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 .
Priority
The present application was filed on 08/27/2023, which claims benefit of the foreign Application China 202211229850X, filed on 10/09/2022.
Claim status
Claims 1, 2, 5, 7-8, and 10 are currently amended; claims 3-4, 6, 9, and 11 are currently canceled; claims 12-13 remain unchanged; and claims 14-15 are newly added. Claims 1, 2, 5, 7-8, 10 and 12-15 are pending and examined.
Claim Objections/Rejections status
The objections of claims 1-2, and 8 are withdrawn in view of the amendments of the claims. The objection of claim 9 is withdrawn in view of the cancellation of the claim.
The rejections of claims 2, and 7-8 under 35 U.S.C.112(b) as being indefinite are withdrawn in view of the amendments of the claims. The rejection of claims 6, 9, and 11 under 112(b) as being indefinite are withdrawn in view of the cancellation of the claim.
The rejections of claims 1-2 under 35 U.S.C.102(a)(1) and (a)(2) are withdrawn in view of the amendments of the claims. The rejections of claims 3-4 under 102(a)(1) and (a)(2) are withdrawn in view of the cancellation of the claims.
The rejections of claims 1-2, 5, 7-8, 10, 12-15 under 35 U.S.C.103 are new.
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 1-2 are rejected under 35 U.S.C. 103 as being unpatentable over Yun et al. (CN111334282) in view of Zhang (CN113234444).
For claim 1, Yun teaches a rare earth detection kit, a detection card and microspheres thereof, as well as a preparation and detection method (see Abstract). The detection card comprises: a sample pad, a nitrocellulose (NC) membrane, an absorbent pad and a plastic backing (see Fig.1 and pages 2-3 and 6: teaching that the card comprises a sample pad, a nitrocellulose membrane, an absorbent paper, a plastic bottom card). The sample pad of the detection card is also a conjugation pad because the end of the sample pad, which is close to a coating film (i.e., nitrocellulose membrane), is sprayed with microsphere line 21 (i.e., detecting reagents) (see page 2 lines 35-40).
Yun teaches the sample pad 2, the conjugation pad 21, the NC membrane 3, and the absorbent pad 4 are superimposed on the plastic backing 1 successively along a horizontal direction (see Example 3 on page 5-6 and Fig.1).
Fig.1 of Yun CN111334282
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Yun teaches that detection antibodies labeled with rare earth nanoparticles (RENPs) are immobilized on the conjugation pad (see page 2 lines 35-40: teaching that a monoclonal antibody labeled with rare earth nano fluorescent microspheres is sprayed on the end of the sample pad, wherein the antibody is specific to a target analyte). The fluorescent microspheres of Yun are rare-earth fluoride near-infrared two-zone light-emitting nanomaterials (see page 2 lines 1-2, page 3 lines 55-60).
Yun teaches that capture antibodies are coated at a test line 31 and quality control antibodies are coated at a control line 32 on the NC membrane (see page 2 lines 35-40, page 4 lines 35-36).
Yun does not teach the RENPs comprise an inner core and outer shell as claimed.
Zhang teaches NIR-II fluorescent RENPs comprising an inner core as LiReF4, wherein Re is Y, Gd, Yb and an outer shell as NaYbF4 or LiYF4 (see page 2 lines 8-16). These RENPs are ideal optical imaging probes because they have significant clinical application prospects, e.g., penetrate into deep tissues and guide doctors to perform surgery. They can emit fluorescent signals for a long time after being charged to avoid the self-fluorescence background of biological tissues caused by real-time excitation, thereby greatly improving the resolution, signal-to-noise ratio and sensitivity of biological imaging technology. See page 1 lines 20-34 and 36-38.
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to replace the RENPs taught by Yun with the RENPs taught by Zhang. The RENPs of Zhang have a long afterglow luminescence, thereby improving the resolution, signal-to-noise ratio and sensitivity of biological imaging technology (see Zhang page 1 lines 36-39).
One of ordinary skill in the art would have had a reasonable expectation of success in combining prior art references, because Zhang uses NIR-II fluorescent RENP as an optical imaging probe, while Yun uses a NIR-II fluorescent RENP as a label for the detecting antibody. Therefore, the RENP of Zhang is capable of being a label for the detection antibody of Yun.
For claim 2, Yun and Zhang teach the test strip as claimed in claim 1. Yun in view of Zhang teaches that the NIR-II fluorescent rare earth nanoprobe is RENPs with fluorescent emission peak in a NIR-II region (see Zhang page 1 lines 50-52, page 2 lines 2-3).
Claims 5 and 7 are rejected under 35 U.S.C. 103 as being unpatentable over Yun in view of Zhang, as applied to claims 1-2, further in view of Yang et al. (Recent advances in design of lanthanide-containing NIR-II luminescent nanoprobes, iScience, Volume 24, Issue 2, 19 February 2021, 102062).
For claim 5, Yun and Zhang teach the test strip as claimed in claim 1. Yun teaches that the surface of the nanoprobe is coated with carboxyl group to obtain water-soluble rare earth nano fluorescent microspheres (see page 2 lines 15-20). Yun does not teach that the surface of the RENPs is modified with one or more molecular polymers.
Yang teaches that to make NIR-II nanoprobes water-soluble and provide reactive groups for subsequent bioconjugation to various biomolecules, a variety of surface modification strategies have been developed over the past decade like organic polymer modification (see page 12 par.2). Polymers comprising carboxyl groups are used to create a hydrophilic surface of NIR-II nanoprobes (see page 13 par.2).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the surface of the nanoprobes taught by Yun in view of Zhang, by coating the nanoprobe with polymers comprising carboxyl group as taught by Yang, because the NIR-II nanoprobe coated with polymer of carboxyl groups is water-soluble and is able to conjugate with various biomolecules subsequently (see Yang page 12 par.2).
