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 .
Claims 1, 3-6, 8, 10, 13-14, 17-19, and 24-28 are pending. Claims 19 and 24-28 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Claims 2, 7, 9, 11-12, 15-16, 20-23, and 29-32 are cancelled. Claims 1 and 19 are amended. Claims 1, 3-6, 8, 10, 13-14, and 17-18 are currently under examination.
Response to Amendment
The Amendment filed 12/19/25 has been entered. Claims 1, 3-6, 8, 10, 13-14, 17-19, and 24-28 are pending. Applicant’s amendments to the sequence listing disclosure, claim 1, and cancellation of claim 2 have overcome the objections, 112(b), and 102 rejections previously set forth in the Non-Final Office Action mailed 9/22/25.
Response to Arguments
Applicant’s arguments, see pages 13-15, filed 12/19/25, with respect to the rejections of claims 1, 3-6, 8, 10, 13-14, and 17-18 under 35 USC 103 have been fully considered are found unpersuasive, and the 103 rejections documented in the Non-Final mailed 9/22/25 have been revised to address claim amendments filed 12/19/25 in this Final Office Action. More detailed responses to Applicant’s arguments are provided at the end of each maintained rejection.
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.
Claims 1, 3-6, 8, 10, 13-14, and 17-18 remain/are rejected under 35 U.S.C. 103 as being unpatentable over Lee et al. (2019; NPL citation U in PTO-892 filed 9/22/25; “Rapid and in-situ detection of fecal indicator bacteria in water using simple DNA extraction and portable loop-mediated isothermal amplification (LAMP) PCR methods”; Water Research, Volume 160, Pages 371-379, https://doi.org/10.1016/j.watres.2019.05.049) in view of Xu et al. (2016; NPL citation 2 in IDS filed 8/23/22; “Paper-Origami-Based Multiplexed Malaria Diagnostics from Whole Blood”. Angew. Chem. Int. Ed. 2016, 55, 15250. https://doi.org/10.1002/anie.201606060, including all supporting information in NPL citation 3 in IDS filed on 8/23/22).
This rejection is revised/updated in response to claim amendments filed 12/19/25.
(i) Lee et al. teaches limitations relevant to claim 1.
Relevant to claim 1, Lee et al. Abstract teaches “In this study, we developed a simple and rapid DNA extraction method based on a syringe filter without any specialized equipment. Furthermore, loop-mediated isothermal amplification (LAMP) PCR for fecal indicator bacteria (FIB) (i.e. E. coli and E. faecalis) was carried out using the DNA extracts from the syringe-filter based DNA extraction method.”
Further relevant to claim 1, Lee et al. teaches “Environmental water samples were collected from freshwater reservoirs and the Singapore coastline, and then immediately transported to the laboratory” (page 372, column 2, paragraph 1 of “2.2. Measurement of FIB in environmental water” section).
These teachings read on claim 1 A method of detecting a pathogen present in a water sample.
Further relevant to claim 1, Lee et al. teaches “Briefly, 100 mL of the sample was passed through a 0.22 µm pore-size syringe filter… in the direction of filtration. 1 mL of TE buffer… was injected into the same filter in the direction of filtration and the flow through pooled into a sterile 15 mL tube. The following solutions were backflushed through the filter in the opposite direction to the filtration direction, and the flow through pooled into the same tube. First, 1 mL of TE/lysozyme buffer (TL)… was backflushed through the filter, and 300 µL of TL was added into the flow-through” (page 372, column 2, paragraph 1 of “2.3. Syringe-filter based DNA extraction method” section).
This teaching reads on claim 1 filtering the water sample through a filter membrane and adding a lysis buffer to the filtered sample to form a nucleic acid solution; extracting nucleic acid of the pathogen from the nucleic acid solution derived from the water sample.
Further relevant to claim 1, Lee et al. teaches “Finally, the genomic DNA was eluted by adding 100 mL of TE buffer. DNA samples were stored at -20°C before (q)PCR and LAMP analyses” (page 372, column 2, paragraph 1 of “2.3. Syringe-filter based DNA extraction method” section).
Further relevant to claim 1, Lee et al. teaches “The products of the LAMP were further analyzed on 1.5% agarose gel” (last sentence of “2.4. Primer design and LAMP reaction conditions” section).
