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 .
Election/Restrictions
Claims 1-15 and 29-30 are withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected Group I and III, there being no allowable generic or linking claim. Applicant timely traversed the restriction (election) requirement in the reply filed on 10/29/25. Applicant traverses on the grounds that all the claims are directed to microfluidic methods of detecting unbound bilirubin. As pointed out in the restriction each group has a different set of method steps.
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.
Claims 16-24 are rejected under 35 U.S.C. 103 as being unpatentable over WO 2016159050 (herein after Iwatani) in view of Millington “Digital microfluidics comes of age: high-throughput screening to bedside diagnostic testing for genetic disorders in newborns” Expert Rev Mol. Diagn. 2018 Augst; 18(8): 701-712.
Regarding claim 16, Iwatani teaches a method of measuring unbound bilirubin in a plasma droplet, the method comprising:
a. providing a plasma sample (1-1 measurement target) comprising a glucose buffer (blood sample (a1) with initial dilution mixed with buffer solution which glucose is included along with PBS, or acetate buffer, or tris buffer; see 2-1 decomposition process) and a surfactant (1-3 disassembly stop process, cationic surfactant, or amphoteric surfactant) compatible with performing a fluorescence-based unbound bilirubin assay using electrowetting-mediated droplet operations to perform assay steps;
b. splitting the plasma into at least three sample and initiating the unbound bilirubin assay (2-1 Decomposition process (i) conditions, controls and a plurality of reaction samples for testing unbound bilirubin), wherein:
i. a first sample is combined with a buffer sample (reagent blank solution, 5. Unbound bilirubin measuring device) to provide a control reaction sample (control container without enzyme provided in incubator control container; unbound bilirubin measuring device);
ii. a second sample is combined with an enzyme reagent (glucose oxidase and hydrogen peroxide) to provide a short reaction (sample with enzyme at time 1); and
iii. a third sample is combined with a second enzyme reagent (glucose oxidase and hydrogen peroxide; the second enzyme does not have to be different from the first enzyme above; further a second enzyme reagent can be a different concentration of enzymes added to create the third sample as seen in 2-1 decomposition process (i) conditions) to provide a long reaction (sample with enzyme at time 2);
c. combining the control reaction and the short reaction with a stop reaction liquid (stopping the decomposition reaction, an antioxidant can be used as an oxidative decomposition stopper. Examples of the antioxidant include glutathione, N-acetylcysteine, ascorbic acid (vitamin C), α-tocopherol (vitamin E), butylhydroxyanisole, catechin, quercetin, uric acid, flavonoids and the like. When ascorbic acid as shown in FIG. 1 is used, the decomposition reaction can be stopped more easily and effectively due to cost, availability, operability (high water solubility), high decomposition stopping effect, and the like) at a time t1 to provide a control reacted sample and a short reacted sample (the control and the short reaction sample are added with the stop reaction liquid at a time, see 2-2 decomposition stop process (ii) conditions), wherein any unbound bilirubin in the short-reacted sample is oxidized (1-2 decomposition process), thereby providing a t1 decomposition product (1-3 disassembly stop process, sample decomposed products seen in 1-4 contact process, B1 and B2);
d. combining the long reaction with a stop reaction (stopping the decomposition reaction, an antioxidant can be used as an oxidative decomposition stopper. Examples of the antioxidant include glutathione, N-acetylcysteine, ascorbic acid (vitamin C), α-tocopherol (vitamin E), butylhydroxyanisole, catechin, quercetin, uric acid, flavonoids and the like. When ascorbic acid as shown in FIG. 1 is used, the decomposition reaction can be stopped more easily and effectively due to cost, availability, operability (high water solubility), high decomposition stopping effect, and the like) at a time t2 to provide a long-reacted (the control and the short reaction sample are added with the stop reaction liquid at a time, see 2-2 decomposition stop process (ii) conditions), wherein any remaining unbound bilirubin in the long-reacted is oxidized, thereby providing a t2 decomposition product (1-3 disassembly stop process, sample decomposed products seen in 1-4 contact process, B1 and B2);
e. diluting the control reacted, t1 decomposition product and t2 decomposition product with a buffer reagent (2-3 contact process (iii) conditions provides a buffer addition to all working samples and diluted to such an extent that the internal blocking effect in the fluorescence detection step (iv) can be ignored), a diluted t1 decomposition product, and a diluted t2 decomposition product for combining with a detection reagent;
f. combining the diluted control reacted, t1 decomposition product, and t2 decomposition product with a detection reagent (fluorescent polypeptide UnaG is added to all working samples in 2-3 contact process (iii) conditons) to provide a control/detection reagent, a short reacted/detection reagent and a long reacted/detection reagent (fluorescent polypeptide UnaG is added to all the final samples before detection 2-3 contact process (iii) conditions); and
g. detecting a reaction product in the control/detection reagent, short reacted/detection reagent, and long reacted/detection reagent to determine the amount of unbound bilirubin in the plasma (2-4 detection process (iv) conditions).