One having an ordinary skill in the art would have had a reasonable expectation of success in modifying the nanoprobe of Yun to obtain water-soluble rare earth nano fluorescent microspheres using Yang’s technique, because Yun is generic to the nanoprobe coated with carboxyl group while Yang is directed to the technique of creating the water-soluble surface on NIR-II nanoprobe by coating polymers comprising carboxyl groups on the surface of the nanoprobe.
For claim 7, Yun, Zhang and Yang teach the test strip as claimed in claim 5. Yun and Yang teaches that the nanoprobe is a carboxylated RENPs (see Yun page 2 lines 15-20: teaches the surface of the probe is modified with the carboxyl group). See discussion in claim 5. Yun also teaches that the particle size of the RENPs is in a range of 20 nm to 200 nm (see Yun page 1 lines 54-55: teaching that the particle size is 40nm~60nm).
Yun does not teach wherein the NIR-II fluorescent rare earth nanoprobe test strip probe is carboxylated RENPs with a surface modified by one or more of sodium citrate, polyacrylic acid (PAA), distearoyl phosphoethanolamine-polyethylene glycol- carboxyl (DSEP-PEG-COOH), poly-dl-lactic-co-glycolic-polyethylene glycol- carboxyl (PLGA-PEG -COOHI), and polyethylene glycol-polylactic acid -carboxyl 12 (PEG-PLA-COOH).
Yang, on page 13 par.2, teaches the carboxyl group comprises DSPE-PEG2000-COOH.
Zhang teaches that the size of NIR-II particles is 20nm-300nm (see page 2 lines 5-6).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to use the specific carboxyl group DSPE-PEG2000-COOH taught by Yang to treat the surface of the probe taught by Yun in view of Zhang. One having an ordinary skill in the art would have been motivated to use DSPE-PEG2000-COOH to treat the surface of the nanoprobe because the PEG2000-COOH treated nanoparticles can markedly improve the water solubility and biocompatibility of NIR-II luminescent nanoprobes (see Yang page 13 par.2).
Since Yun is generic to the carboxyl group and Yang is specific for one carboxyl group composition that can be used to treat the nanoprobe’s surface. Therefore, the combination of Yun and Yang would yield a predictable result.
Claims 8 and 10 are rejected under 35 U.S.C. 103 as being unpatentable over Yun and Zhang, as applied to claim 1, in view of Merck (Rapid Lateral Flow Test Strips, Considerations for Product Development, 2013, PTO-892 dated 01/28/2026), Hao et al. (US 20160349251), Han et al. (Low-cost, open-source 3D printed antibody dispenser for development and small-scale production of lateral flow assay strips, 2021, PTO-892 dated 01/28/2026), Zhong et al. (In vivo molecular imaging for immunotherapy using ultra-bright near-infrared-IIb rare-earth nanoparticles, Nature Biotechnology volume 37, pages 1322-1331 (2019), PTO-892 dated 01/28/2026), Delport et al. (Use of nano-particles in biosensing, Comm. Appl. Biol. Sci, Ghent University, 72/1, 2007, PTO-892 dated 01/28/2026), and Life Science network (10x PBS buffer (10x Phosphate Buffered Saline), 2013, PTO-892 dated 01/28/2026).
For claim 8, Yun and Zhang teach the test strip as claimed in claim 1. Yun also teaches a preparation method of the NIR-Il fluorescent rare earth nanoprobe test strip comprising the following steps:
Step 1 preparation of the sample pad: the sample pad is made of glass fiber (see page 8 lines 41-43), treating the glass fiber with a sample pad treatment solution, and finally drying in an oven (see page 8 lines 20-30: teaching that a sample pad treatment solution is sprayed on one end of the sample pad, then the sample pad is placed in an oven and dry overnight).
Step 2 preparation of the conjugation pad: diluting an RENPs labeling solution with a microsphere diluent to obtain a diluted RENPs labeling solution, spreading the diluted RENPs labeling solution on the glass fiber, and then drying in the oven (see page 8 lines 39-41: teaching that sample pad is made of glass fiber; page 8 lines 20-30: teaching that a diluted solution of microsphere diluent and rare earth nano-fluorescent microspheres labeled antibody in HCl solution is sprayed on the other end of the sample pad, then the sample pad is placed in an oven and dry overnight).
Step 2 further comprises:
Performing ultrasonic resuspension on carboxylated RENPs, and then discarding a supernatant after high-speed centrifugation to obtain a precipitate (see page 2 lines 20-25); adding a 2-morpholinoethanesulphonic acid (MES) buffer into the precipitate, ultrasonically dispersing the precipitate added with the MES buffer, followed by adding an activator of 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydro (EDC) and a coupling agent of N- hydroxysulfosuccinimide (Sulfo-NHS), oscillating and performing a reaction to obtain a first product (see page 2 lines 20-25); discarding a supernatant of the first product and obtaining a precipitation of the first product after high-speed centrifugation (see page 2 lines 20-25);
adding the detection antibodies in the activated RENPs solution, oscillating for a reaction, then adding a bovine serum albumin (BSA) solution for sealing (see page 6 lines 19-25); discarding a supernatant of the second product and obtaining a precipitate of the second product after high-speed centrifugation, followed by adding microsphere washing liquid into the precipitate of the second product and ultrasonically suspending for 2-3 times; then adding a microsphere protective solution and storing at 4 °C for later use (see page 6 lines 19-25).
Yun teaches the microsphere washing solution containing 0.5% BSA and 0.1% Tween-20 and the microsphere protective solution containing 0.5% BSA (see page 6 lines 20-25: teaching that a 0.5%BSA, 0.1% Tween-20 10~50mM, pH7.5~8.5 Tris-HCl preservation solution works as 2 solutions for washing and storing after conjugating antibody on RENPs).