These teachings read on claim 1 eluting the extracted nucleic acid… performing LAMP reactions… to obtain LAMP products; and detecting the LAMP products via an amplicon detection test.
(ii) Lee et al. is silent to specifics regarding multilayer devices with paper-based fluid flow channels (relevant to claims 1, 3-6, 8, 10, 13-14, and 17-18). However, these limitations were known in the prior art and taught by Xu et al.
Relevant to claim 1, Xu et al. Abstract teaches “We demonstrate, for the first time, the multiplexed determination of microbial species from whole blood using the paper-folding technique of origami to enable the sequential steps of DNA extraction, loop-mediated isothermal amplification (LAMP), and array-based fluorescence detection. A low-cost handheld flashlight reveals the presence of the final DNA amplicon to the naked eye, providing a ‘sample-to-answer’ diagnosis from a finger-prick volume of human blood, within 45 min, with minimal user intervention.”
There is no instant limiting definition of a water sample. Therefore, the Xu et al. blood sample is analogous to the instant water sample, as the broadest reasonable interpretation of a water sample includes blood samples, which are samples largely comprised of water.
Further relevant to claim 1, Xu et al. Figure 1 and associated caption depicts the extraction process, wherein “The sample is dispensed onto the device (panel 3) and extracted using capillary flows vertically (flow of liquid from Panel 3 to Panel 1).”
Further relevant to claim 1, Xu et al. teaches “After addition of the sample onto the device (Panel 3, Figure 2 A), the paper was folded (structure S1, Figure 2B) to enable the first steps of the assay, involving cell lysis and DNA extraction, to yield purified DNA (Figure 2 B–D) on the glass-fiber paper. To transfer the DNA from the extraction panel to the amplification panel, the fold S1 is flipped on the opposite side (Figure 2E), allowing elution (Panels 4-5 of Figure 2A and F)” (page 15251, column 1, paragraph 2).
These teachings read on claim 1 at a solid phase extraction structure mounted at a first layer of a multilayer device; eluting the extracted nucleic acid from the solid phase extraction structure to at least a second layer of the multilayer device having paper-based fluid flow channels connected together via folded regions.
Further relevant to claim 1, Xu et al. Supporting Information teaches that “Each device contained three components, namely: a filter paper based fluidic device where the liquid was constrained by printed hydrophobic wax; a single sided adhesive acetate film sealed plastic plate with 4 glass fibre spots (diameter 3mm), which form chambers for LAMP reaction (Figure 1B); and one glass fiber spot (Figure 2A) (GFF, Whatman)[citation] with diameter 4 mm for absorbing nucleic acids from the sample in the presence of high concentration of the chaotropic agent…” (page 2, paragraph 1 of Section “Paper devices”).
This teaching reads on claim 1 allowing the nucleic acid to flow through the paper-based fluid flow channels to further layers of the multilayer device having discrete reaction chambers, each of the chambers fed respectively by at least one of the fluid flow channels.
Further relevant to claim 1, Xu et al. Supporting Information Figure S1 (A) and Figure S2 depict that the nucleic acid is divided into 4 channels as it transfers between the respective layers, reading on claim 1 wherein the flow of the extracted nucleic acid in the paper-based fluid flow channels is divided at the folded regions as the flow transfers between the respective layers.
Further relevant to claim 1, Xu et al. Figure 2 and associated caption teach “Paper-folding steps for fluidic manipulation of assay steps. The broken arrows indicate folding direction. Panels numbered as in Figure 1. A) A hole from the center of the third Panel has a glass-fiber disc onto which the sample is dispensed; Numbers in the last panel indicate the different reagents placed onto the four different spots for amplification of different species. 1. Internal control (IC); 2. Plasmodium pan; 3. P. falciparum; 4. P. vivax. B) The second/third Panel are folded and clamped to form structure 1 (S1); C) The fifth Panel is folded onto the back of the fourth frame to form structure 2 (S2); D) S1 is folded onto the first Panel before adding lysis/washing buffer for DNA extraction and purification; E–F) S1 is folded onto S2 for elution and elution buffer added.”
The Xu et al. plurality of primary layers (S1 and S2) are divided into a plurality of secondary layers (panels) through folding.
