.png
798
680
media_image1.png
Greyscale
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:
PNG
media_image2.png
690
642
media_image2.png
Greyscale
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.
Conclusion
No claims are allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to TIGABU KASSA whose telephone number is (571)270-5867. The examiner can normally be reached on 8 AM-5 PM.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, David Blanchard can be reached on 571-272-0827. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of an application may be obtained from the Patent Application Information Retrieval (PAIR) system. Status information for published applications may be obtained from either Private PAIR or Public PAIR. Status information for unpublished applications is available through Private PAIR only. For more information about the PAIR system, see http://pair-direct.uspto.gov. Should you have questions on access to the Private PAIR system, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative or access to the automated information system, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/TIGABU KASSA/
Primary Examiner, Art Unit 1619