Step 3 coating antibody of the NC membrane: preparing a capture antibody solution and a quality control antibody solution with a buffer, respectively; simultaneously spraying the capture antibody solution and the quality control antibody solution on the NC membrane to form the capture antibodies set as the test line (T line) and the quality control antibodies as the control line (C line) on the NC membrane, and then drying in the oven (see page 8 lines 30-35: teaching that a coating buffer (20mM pH8.0 Tris-HCl buffer containing 2.5% (w/w) sucrose) is used to adjust the concentration of PTH monoclonal antibody and goat anti-mouse IgG antibody to 1mg/ml, 1μl of coating volume of capture or control antibody buffer is used for a centimeter coating film, as the test line and quality control line, respectively, and dried in an oven; see page 8 lines 39-41: teaching that coating film is made of nitrocellulose).
Step 4 preparation of the absorbent pad: cutting the absorbent pad to a target size (see page 8 line 40: teaching that an absorbent paper is placed on the backing (size 80*300 mm) (the size is 28*300mm), which means the absorbent paper is cut to a target size).
Step 5 assembling a detection card: superimposing the sample pad, the conjugation pad, the NC membrane, and the absorbent pad on the plastic backing successively along the horizontal direction (see Fig.1, Example 3 on page 5-6 and page 8 line 39-41); cutting the assembled pad and membrane into test strips, then putting the test strips into card slots to make detection cards, finally storing the detection cards in a dry environment (see page 6 lines 8-11, page 8 line 39-50).
Yun does not teach: soaking the pad in the solution, then shaking at room temperature (RT) for 2-3 hours as in step 1;
using a three-dimensional point spray platform as in step 3.
Using an automatic cutting machine to cut the test paper board into test strips as in step 5.
Yun does not clearly teach adding a phosphate buffer for ultrasonic dispersion to obtain an activated RENPs solution and adding an ethanolamine solution to terminate the reaction to obtain a second product as in step 2 last paragraph.
Merck is generic about the method of producing a lateral test strip, where the sample pad can be impregnated with a variety of chemical agents designed to make the full range of samples compatible with the functioning of the test strip, and once impregnated with the treatment solution, the sample pads should be dried and stored at a certain condition (see page 27 Preparing Sample Pads section, see page 27 col.2 Storage section).
Merck also teaches using automatic cutting machine to cut the test paper board into test strips (see page 29 Slitting section).
Hao teaches the preparation and assemblage of the immunoassay test immunochromatographic strip (see par.94), which comprises submerging the sample pad of glass fiber with a treatment solution and then drying at 37° C. for 1.5 hours (see par.94).
Han teaches a 3D printed open-source antibody dispenser that can be easily built and used for the development of lateral flow assay (LFA) strips (see Abstract). Han demonstrates uniform dispensing of capture antibody and control antibody to draw a test line and a control line on nitrocellulose membrane for the lateral flow test strip (see Abstract, page 2 par.4).
Zhong teaches that the carboxylated RENPs are dispersed in 1xPBS solution at 4 °C for long-term storage to obtain an activated RENPs solution (page 1332 col.2 par.1). This teaching discloses the application of a PBS solution in the activating treatment of RENPs. Zhong also teaches the process of conjugating antibody onto the surface of activated RENPs (see page 1332 col.2 par.2-3), wherein all the steps from dispersing, mixing, to washing are done with PBS solution (see page 1332 col.2 par.3). This teaching discloses the application of a PBS solution to RENPs dispersion during the conjugation of antibodies on the surface of RENPs.
Zhong teaches that the hydrophilic functionalized RENPs showed remarkable stability in aqueous buffers (1xPBS) and serum without aggregation and exhibited zero photo-bleaching (see page 1325 col.1).
Delport teaches techniques for the immobilization of different bio-molecules on nanoparticle NP (see Introduction). Delport teaches that for all protein bioconjugation experiments, EDC chemistry is applied for activating the carboxyl groups on NP before mixing protein with NP (see page 1 last paragraph). To prevent unspecific binding, an excess of ethanolamine is added after a bio-molecule immobilization step to block the activated carboxyl groups on the surface of the NP, wherein ethanolamine binds all activated groups left on the surface and changes a charged carboxyl surface into a hydrophilic hydroxyl surface (see page 2 par.3).
Life Science network teaches that 1xPBS is 10mM PBS buffer (i.e., 1x buffer will contain 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM K2HPO4).
Yun, Merck, and Hao do not teach the same method of treating the sample pad with the treatment solution as claimed, which is soaking and shaking the sample pad in the treatment buffer for 2-3 hrs at RT. However, Yun, Merck and Hao suggest a finite number of ways to prepare the sample pad for the test strip: Merck teaches that the sample pad is impregnated with the treatment solution; Yun teaches that the treatment solution is spraying to the sample pad; Hao teaches that the sample pad is submerged in the treatment solution. At the time of invention, it would have been obvious to one of ordinary skill in the art to try one or another way to apply the treatment solution on the sample pad and arrive the claimed method. A person of ordinary skill has good reason to pursue the known options within his or her technical grasp. The results of the combination are predictable (i.e., the sample pad is treated with the treatment buffer and is dried and stored at a certain condition) with a reasonable expectation of success.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to use the automatic cutting machine to cut the test paper board into test strips taught by Merck in the preparation method of the NIR-II nanoprobe test strip taught by Yun. It is because Yun is generic about the cutting method and Merck provides one specific method to cut the board into strips. The method of Merck makes the cutting process quicker and more convenient.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to use 3D printed open-source antibody dispenser to draw a test line and a control line on the test strip of Yun, because the dispenser can dispense capture antibodies and control antibodies uniformly as taught by Han.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the activating treatment step of Yun, by using PBS solution in dispersion step, e.g., by ultrasonicating, to obtain an activated RENPs solution or to obtain an antibody conjugated RENPs as taught by Zhong because the hydrophilic functionalized RENPs showed remarkable stability in aqueous buffers (1xPBS) without aggregation and exhibited zero photo-bleaching (see Zhong page 1325 col.1 and page 1332 col.1-3). Life Science network states that the 1xPBS comprises 10mM PBS.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the preparation step for antibody-conjugated RENPs of Yun, by using ethanolamine solution to terminate the conjugating reaction, because Delport teaches that ethanolamine can block the activated carboxyl groups on the surface of the NP to prevent unspecific binding after the conjugation step is completed.