Thus, this teaching reads on claim 1 wherein the layers comprise a plurality of primary layers, each of the primary layers divided into a plurality of secondary layers, and wherein the primary layers and secondary layers are integrally formed and coupled to one another, the folded regions positioned at respective edges of the primary and secondary layers, and wherein the fluid flow channels within each primary layer are divided respectively at the folded regions that divide respectively the primary layers into the secondary layers.
Further relevant to claim 1, Xu et al. teaches that “Multiplexing analysis was enabled by using capillarity to guide the sample to four independent locations on the paper within hot wax printed channels, where species-specific LAMP reagents were deposited (Figure 2A, panel 5). The system was sealed by an acetate film to prevent evaporation during incubation (Figure 1B and D) and amplification was carried out 63 °C for up to 45 min on a simple hotplate. The results of species-specific LAMP were initially read-out by the naked eye with a handheld UV lamp (365 nm; Figure 3A)” (page 15251, column 1, paragraph 3).
This teaching reads on claim 1 performing LAMP reactions within each reaction chamber to obtain LAMP products.
Further relevant to claim 1, Xu et al. teaches “We also showed that the technique was amenable to quantification (Figure 3 B,C)” (page 15251, column 1, paragraph 4). The Xu et al. Figure 3 and associated caption teaches “B) Real-time amplification curve of Plasmodium pan LAMP”.
These teachings read on claim 1 detecting the LAMP products via an amplicon detection test.
Relevant to claims 3-4, Xu et al. Supporting Information teaches "Subsequently, 100 μl washing buffer containing 70% ethanol and 30 mM NaOAc was used to wash cell residues away. After washing, 40 μl elution buffer (10 mM TE buffer, pH 8.0) was used to elute the nucleic acid from the glass fiber and onto the printed LAMP reagents (Figure 1A, S2)" (page 2, column 2, "DNA extraction from blood" section).
This teaching reads on claim 3 wherein prior to said step of eluting the nucleic acid, the method comprises washing the nucleic acid at the solid phase extraction structure with a washing buffer; and claim 4 wherein the solid phase extraction structure comprises glass fibre.
Relevant to claims 5-6, Xu et al. Supporting Information Figure S1 (A) and associated caption teach "Illustration of the pathway taken by DNA molecule through the folded device during elution. 1. Structure S1 clamped with glass fibre (white); 2. Structure S2 (see Figures 1&2); 3. Acetate film with filter paper spots (white); 4. Plastic plate with holes (grey); 5. Acetate film. The red dotted line denotes the route of DNA movement at dilution step." The figure depicts the nucleic acid flow through several adjacent layers, including the third and fourth layers.
These teachings read on claim 5 wherein the step of allowing the nucleic acid to flow comprises allowing the nucleic acid to flow along the paper-based fluid flow channels of the second layer into paper-based fluid flow channels of a third layer positioned adjacent the second layer; and claim 6 allowing the nucleic acid to flow from the paper-based fluid flow channels of the third layer into paper-based fluid flow channels of a fourth layer positioned adjacent the third layer.
Relevant to claim 8, Xu et al. Supporting Information Figure S1 (A) and associated caption teach "3. Acetate film with filter paper spots (white); 4. Plastic plate with holes", reading on claim 8 wherein the discrete reaction chambers at the further layer comprises paper inserts positioned within respective holes in the third layer, the further layer comprising a plastic material.
Relevant to claims 10 and 13-14, Xu et al. teaches "Multiplexing analysis was enabled by using capillarity to guide the sample to four independent locations on the paper within hot wax printed channels, where species-specific LAMP reagents were deposited (Figure 2 A, panel 5). The system was sealed by an acetate film to prevent evaporation during incubation (Figure 1 B and D) and amplification was carried out 63°C for up to 45 min on a simple hotplate. The results of species-specific LAMP were initially read-out by the naked eye with a handheld UV lamp (365 nm; Figure 3 A)" (page 15251, column 1, paragraph 3).
