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 .
Remarks
The amendments and remarks filed on 04/16/2026 have been entered and considered. The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior office action. The rejections and/or objections presented herein are the only rejections and/or objections currently outstanding. Any previously presented objections or rejections that are not presented in this Office Action are withdrawn. Claims 20-38 are pending; Claims 1-19 are cancelled; Claims 20, 22, 24, 26, 28-31, 34, and 38 are amended; and Claims 20-38 are under examination.
Withdrawal of Objections
The objection to Claims 20, 22, 24, 26, and 38 are withdrawn due to the amendment of the claims filed on 04/16/2026.
The objection to the specification for not complying with sequence rules is withdrawn due to the amendment of the specification filed on 04/16/2026.
Withdrawal of Rejections
The rejection of claims 26-28, 31, 34, and 38 under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph is withdrawn due to the amendment of the claims and Examiner’s reconsideration.
Claim Rejections - 35 USC § 102/103
Claims 20-22, 25-27, 31-33 and 35-38 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by or, in the alternative, under 35 U.S.C. 103 as obvious over Pollack et al. (US 2009/0155902, 2009, cited in IDS). This rejection is maintained.
Pollack et al. teach a method for manipulating a cell (i.e. an adherent cell) in a droplet/microdroplet system by conjugating the cell to a bead/microbead, comprising steps: (i) providing a droplet actuator; (ii) loading sample droplets/microdroplets onto the droplet actuator, the sample droplets comprising cells and a fluid; (iii) loading bead droplets/microdroplets onto the droplet actuator, the bead droplets comprising one or more beads/microbead having affinity for cells of a specific cell type as well as a fluid; (iv) conducting one or more droplet operations to combine/merge the bead droplets with the sample droplets to obtain merged droplets/microdroplets comprising the cells, beads and fluids, thereby permitting cells of the specific cell type to bind to the beads/microbeads; (v) conducting a droplet-based washing protocol to separate the cells bound to the beads from cells not bound to the beads (when there are cells of other cell type(s) not bound to the beads); and (vi) growing cells bound to the beads in merged droplets/microdroplets (Figs. 7-8, Claim 4, paras 0051-0054, 0017-0021, and 0006); wherein the droplet actuator is a micro-actuator comprised in a microfluidic system/chip (first half of para 0019), thus the sample and bead droplets being loaded into a microfluidic space of the system; wherein the one or more droplet operations comprise merging the bead droplets and the sample droplets by transporting these two sets of droplets/microdroplets into contact with each other on the droplet actuator (paras 0020: right col/lines 1-8, and 0054); wherein the droplets/microdroplets are aqueous droplets (i.e. containing a fluid of water) and filler fluid associated with droplets is oil (paras 0018/line 5-6 and 0021/lines 5-6) and the fluid in the droplets includes water, saline solutions, acidic solutions, basic solutions, buffers and/or other reagent involved with biochemical protocol or biological fluids (para 0018: page 2/right col/last 4 lines and page 3/first 4 lines); wherein the manipulation of droplets/microdroplets by the droplet actuator is electrowetting mediated (paras 0019, 0052/lines 3-4, and 0054/lines 1-4; Figs. 7-8); and wherein the beads/microbeads are magnetically responsive or non-magnetically responsive (paras 0017, 0051, 0053, and 0056).
Regarding the step of “agitating each of the merged microdroplets …” recited in the claim 20, the specific embodiment and claims of Pollack et al. do not expressively teach a step of agitating merged microdroplets. However, they teach that conducting droplet operations to merge the bead droplets and sample droplets to obtain merged microdroplets, thereby permitting cells to bind to microbeads; and the droplet operations comprise moving the bead droplets and sample droplets into contact with each other on the droplet actuator for merging these droplets, as indicated above. It is noted that the movement of microdroplets into contact with each other would cause agitation of moved microdroplets and agitation of the microdroplets being merged, and such agitation would be carried over to the merged microdroplets. Thus, it appears that the method of Pollack et al. inherently comprises a step of agitating each of the merged microdroplets, as required by the claim 20. However, even if the droplet movement does not cause agitation of each of merged microdroplets, in which case there is no anticipation, it would at least have been obvious to include agitating each of merged microdroplets as one of the droplet operations in the method of Pollack et al. for promoting cells and microbeads to move and contact with each other, thus the binding/adhering the cells to the microbeads, because Pollack et al. (in para 0020/lines 1-2 and 8-9) specifically teach that their droplet operations comprise agitating droplets on the droplet actuator.