One having an ordinary skill in the art would have had a reasonable expectation of success in combining Yun, Zhong and Delport because they are directed to prepare nanoparticle probes for biosensing assay, where the nanoparticle probes need to be activated to conjugate with the biomolecule on their surface and the probes are used in immunoassay.
For claim 10, Yun, Zhang, Merck, Hao, Han, Zhong, and Delport teach the preparation method as claimed in claim 8. Yun teaches activating the RENPs by incubating 200ul of the carboxylated RENPs with 50 μl of 100 mg/ml EDC and 100 μl of 100 mg/ml Sulfo-NHS (see Yun page 8 lines 5-10).
Delport teaches that “to maximize the efficiency of the NP's, the conjugation chemistry and coating have to be optimized for every material, shape or size of the NP and every type of biomolecule” (see Introduction). Delport teaches activating the RENPs by incubating a quantity of NP with 0.15 mg/ml EDC (see page 1 last paragraph). To further improve efficiency, the activated carboxyl groups stabilized with NHS before reaction with the primary amines (see page 2 par.2). The optimal concentrations were found to be 12.5 mg/ml for both the EDC and NHS (see page 2 par.2).
Yun and Delport do not teach the same molar ratio of the carboxylated RENPs: the EDC: the Sulfo-NHS is 1: 5: 10 as claimed. However, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to define the optimal concentrations of each chemical used in the activating treatment step to maximize the efficiency of the NPs, as taught by Delport. It is obvious that the varying amounts of RENPs, EDC and Sulfo-NHS used for activating the carboxyl groups on the surface of NP taught by Yun and Delport is the consequence of the routine optimization process based on the material, shape or size of the NP and every type of biomolecule (see Delport Introduction). Absent unexpected results, it would have been obvious to one of ordinary skill to have arrived at the claimed molar ratio by routine optimization in order to uncover the optimum workable ranges of the method of preparing conjugated RENPs.
Claim(s) 12 is/are rejected under 35 U.S.C. 103 as being unpatentable over Yun, Zhang, Merck, Hao, Han, Zhong, Delport, as applied to claim 8, and further in view of Trenkenschuh et al. (Freeze-thaw stability of aluminum oxide nanoparticles, International Journal of Pharmaceutics 606 (2021) 120932, PTO-892 dated 01/28/2026).
For claim 12, Yun, Zhang, Merck, Hao, Han, Zhong, and Delport teach the preparation method as claimed in claim 8. Yun teaches that the microsphere diluent comprises a solution containing sucrose (see page 8 lines 24-25: teaching that a microsphere diluent comprises 20mM Tris containing 0.5% (w/w) BSA, 25% (w/w) sucrose).
Yun does not teach a 10 mM citric acid solution containing 1% sucrose
Merck teaches that the microsphere diluent comprises a solution containing 1%-10% sucrose which serves as a preservative and a re-solubilization agent (see page 24 Preparing conjugate pads).
Trenkenschuh teaches that NPs preserved in 10 mM Na-citrate buffer pH 5 and 8 (i.e., 10mM citric acid solution) are good. However, the size of NPs is still increasing in the absence of further additives, e.g., sucrose (see page 8 col.1 par.3). The additives, e.g., sucrose improved NPs in preserved condition due to enhanced particle isolation and the formation of a stabilizing matrix (see page 8 col.1 par.3).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the microsphere diluent of Yun, using a solution which comprises 10 mM citric acid solution containing 1% sucrose because Trenkenschuh teaches that the solution comprising 10 mM citric acid, and 1% sucrose improves the NPs isolation and stabilization. Moreover, the solution with 1% sucrose also serves as a preservative and a re-solubilization agent when applying the RENPs on the conjugation pad of the test strip as taught by Merck.
One having an ordinary skill in the art would have had a reasonable expectation of success in combining Yun, Merck and Trenkenschuh because they are directed to prepare nanoparticle probes for biosensing assay, where the nanoparticle probes are preserved for use later.
Claim(s) 13 is/are rejected under 35 U.S.C. 103 as being unpatentable over Yun, Zhang, Merck, Hao, Han, Zhong, Delport, as applied to claim 8, and further in view of Li et al. (Lateral Flow Immunochromatography Assay for Detection of Furosemide in Slimming Health Foods, Foods 2021, 10, 2041, PTO-892 dated 01/28/2026).
For claim 13, Yun, Zhang, Merck, Hao, Han, Zhong, and Delport teach the preparation method as claimed in claim 8. Yun teaches wherein the sample pad treatment solution comprises a 20 mM tris(hydroxymethyl)aminomethane (Tris) buffer containing 0.5% surfactant, BSA (see page 8 lines 20-27: teaching that a solution comprises 20mM, pH8.0 Tris-HCl containing 0.5% S17 (i.e., surfactant), 0.1%BSA).