Iwatani provides the above method steps with samples provided in wells and samples, buffers, detection reagents added to wells by hand. Iwatani does teach (unbound bilirubin measuring device) the mixing and divided addition step can be performed manually. However, the sample preparation apparatus for measuring unbound bilirubin according to the present invention does not exclude that it is configured to automate the mixing and divided addition steps.
Iwatani does not teach using electrowetting droplet operations to perform mixing and dividing sample/buffer steps.
Millington teaches an automated droplet based microfluidic device/method that performs collecting a blood sample, performing droplet splitting on an electrowetting device. Samples, buffers, reagents, oils (separating droplets) are loaded into the device, then the device is activated which runs a computer program that performs sample splitting into droplets, addition of buffers, detection reagents, and finally detection of a product in an automated fashion. Millington states DMF (digital microfluidics) have many benefits with the primary being the overall simplicity of the device, reduction of the volume of sample and reagent solution that are required compared to typical benchtop assay methods (page 4), the ability to perform enzymatic reactions with discrete droplets permits optimization of the reaction conditions (page 4), and further reduces time to generate results (page 4). Therefore it would have been obvious to one having an ordinary skill in the art at the time of the invention to modify Iwatani to employ a DMF platform for the manipulation of fluids and sample detection due to the overall all simplicity of the device, reduction of sample/reagent size, optimization of reaction conditions and reduction of time to generate results as taught by Millington (page 3-4).
Regarding claim 17, Iwatani teaches the method of claim 16 wherein the enzyme reagent droplet comprises glucose oxidase (GOD) and peroxidase (POD) (1-1 measurement target, enzyme GOD and POD are employed).
Regarding claim 18, Iwatani teaches the method of claim 16 wherein the stop reaction droplet comprises ascorbic acid (1-3. Disassembly stop process).
Regarding claim 19, Iwatani teaches the method of claim 16 wherein time t1 is about 48 seconds (2-2 decomposition stop process (ii) conditions, 10-60 seconds).
Regarding claim 20, Iwatani the method of claim 16 wherein the time t2 is about 120 seconds (2-2 decomposition stop process (ii) conditions, 10-60 seconds). Iwatani states the decomposition stop time is determined by the time is assumed to be a system in which the degree of decrease in the total amount of bilirubin and the degree of decrease in the amount of unconjugated bilirubin can be equated with a normal amount of conjugated bilirubin D-Bil. This may correspond to the time taken for the total bilirubin amount to decrease by about 20% (specifically, 18% or more and 25% or less) from its initial concentration. Considering that the total bilirubin concentration decreases by about 5% before the reaction system in the decomposition step (i) becomes uniform due to complete mixing, specifically, when the initial concentration of total bilirubin is 100%, it may be equivalent to the time taken to decrease from% to 76%. Therefore it would have been obvious to one having an ordinary skill in the art at the time of the invention to modify the modified Iwatani to increase the T2 time based upon the time taken to decrease to 76% or more during the decomposition step based on the amount of unbound bilirubin to be decomposed.
Regarding claim 21, the method of claim 16, the modified Iwatani teaches wherein combining each diluted reaction droplet with a detection reagent comprises transporting a diluted reaction droplet to a certain droplet operations electrode comprising a dried detection reagent and reconstituting the dried detection reagent (liquids are moved to interact with reagents on the DMF).
Regarding claim 22, Iwatani teaches the method of claim 16 wherein the dried detection reagent for detecting unbound bilirubin is UnaG (2-3 contact process (iii) conditions).
Regarding claim 23, Iwatani teaches the method of claim 16 wherein detecting a reaction product comprises measuring a UnaG fluorescence signal (2-3 contact process (iii) conditions-2-4 detection process (iv) conditions).
Regarding claim 24, Iwatani teaches the method of claim 16 wherein determining the amount of unbound bilirubin in the plasma droplet comprises determining the difference in the UnaG fluorescence signal between the control/detection reagent droplet, and short and long reacted/detection reagent droplets (2-4 detection process (iv)conditions, unbound bilirubin measuring device).
Conclusion
Any inquiry concerning this communication or earlier communications from the examiner should be directed to SAMUEL P SIEFKE whose telephone number is (571)272-1262. The examiner can normally be reached Monday, Tuesday, Thursday, Friday, 8-6.
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/SAMUEL P SIEFKE/Primary Examiner, Art Unit 1758