Regarding the claim 21, Pollack et al. teach performing a culturing or assaying process on cells adhered/bound to the microbeads (Claims 16 and 19; paras 0054, 0060, and 0018/last 4 lines).
Regarding the claim 22, the microfluidic space used in the method of Pollack et al. is a part of a microfluidic chip, which is configured to manipulate the bead droplets (reading on the claimed “first droplets”) and sample droplets (reading on the claimed “second droplets”) via electrowetting mediation, as indicated above. Pollack et al. further teach the electrowetting mediation is opto-electrowetting mediated (i.e. optically mediated electrowetting) (para 0019: lines 10-11 from the bottom), thus meeting the claimed limitation.
Regarding the claim 25, the cells in the method of Pollack et al. are adherent cells in their native adherent state, which grow on the support of beads (para 0054).
Regarding the claims 26 and 27, Pollack et al. teach inspecting the microdroplets prior to the merging by sorting the droplets, determining an cell count (i.e. an amount of cells) in droplet, and discarding droplets not having a desired cell count (paras 0038, 0039/lines 1-4, and 0040).
Regarding the claim 31, the recited limitation “coupling promoter” is not defined in the specification. Examiner notes that any component that facilitates merging of the first and second droplets can be considered as a coupling promoter. As such, even the water comprised in the aqueous droplets of Pollack et al. can be considered as a coupling promoter, because it provides an aqueous phase that facilitates contacting and merging of the bead/first droplets and cell/second droplets of Pollack et al. Thus, the teachings of Pollack et al. meet the claimed limitation.
Regarding the claims 32 and 33, Pollack et al. teach cell-containing droplets are provided with a fluid comprising cell growth/culture medium for culturing cells (paras 0008/line 4-5, 0054-55); and Pollack et al. further teach introducing a reagent droplet (i.e. a carrier phase) comprising a lysis reagent into the microfluidic space for treating cells in cell-containing droplets and further performing analysis on the cells, such as nucleic acid amplification, affinity-based or enzymatic assay, and sequencing (paras 0048/lines 5-6 and last 3 lines, and 0018/last 6 lines) (Note: the lysis reagent of Pollack et al. can be considered as a releasing agent recited in the claim 32 because it lyses and releases adherent cells from beads and the instant claim does not require intact cells are released from beads). Given Pollack et al. teach that a fluid in cell-containing droplets comprising cell-bound beads can be readily depleted and exchanged/replaced with a reagent fluid in reagent droplets (paras 0056-57), it would have been obvious to deplete the cell growth medium from the microdroplets containing cells adhering to microbeads and then exchange and replace it with a lysis reagent from the reagent droplets/carrier phase in the method of Pollack et al., such that the cells adhered to the beads can be released and lysed for performing analysis on the cells.
Regarding the claim 35, Pollack et al. teach incubating the merged microdroplets at a controlled temperature and monitoring growth of cells bound to the microbeads of each of the merged microdroplets (para 0054, Fig. 8).
Regarding the claim 36, Pollack et al. teach performing an on-chip reporter assay on merged microdroplets by using material extract from the cells with a droplet-based protocol, for determining gene expression, presence of proteins produced, and enzymatic activity of the proteins of the cells (para 0060).