Yun does not teach the BSA used at 0.05%. Yun does not teach the Tris buffer containing 0.05% Tween-20, 0.3% polyvinylpyrrolidone K30 (PVP-K30) and 0.05% PROCLIN-300.
Li teaches a preparation method of a lateral test strip (see pages 2-4). Li teaches the sample pad treatment solution comprising 0.5% BSA, 0.5% Tween-20, 0.3% PVP and 0.03% ProClin 300 (see page 4 par.2). Tween-20 is for a better release AuNPs–Abs probe and to adjust the chromatography speed, PVP is a steric stabilizer or capping agent to protect the AuNPs–Abs against agglomeration, and ProClin 300 is used as a preservative composition to prevent metamorphism (see page 3 par.3).
Li teaches that the sample pad plays a crucial role in reducing the interference of the sample matrix and affects the binding of the labeled probe on the nitrocellulose membrane, thereby affecting the color intensity and sensitivity of the test strip (see page 7 section 3.2.5). Li teaches an optimizing process to define the formula and the concentration of each component of the treating solution to achieve optimal results for the nanoparticles lateral flow immunochromatography (see pages 5-7).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the sample pad treatment solution of Yun, by adding Tween-20, polyvinylpyrrolidone K30 (PVP-K30) and PROCLIN-300 as taught by Li because Tween-20 is for a better release AuNPs–Abs probe and to adjust the chromatography speed, PVP is a steric stabilizer or capping agent to protect the AuNPs–Abs against agglomeration, and ProClin 300 is used as a preservative composition to prevent metamorphism (see Li page 3 par.3). One having an ordinary skill in the art would have been motivated to add Tween-20, polyvinylpyrrolidone K30 (PVP-K30) and PROCLIN-300 in the treating solution for obtaining a rapid, convenient and sensitive lateral flow immunochromatography (LFIA) based on nanoparticles (see Li in Abstract).
It would have been obvious to one of ordinary skill to discover the workable ranges of the concentration of each component of the treating solution by normal optimization procedures known in the art (e.g., taught by Li in pages 5-7) to achieve a rapid, convenient and sensitive lateral flow immunochromatography (LFIA) based on nanoparticles.
One having an ordinary skill in the art would have had a reasonable expectation of success in combining Yun and Li because they are directed to prepare a sample pad for a lateral flow assay.
Claim(s) 14 is/are rejected under 35 U.S.C. 103 as being unpatentable over Yun (CN111334282) in view of Merck (Rapid Lateral Flow Test Strips, Considerations for Product Development, 2013), Hao et al. (US 20160349251), Han et al. (Low-cost, open-source 3D printed antibody dispenser for development and small-scale production of lateral flow assay strips, 2021), Zhong et al. (In vivo molecular imaging for immunotherapy using ultra-bright near-infrared-IIb rare-earth nanoparticles, Nature Biotechnology volume 37, pages 1322-1331 (2019)), Delport et al. (Use of nano-particles in biosensing, Comm. Appl. Biol. Sci, Ghent University, 72/1, 2007), and Life Science network (10x PBS buffer (10x Phosphate Buffered Saline), 2013).
For claim 14, Yun teaches a preparation method of the NIR-II fluorescent rare earth nanoprobe test strip comprising the following steps:
(1) preparation of a sample pad: choosing a glass fiber, soaking the glass fiber in a sample pad treatment solution, then shaking at RT for 2-3 h, and finally drying in an oven to thereby obtain the sample pad; (see Yun page 8 lines 20-30: teaching that a sample pad treatment solution is sprayed on one end of the sample pad, then the sample pad is placed in an oven and dry overnight)
(2) preparation of a conjugation pad:
performing ultrasonic resuspension on carboxylated RENPs, and then discarding a supernatant after high-speed centrifugation to obtain a precipitate; adding a MES buffer into the precipitate, ultrasonically dispersing the precipitate added with the MES buffer, followed by adding an activator of EDC and a coupling agent of Sulfo-NHS, oscillating and performing a reaction to obtain a first product; discarding a supernatant of the first product and obtaining a precipitate of the first product; (see Yun page 2 lines 20-25: disclosing “Activate rare earth nano fluorescent microspheres: Ultrasonic treatment and centrifugation are performed on the rare earth nano fluorescent microspheres in step 2, and the precipitate is washed with 10-100mM MES solution with a pH of 5.0-7.0; adding carbodiimide, N-Hydroxysulfosuccinimide was mixed and then centrifuged at high speed, and the precipitate was washed with MES solution with a pH of 5.0~7.0 to obtain activated rare earth nano fluorescent microspheres”)
adding the detection antibodies into the activated RENPs solution, oscillating for a reaction, then adding a bovine serum albumin (BSA) solution for sealing (see Yun page 6 lines 19-25); discarding a supernatant of the second product and obtaining a precipitate of the second product after high-speed centrifugation, followed by adding microsphere washing liquid into the precipitate of the second product and ultrasonically suspending for 2-3 times; then adding a microsphere protective solution and storing at 4 °C for later use (see Yun page 6 lines 19-25).
Yun teaches the microsphere washing solution containing 0.5% BSA and 0.1% Tween-20 and the microsphere protective solution containing 0.5% BSA (see page 6 lines 20-25: teaching that a 0.5%BSA, 0.1% Tween-20 10~50mM, pH7.5~8.5 Tris-HCl preservation solution works as 2 solutions for washing and storing after conjugating antibody on RENPs).
Yun teaches diluting the RENPs labeling solution with a microsphere diluent to obtain a diluted RENPs labeling solution, spreading the diluted RENPs labeling solution on the glass fiber, and then drying in the oven (see page 8 lines 39-41: teaching that sample pad is made of glass fiber; page 8 lines 20-30: teaching that a diluted solution of microsphere diluent and rare earth nano-fluorescent microspheres labeled antibody in HCl solution is sprayed on the other end of the sample pad, then the sample pad is placed in an oven and dry overnight).