This teaching reads on claim 10 wherein the step of performing the LAMP reactions comprises adding at least one set of LAMP primers to the discrete reaction chambers to create respective LAMP assays; claim 13 wherein the step of performing the LAMP reactions further comprises heating the further layer and the LAMP assays at a predetermined temperature and for a predetermined time and wherein the predetermined temperature is in a range 40 to 80°C and the predetermined time is in a range 10 to 90 minutes; and claim 14 wherein the step of detecting the LAMP products comprises monitoring and capturing a signal from the LAMP products emitted from the reaction chambers.
Relevant to claim 17, Xu et al. Figure 3 and associated caption teach "Results of multiplex LAMP amplification under UV light. Under UV excitation, green calcein emission occurs in the presence of pyrophosphate A) Numbers denote different species-specific LAMP reaction. 1. Internal control (IC)… and ddH2O as a negative control (5): 1. 10^8 IU/mL (red up-triangle); 2. 10^7 IU/mL (green right-triangle); 3. 10^6 IU/mL (cyan down-triangle); 4. 10^5 IU/mL (magenta circle). 5. Negative control (black square: no target DNA)."
These teachings read on claim 17 wherein the step of detecting the LAMP products comprises using one of the discrete reaction chambers as an internal positive control containing a predetermined genomic nucleic acid as a template and using one of the discrete reaction chambers as an internal negative control containing a predetermined genomic nucleic acid as a template.
Relevant to claim 18, Xu et al. Supporting Information teaches "Quantification: The LAMP reaction was quantified in real-time using a fluorescence microscope (Axio Scope A1, Zeiss) with a 10x objective and a FITC filter set (490 nm excitation, 515 nm emission). Data was collected every minute for 45 minutes (Hamamatsu) and quantified using the Wasabi software (Hamamatsu Photonics)" (page 2, column 2, "Quantification" section).
Further relevant to claim 18, Xu et al. Supporting Information teaches "Analytical sensitivity analysis: We studied the analytical sensitivity of our method through serial dilutions of cultured P. falciparum into uninfected whole blood, quantified through microscopy. Figure S2 below shows that the origami device was able to detect concentrations down to 5 parasites/μl by simple visual observation. We further quantified the intensity of color green in each spot of Figure S2 and normalised these to the controls (subtracted the intensity of the negative spot and then divided by the intensity of the positive control – IC), such that a value around 1 indicated a positive spot, while values around 0 indicated a negative spot. Using this methodology, the sensitivity of the origami test was also at 5 parasites/μl (Figure S3), using a t-test to differentiate from the negative control (0 parasites/μl – p-value of 10^-5 for 5 parasites/μl and 3.10^-3 for 10 parasites/μl)" (page 3, column 2, "Analytical sensitivity analysis" section).
Further relevant to claim 18, Xu et al. Supporting Information Figure S2 associated caption teaches that “The pictures were taken under UV illumination.”
Thus, these Xu et al. Supporting Information Figure S2 photographic images, LAMP product quantification, and normalization read on claim 18 wherein the step of detecting the LAMP products comprises monitoring and capturing a signal from the LAMP products emitted from the reaction chambers, wherein the step of capturing the signal comprises recording a fluorescent or UV signal as a photographic image, wherein the method further comprises analysing the at least one photographic image using software to obtain an average fluorescent or UV signal intensity of the LAMP products emitted from the respective reaction chambers, and wherein the method further comprises normalising the average fluorescent or UV signal intensity of the LAMP products using an average fluorescent or UV signal intensity of the positive control and the negative control respectively.
(iii) Although Lee et al. extraction methodology does not explicitly teach the Xu et al. multilayer device, it would have been prima facie obvious to the skilled artisan. It is noted that Lee et al. and Xu et al. are analogous disclosures to the instant nucleic acid detection methodology.
The skilled artisan would have been motivated to combine the analogous art. Xu et al. teaches that “identification of microbial species in resource-limited environments requires low cost, simple tests that do not need external or fixed power supplies” (page 15250, column 1, paragraph 1 of Introduction). Xu et al. further teaches that “As an additional feature of paper-based devices for disease diagnostics, we noted that samples can be readily disposed of by incineration” (page 15253, column 1, paragraph 1). Contrastingly, the Lee et al. portable LAMP device contains “external or fixed power supplies”, and the additional features and components (see Lee et al. Fig. 6) would not be as readily – or cheaply – disposed of by incineration. Thus, the skilled artisan would have been motivated to include the Xu et al. LAMP device within the Lee et al. pathogen detection methodology in order to provide low cost, simple, and readily disposable pathogen detection. The skilled artisan would have a reasonable expectation of success based on the disclosures of Lee et al. in view of Xu et al., as discussed in the preceding paragraphs.