Regarding the claims 37 and 38, Pollack et al. teach that cells in the merged microdroplets bind/adhere to the microbeads because the beads have an affinity for cells of a particular cell type (para 0054/lines 8-11) (i.e. at least one adherent cell adheres to at least one microbead in a merged microdroplet); and cells-containing droplets are transported/deposited into a cell culture reservoir or a well for culturing cells (para 0055/lines 1-6) (Note: the cell culture reservoir and the well have internal surfaces, thus reading on a receptacle and a surface of the receptacle in the claims). As such, the claims would have been obvious over Pollack et al.
Therefore, the invention as a whole would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention.
Claim Rejections - 35 USC § 103
Claims 23 and 24 are rejected under 35 U.S.C. 103 as obvious over Pollack et al. (US 2009/0155902, 2009, cited in IDS), as applied to Claims 20-22, 25-27, 31-33 and 35-38, further in view of Ingber et al. (WO 94/25487, 1994, cited in IDS). This rejection is maintained.
The teachings of Pollack et al. are described above.
Regarding Claims 23 and 24, Pollack et al. do not teach that a surface of the microbeads is coated with a peptide comprising the amino acid sequence RGD to form a surface functionalization for facilitating cell adhesion. However, Pollack et al. further teach the beads are coated on surface and they have an affinity to cells of a particular cell type and specifically adhere/bind to the cells (page 2: left col/last line and right col/first line; paras 0052/lines 9-11 and 0054/lines 9-11).
It would have been obvious to coat the microbeads of Pollack et al. with a RGD-containing peptide for modifying surface functionalization of the beads for facilitating cell adhesion, thus arriving at the beads having an affinity to a particular cell type required by the method of Pollack et al. for binding cells of the particular cell type in merged microdroplets. This is because it is a common practice in the art to coat beads for modifying their surface functionalization and it is well known in the art that microbeads coated with a RGD-containing peptide adhere/bind to cells of a particular cell type. Furthermore, the RGD-coated microbeads have an advantage of exhibiting less non-specific clumping during magnetic pelleting. In support, Ingber et al. teach using microbeads coated a RGD-containing peptide for binding adherent cells of a specific cell type, i.e. eukaryotic cells such as mammalian, insect, or plant cells, for isolating adhesion complex (FACs) formed between the RGD and cells from the surface of the beads, wherein the RGD-containing peptide/protein provide an attachment site for the beads to bind/ligate integrins of the cells (page 4/lines 13-22, page 12/lines 10-32, page 10/lines 8-13, page 26/lines 6-13, page 30/lines 19-21). Ingber et al. further teach RGD-coated microbeads are chosen because they exhibit less non-specific clumping during magnetic pelleting (pages 11/lines 15-18, and 27/lines 9-11).
Examiner notes that the mammalian, insect, and plant cells taught by Ingber et al. are cells in their inactive adherent state, meeting the limitation of Claim 25.
Therefore, the invention as a whole would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention.
Claims 28-30 and 34 are rejected under 35 U.S.C. 103 as obvious over Pollack et al. (US 2009/0155902, 2009, cited in IDS), as applied to Claims 20-22, 25-27, 31-33 and 35-38, further in view of Pollack-2 et al. (US 2015/0329891, 2015, cited in IDS). This rejection is maintained.
The teachings of Pollack et al. are described above.
Regarding Claims 28-30, Pollack et al. do not expressively teach discarding microdroplets not having a desired bead count, or merging and splitting, respectively, two or more microdroplets not having a desired bead count (below and at a threshold of bead count) for increasing and decreasing the cell count. However, Pollack et al. teach merging and splitting the droplets for changing contents of the droplets (e.g. changing a concentration or number of cells or beads), as described above and also see paras 0007, 0020, 0022, and 0046.