(3) coating antibody of the NC membrane: preparing a capture antibody solution and a quality control antibody solution with a buffer, respectively; simultaneously spraying the capture antibody solution and the quality control antibody solution on the NC membrane to form the capture antibodies set as the test line (T line) and the quality control antibodies as the control line (C line) on the NC membrane, and then drying in the oven (see Yun page 8 lines 30-35: teaching that a coating buffer (20mM pH8.0 Tris-HCl buffer containing 2.5% (w/w) sucrose) is used to adjust the concentration of PTH monoclonal antibody and goat anti-mouse IgG antibody to 1mg/ml, 1μl of coating volume of capture or control antibody buffer is used for a centimeter coating film, as the test line and quality control line, respectively, and dried in an oven; see page 8 lines 39-41: teaching that coating film is made of nitrocellulose).
(4) preparation of the absorbent pad: cutting the absorbent pad to a target size (see Yun page 8 line 40: teaching that an absorbent paper is placed on the backing (size 80*300mm) (the size is 28*300mm), which means the absorbent paper is cut to a target size).
(5) assembling a detection card: superimposing the sample pad, the conjugation pad, the NC membrane, and the absorbent pad on the plastic backing successively along the horizontal direction (see Yun Fig.1, Example 3 on page 5-6 and page 8 line 39-41); cutting the assembled pad and membrane into test strips, then putting the test strips into card slots to make detection cards, finally storing the detection cards in a dry environment (see Yun page 6 lines 8-11, page 8 line 39-50).
Yun does not teach: soaking the pad in the solution, then shaking at room temperature (RT) for 2-3 hours as in step 1;
using a three-dimensional point spray platform as in step 3.
Using an automatic cutting machine to cut the test paper board into test strips as in step 5.
Yun does not clearly teach adding a phosphate buffer for ultrasonic dispersion to obtain an activated RENPs solution and adding an ethanolamine solution to terminate the reaction to obtain a second product as in step 2 second paragraph.
Merck is generic about the method of producing a lateral test strip, where the sample pad can be impregnated with a variety of chemical agents designed to make the full range of samples compatible with the functioning of the test strip, and once impregnated with the treatment solution, the sample pads should be dried and stored at a certain condition (see page 27 Preparing Sample Pads section, see page 27 col.2 Storage section).
Merck also teaches using automatic cutting machine to cut the test paper board into test strips (see page 29 Slitting section).
Hao teaches the preparation and assemblage of the immunoassay test immunochromatographic strip (see par.94), which comprises submerging the sample pad of glass fiber with a treatment solution and then drying at 37° C. for 1.5 hours (see par.94).
Han teaches a 3D printed open-source antibody dispenser that can be easily built and used for the development of lateral flow assay (LFA) strips (see Abstract). Han demonstrates uniform dispensing of capture antibody and control antibody to draw a test line and a control line on nitrocellulose membrane for the lateral flow test strip (see Abstract, page 2 par.4).
Zhong teaches that the carboxylated RENPs are dispersed in 1xPBS solution at 4 °C for long-term storage to obtain an activated RENPs solution (page 1332 col.2 par.1). This teaching discloses the application of a PBS solution in the activating treatment of RENPs. Zhong also teaches the process of conjugating antibody onto the surface of activated RENPs (see page 1332 col.2 par.2-3), wherein all the steps from dispersing, mixing, to washing are done with PBS solution (see page 1332 col.2 par.3). This teaching discloses the application of a PBS solution to RENPs dispersion during the conjugation of antibodies on the surface of RENPs.
Zhong teaches that the hydrophilic functionalized RENPs showed remarkable stability in aqueous buffers (1xPBS) and serum without aggregation and exhibited zero photo-bleaching (see page 1325 col.1).
Delport teaches techniques for the immobilization of different bio-molecules on nanoparticle NP (see Introduction). Delport teaches that for all protein bioconjugation experiments, EDC chemistry is applied for activating the carboxyl groups on NP before mixing protein with NP (see page 1 last paragraph). To prevent unspecific binding, an excess of ethanolamine is added after a bio-molecule immobilization step to block the activated carboxyl groups on the surface of the NP, wherein ethanolamine binds all activated groups left on the surface and changes a charged carboxyl surface into a hydrophilic hydroxyl surface (see page 2 par.3).
Life Science network teaches that 1xPBS is 10mM PBS buffer (i.e., 1x buffer will contain 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM K2HPO4).
Yun, Merck, and Hao do not teach the same method of treating the sample pad with the treatment solution as claimed, which is soaking and shaking the sample pad in the treatment buffer for 2-3 hrs at RT. However, Yun, Merck and Hao suggest a finite number of ways to prepare the sample pad for the test strip: Merck teaches that the sample pad is impregnated with the treatment solution; Yun teaches that the treatment solution is spraying to the sample pad; Hao teaches that the sample pad is submerged in the treatment solution. At the time of invention, it would have been obvious to one of ordinary skill in the art to try one or another way to apply the treatment solution on the sample pad and arrive the claimed method. A person of ordinary skill has good reason to pursue the known options within his or her technical grasp. The results of the combination are predictable (i.e., the sample pad is treated with the treatment buffer and is dried and stored at a certain condition) with a reasonable expectation of success.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to use the automatic cutting machine to cut the test paper board into test strips taught by Merck in the preparation method of the NIR-II nanoprobe test strip taught by Yun. It is because Yun is generic about the cutting method and Merck provides one specific method to cut the board into strips. The method of Merck makes the cutting process quicker and more convenient.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to use 3D printed open-source antibody dispenser to draw a test line and a control line on the test strip of Yun, because the dispenser can dispense capture antibodies and control antibodies uniformly as taught by Han.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the activating treatment step of Yun, by using PBS solution in dispersion step, e.g., by ultrasonicating, to obtain an activated RENPs solution or to obtain an antibody conjugated RENPs as taught by Zhong because the hydrophilic functionalized RENPs showed remarkable stability in aqueous buffers (1xPBS) without aggregation and exhibited zero photo-bleaching (see Zhong page 1325 col.1 and page 1332 col.1-3). Life Science network states that the 1xPBS comprises 10mM PBS.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the preparation step for antibody-conjugated RENPs of Yun, by using ethanolamine solution to terminate the conjugating reaction, because Delport teaches that ethanolamine can block the activated carboxyl groups on the surface of the NP to prevent unspecific binding after the conjugation step is completed.