Applicant’s Arguments
Applicant argues “There is no disclosure in either of Lee or Xu of a combination of primary and secondary layers having respective folded regions that are configured to divide the fluid flow as presently claimed” (Remarks 12/19/25, page 13, last sentence of penultimate paragraph; emphasis added).
Applicant further argues “Further, neither Lee nor Xu, either alone or in combination, discloses or fairly suggests a method including filtering of the sample at a filter membrane and the extraction and processing at a solid phase support structure, with the subsequent parallel delivery of extracted nucleic acid to the discreet multiple reaction chambers provided at a multilayer device as presently claimed” (Remarks 12/19/25, last sentence of page 13 continued to page 14; emphasis added).
Applicant further argues “Further, the technical effect of the division of fluid flow via primary and secondary layers having folded regions enables processing and pathogen detection at very low concentrations as described in the present application at page 1, lines 19-21 and page 3, lines 3-14. This is in contrast to the method of Xu that describes multiplexing of blood samples. As will be appreciated, target analytes in blood are at significantly high concentrations relative to very dilute aqueous sample containing pathogens. Therefore, Xu is not presented with the same technical problem as in the presently claimed invention” (Remarks 12/19/25, last paragraph of page 14; emphasis added).
Response to Applicant’s Arguments
The Examiner respectfully disagrees with the assertion that Xu et al. does not disclose a “combination of primary and secondary layers having respective folded regions that are configured to divide the fluid flow as presently claimed”. Absent limiting definitions of primary and secondary layers, the broadest reasonable interpretation of the configuration includes the Xu et al. folding of panels into structures as applied to above rejection of claim 1.
The Examiner further respectfully disagrees with the assertion that Lee et al. and Xu et al. do not disclose “filtering of the sample at a filter membrane and the extraction and processing at a solid phase support structure, with the subsequent parallel delivery of extracted nucleic acid to the discreet multiple reaction chambers provided at a multilayer device as presently claimed”.
For clarity, the rejections of claims 1 and 4 are expanded: Lee et al. teaches “Briefly, 100 mL of the sample was passed through a 0.22 µm pore-size syringe filter… in the direction of filtration. 1 mL of TE buffer… was injected into the same filter in the direction of filtration and the flow through pooled into a sterile 15 mL tube. The following solutions were backflushed through the filter in the opposite direction to the filtration direction, and the flow through pooled into the same tube. First, 1 mL of TE/lysozyme buffer (TL)… was backflushed through the filter, and 300 µL of TL was added into the flow-through” (page 372, column 2, paragraph 1 of “2.3. Syringe-filter based DNA extraction method” section).
This teaching reads on claim 1 filtering the water sample through a filter membrane and adding a lysis buffer to the filtered sample to form a nucleic acid solution. Absent a limiting definition for a filter membrane, Lee et al. teaches filtering water samples through filter membranes for a nucleic acid extraction method.
Additionally, the Xu et al. glass-fiber disc (seen in Xu et al. Figure 2) fulfills the structure and function of claim 1 a solid phase extraction structure mounted at a first layer of a multilayer device and claim 4 wherein the solid phase extraction structure comprises glass fibre.
The Examiner notes that the argued very low concentration of pathogen detection is not claimed. As discussed above in rejection claim 1, there is no instant limiting definition of a water sample. Therefore, the Xu et al. blood sample is analogous to the instant water sample, as the broadest reasonable interpretation of a water sample includes blood samples, which are samples largely comprised of water. Additionally, Xu et al. Supplemental section “Analytical sensitivity analysis” teaches detection of 5 parasites/µL, indicating “pathogen detection at very low concentrations”. Thus, the Examiner disagree with the assertion that Xu et al. is not analogous to the instant invention.
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 nonprovisional extension fee (37 CFR 1.17(a)) 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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/SARAH JANE KENNEDY/Examiner, Art Unit 1682
/WU CHENG W SHEN/Supervisory Patent Examiner, Art Unit 1682