It would have been obvious to sort the bead microdroplets and exclude or discard the droplets not having a desired bead count prior to conducting the merging step in the method of Pollack et al. for ascertaining the bead droplets having a desired bead count are used for forming merged droplets, thus facilitating proper droplet operation and binding of cells to beads, wherein the bead droplets are either merged to increase the bead count (when the bead count is below a threshold level) or split to increase the bead count (when the count is at a threshold level), because it is well known in the art that sorting bead droplets allows bead droplets having a desired bead count to be selected and used in the merging step, and bead numbers in the droplets need to be maintained at a desired level for conducting proper droplet operations. In support, Pollack-2 et al. teach a method of manipulating droplets on a droplet actuator for separating and analyzing target substance, comprising a step of merging a bead-containing droplet with a target substance-containing sample droplet, wherein the target substance include cells (abstract, paras 0007 and 0022); and a step of sorting bead-containing droplets before the merging step to determine the bead content of the droplets, wherein droplets having a desired bead count are selected and droplets not having any beads or a desired bead count (a predetermined number of beads) are excluded (paras 0092: last 6 lines; and 0094). Furthermore, the techniques for merging droplets and splitting a single droplet are well established in the art, which would allow two or more bead droplets to be merged into a single droplet with an increased bead count as well as a bead droplet to be split into two or more daughter droplets with a decreased bead count, as supported by Pollack et al. described above as well as by Pollack-2 et al. (see paras 0006/lines 10-11, and 0024/lines 1-8).
Regarding Claim 34, Pollack et al. do not expressively teach that the releasing agent (lysis reagent) is a protease. However, it would have been obvious to apply a protease as the releasing agent (lysis reagent) in the method of Pollack et al. for lysing and releasing the cells from the microbeads and further analyzing cell components, because it is a common practice in the art to use the protease as a lysis reagent, as supported by Pollack-2 et al., who further teach using a lysis reagent for lysing target substance including cells and virus, wherein the lysis reagent is Proteinase K (paras 0099 and 0155/lines 2-5).
Therefore, the invention as a whole would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention.
Response to Arguments
Applicant's arguments about the claim objection and claim rejection under 35 U.S.C. 112(b) in the response filed on 04/16/2026 (pages 7-8) have been fully considered but they are moot, because the objection and rejection have been withdrawn, as indicated above. Regarding the coupling promoter in the claim 31, this term is interpreted as being broad but not indefinite after Examiner’s reconsideration. With regard to the specification in page 17, it is noted that the disclosure of the specification cannot read on the claim. Even assuming that EDC, the specifically exampled crosslinking agent (disclosed in page 17 of the specification), reads on the claimed term, it still has no novelty because EDC is a well-known crosslinking agent, which has been commonly used in the prior art for crosslinking carboxyl groups (beads) and amine groups (proteins/cells), as evidenced by Ziraldo et al. (WO 2020167862 A1, 2020, effective filing date: 2/12/2019), who teach that COOH groups on beads are activated by EDC (1-ethyl-3-(3-dimethylamino propyl)carbodiimide) such that they are reactive to amine functional groups, and the activated COOH groups can then react with a target comprising an amine functional group (para 00198, last 8 lines).
Applicant's arguments about the rejections under 35 U.S.C. 102 and 103 in the 04/16/2026 response (pages 10-11) have been fully considered but they are not persuasive for the following reasons.