One having an ordinary skill in the art would have had a reasonable expectation of success in combining Yun, Zhong and Delport because they are directed to prepare nanoparticle probes for biosensing assay, where the nanoparticle probes need to be activated to conjugate with the biomolecule on their surface and the probes are used in immunoassay.
Claim(s) 15 is/are rejected under 35 U.S.C. 103 as being unpatentable over Yun (CN111334282) in view of Merck (Rapid Lateral Flow Test Strips, Considerations for Product Development, 2013), Hao et al. (US 20160349251), Han et al. (Low-cost, open-source 3D printed antibody dispenser for development and small-scale production of lateral flow assay strips, 2021), Zhong et al. (In vivo molecular imaging for immunotherapy using ultra-bright near-infrared-IIb rare-earth nanoparticles, Nature Biotechnology volume 37, pages 1322-1331 (2019)), Life Science network (10x PBS buffer (10x Phosphate Buffered Saline), 2013), Delport et al. (Use of nano-particles in biosensing, Comm. Appl. Biol. Sci, Ghent University, 72/1, 2007) and Hasanzadeh et al. (Nanomaterials for use in immunosensing of carcinoembryonic antigen (CEA): Recent advances, Trends in Analytical Chemistry 86 (2017) 185e205).
For claim 15, Yun teaches a preparation method of the NIR-II fluorescent rare earth nanoprobe test strip comprising the following steps:
(1) Preparation of a sample pad: choosing a glass fiber, soaking the glass fiber in a sample pad treatment solution, then shaking at RT for 2-3 h, and finally drying in an oven to thereby obtain the sample pad; (see Yun page 8 lines 20-30: teaching that a sample pad treatment solution is sprayed on one end of the sample pad, then the sample pad is placed in an oven and dry overnight).
(2) Preparation of a conjugation pad:
Yun teaches a step of activating RENPs on page 2 lines 20-25: Ultrasonic treatment and centrifugation are performed on the rare earth nano fluorescent microspheres, and the precipitate is washed with 10-100mM MES solution with a pH of 5.0-7.0; adding carbodiimide, N-Hydroxysulfosuccinimide was mixed and then centrifuged at high speed, and the precipitate was washed with MES solution with a pH of 5.0~7.0 to obtain activated rare earth nano fluorescent microspheres (see Yun page 2 lines 20-25).
Yun also teaches a step of conjugating antibody to RENPs in Example 4 on page 6 (see at least on lines 19-25) as follows: Ultrasonicate the activated rare-earth nano-fluorescent microspheres in the previous step for 1-2 minutes, then add PTH monoclonal antibody at 50-200μg/200μl, mix for 1-3 hours, Use 10~50mM, pH7.5~8.5 Tris-HCl blocking solution containing 0.5%BSA to block for 0.5~1 hour, then centrifuge at 12000~14000rpm for 5~15min at high speed, and use 1% NaCl, 0.5%BSA, 0.1% Tween-20 10~50mM, pH7.5~8.5 Tris-HCl preservation solution, washed and resuspended, and stored at 4°C in the dark.
Yun teaches diluting a RENPs labeling solution with a microsphere diluent to obtain a diluted RENPs labeling solution, spreading the diluted RENPs labeling solution on the glass fiber, and then drying in the oven (see page 8 lines 39-41: teaching that sample pad is made of glass fiber; page 8 lines 20-30: teaching that a diluted solution of microsphere diluent and rare earth nano-fluorescent microspheres labeled antibody in HCl solution is sprayed on the other end of the sample pad, then the sample pad is placed in an oven and dry overnight).
(3) Coating antibody of the NC membrane: preparing a capture antibody solution and a quality control antibody solution with a buffer, respectively; simultaneously spraying the capture antibody solution and the quality control antibody solution on the NC membrane to form the capture antibodies set as the test line (T line) and the quality control antibodies as the control line (C line) on the NC membrane, and then drying in the oven (see Yun page 8 lines 30-35: teaching that a coating buffer (20mM pH8.0 Tris-HCl buffer containing 2.5% (w/w) sucrose) is used to adjust the concentration of PTH monoclonal antibody and goat anti-mouse IgG antibody to 1mg/ml, 1μl of coating volume of capture or control antibody buffer is used for a centimeter coating film, as the test line and quality control line, respectively, and dried in an oven; see page 8 lines 39-41: teaching that coating film is made of nitrocellulose).
(4) Preparation of the absorbent pad: cutting the absorbent pad to a target size (see Yun page 8 line 40: teaching that an absorbent paper is placed on the backing (size 80*300 mm) (the size is 28*300mm), which means the absorbent paper is cut to a target size).