First, it is noted that the disclosure about the agitating merged microdroplets in page 5/para 2 of the specification does not read on the instant claims. With regard to the stirring and shaking microdroplets disclosed in page 5/para 2, they are specific examples of agitation, not the definition of agitation, thus not reading on the limitation “agitation” in the claim 20. Second, even assuming the disclosure “cause sufficient fluid flow for the adherent cell and the microbeads to come together …” in page 5/para 2 provides the definition, this disclosure does not effectively define the term “agitation” in the claim because the recited “sufficient” is a relative term and the disclosure does not define which specific levels of fluid flow can be considered as being “sufficient fluid flow”. Third, cells and beads handled by the method of Pollack are respectively comprised in two different types of droplets (i.e. sample droplets and bead droplets), and these two types of droplets must be merged together to allow the cells in sample droplets to bind to the beads in bead droplets. As such, in contrary to Applicant’s arguments, the binding of the cells to the beads in the method of Pollack takes place after the two types of droplets have merged (i.e. combined). Fourth, the Examiner has not misinterpreted the movement of droplets vs. mixing of contents; and it is Examiner’s position that the movement of droplets comes along with mixing of their contents (in unmerged and merged droplets), and the moving action (i.e. agitation) of droplets in the method of Pollack is not limited to a time phrase of “before the bead droplet and sample droplet have merged”. As indicated above, Pollack teaches moving (with agitation) a sample droplet and a bead droplet from different directions toward each other to merge them together. The two agitating droplets would collide when the merging occurs, and such a merged droplet is under the action of agitating forces from two different directions, which would cause the merged droplet to agitate, thus allowing the contents (cells and beads) in the merged droplet to be mixed. In fact, the disclosure of page 5/para 2 pointed by Applicant provides evidence to support that merged droplets of Pollack agitate. See: “cells and carrier beads are both slow-diffusing large particles and are unlikely to encounter each other through random diffusion and in stationary droplets there is minimal internal flow”. Given cells and beads in merged droplets of Pollack effectively encounter/contact and bind each other, their movement is not through random diffusion in stationary droplets. Rather, active movement of cells and beads as well as sufficient fluid flow of merged droplets, driven by agitation, must have occurred after the droplets are merged. Thus, a step of agitating merged droplets appears to be inherently comprised in the method of Pollack. Furthermore, Pollack expressively teach agitating droplets as a part of droplet operation. In view of that the instant claims do not recite any limitation to define how the agitating step in the claim 20 is specifically performed, the claimed method would be anticipated by or, in the alternative, obvious over Pollack et al., for all the reasons indicated above.
In response to Applicant’s arguments about the teachings of para 0020 of Pollack in the 04/16/2026 response (page 11/para 3), this paragraph expressively teach agitating droplets, and the droplet to be agitated encompasses a merged droplet. As such, the para 0020 of Pollack renders the agitating step in the claim 20 to be obvious, as indicated above.
Moreover, Examiner notes that it is well known in the art to agitate merged droplets to provide benefits such as improving efficiency of mixing contents and increasing reaction efficiency, as evidenced by Koyama et al. (Transducers & Eurosensors, 2007, 3E016.P, 2385-2388), who teach fusing/merging bead droplets carrying an AP enzyme and substrate droplets carrying AP substrates for performing an enzyme reaction, and demonstrate that the reaction efficiency is increased 4 times in merged droplets with agitation, compared to that without agitation (abstract/last 3 lines, page 2386/last para – page 2387/para 1, the para spanning both cols in page 2387); and as further evidenced by Hu et al. (Transducers & Eurosensors, 2007, 3E018.P, 1869-1872), who teach merging sample droplets and reagent droplets by moving them toward each other for trigging a biochemical reaction (page 1871/para 1); and applying vortex/shaking to merged droplets greatly increases a mixing efficiency of contents in the merged droplets and accelerating the reaction in a short time (abstract, page 1869/col 2/para 1, page 1871/col 2/para 1). In view of the prior art, the claimed method comprising a step of agitating merged droplets has no novelty.
Overall, the conclusion of the anticipation or obviousness of the claims 20-38 has been established for all the reasons indicated above.
Conclusion
THIS ACTION IS MADE FINAL. Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any extension fee pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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Any inquiry concerning this communication or earlier communications from the examiner should be directed to Qing Xu, Ph.D., whose telephone number is (571) 272-3076. The examiner can normally be reached on Monday-Friday from 9:30 AM to 5:00 PM. If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Manjunath N. Rao, can be reached at (571) 272-0939. Any inquiry of a general nature or relating to the status of this application or proceeding should be directed to the receptionist whose telephone number is (571) 272-1600.
/Qing Xu/
Patent Examiner
Art Unit 1656
/MANJUNATH N RAO/Supervisory Patent Examiner, Art Unit 1656
Prosecution Insights