(5) Assembling of a detection card: superimposing the sample pad, the conjugation pad, the NC membrane, the absorbent pad on the plastic backing successively along the horizontal direction (see Yun Fig.1, Example 3 on page 5-6 and page 8 line 39-41); cutting the assembled pad and membrane into test strips, then putting the test strips into card slots to make detection cards, finally storing the detection cards in a dry environment (see Yun page 6 lines 8-11, page 8 line 39-50).
Yun does not teach: soaking the pad in the solution, then shaking at room temperature (RT) for 2-3 hours as in step 1;
using a three-dimensional point spray platform as in step 3.
using an automatic cutting machine to cut the test paper board into test strips as in step 5.
Yun does not teach the same amounts of reagents in the preparation steps, or the same mixing and centrifuging time; using PBS in ultrasonic dispersion or in the activating treatment of RENPs; conjugating carcinoembryonic antigen (CEA) detection antibody as in step 2.
Merck is generic about the method of producing a lateral test strip, where the sample pad can be impregnated with a variety of chemical agents designed to make the full range of samples compatible with the functioning of the test strip, and once impregnated with the treatment solution, the sample pads should be dried and stored at a certain condition (see page 27 Preparing Sample Pads section, see page 27 col.2 Storage section).
Merck also teaches using automatic cutting machine to cut the test paper board into test strips (see page 29 Slitting section).
Hao teaches a preparation and assemblage of the immunoassay test immunochromatographic strip (see par.94), which comprises submerging the sample pad of glass fiber with a treatment solution and then drying at 37° C. for 1.5 hours (see par.94).
Han teaches a 3D printed open-source antibody dispenser that can be easily built and used for the development of lateral flow assay (LFA) strips (see Abstract). Han demonstrates a uniform dispensing of capture antibody and control antibody to draw a test line and a control line on nitrocellulose membrane for lateral flow test strip (see Abstract, page 2 par.4).
Zhong teaches that the carboxylated RENPs are dispersed in 1xPBS solution at 4 °C for long-term storage to obtain an activated RENPs solution (page 1332 col.2 par.1). This teaching discloses the application of a PBS solution in the activating treatment of RENPs. Zhong also teaches the process of conjugating antibody onto the surface of activated RENPs (see page 1332 col.2 par.2-3), wherein all the steps from dispersing, mixing, to washing are done with PBS solution (see page 1332 col.2 par.3). This teaching discloses the application of a PBS solution to RENPs dispersion during the conjugation of antibodies on the surface of RENPs. Zhong teaches that the hydrophilic functionalized RENPs showed remarkable stability in aqueous buffers (1xPBS) and serum without aggregation and exhibited zero photo-bleaching (see page 1325 col.1).
Zhong teaches a method for activating nanoparticle before the protein conjugation step, e.g., mixing a 0.25mg nanoparticle in 1xPBS with EDC 0.75mg and 500ul MES (10mM, pH=6.5). The solution was stirred at room temperature for 3 h. Zhong teaches the solution of nanoparticles is washed by a centrifugal filter (100 kDa) (see page 1332 col.2 par.1-2). Zhong teaches incubating the activated RENPs solution with 150ug antibody in 1xPBS solution. (See page 1332 col.2 par.3)
Life Science network teaches that 1xPBS is 10mM PBS buffer (i.e., 1x buffer will contain 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM K2HPO4).
Delport teaches techniques for the immobilization of different bio-molecules on nanoparticle NP (see Introduction). Delport teaches that “to maximize the efficiency of the NP's, the conjugation chemistry and coating have to be optimized for every material, shape or size of the NP and every type of biomolecule” (see Introduction). All immobilization reactions were performed in a 100 mM MES (2-(N-morpholino)ethane sulfonic acid) buffer with pH=5 (see page 1 par.3). Delport teaches a method for activating the carboxyl groups on nanoparticles before the protein conjugation step, e.g., mixing a quantity of nanoparticle with 0.15 mg/ml EDC, then adding 100pM to 1nM proteins (see page 1 last paragraph). Delport also teaches the amounts of EDC and NHS are optimized to improve the efficiency of protein conjugation (see page 2 par.2).
Hasanzadeh teaches that carcinoembryonic antigen (CEA) is an important tumor biomarker for colorectal. CEA detecting assay is widely accepted in oncology practice as a useful and cost-effective tool for monitoring colon cancer after surgery. See page 185 column 1 paragraph 1. The applications of anti-CEA antibody conjugated nanoparticles to provide sensitive detection tools are well known in the art (see page 189 col.1 par.2, and page 192 col.1 par.3). Hasanzadeh states that nano material based immunosensors can be used as quantitative analytical tools for detection of CEA in different cancers (see page 203 col.2 par.2).
Yun, Merck, and Hao do not teach the same method of treating the sample pad with the treatment solution as claimed, which is soaking and shaking the sample pad in the treatment buffer for 2-3 hrs at RT. However, Yun, Merck and Hao suggest a finite number of ways to prepare the sample pad for the test strip: Merck teaches that the sample pad is impregnated with the treatment solution; Yun teaches that the treatment solution is spraying to the sample pad; Hao teaches that the sample pad is submerged in the treatment solution. At the time of invention, it would have been obvious to one of ordinary skill in the art to try one or another way to apply the treatment solution on the sample pad and arrive the claimed method. A person of ordinary skill has good reason to pursue the known options within his or her technical grasp. The results of the combination are predictable (i.e., the sample pad is treated with the treatment buffer and is dried and stored at a certain condition) with a reasonable expectation of success.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to use the automatic cutting machine to cut the test paper board into test strips taught by Merck in the preparation method of the NIR-II nanoprobe test strip taught by Yun. It is because Yun is generic about the cutting method and Merck provides one specific method to cut the board into strips. The method of Merck makes the cutting process quicker and more convenient.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to use 3D printed open-source antibody dispenser to draw a test line and a control line on the test strip of Yun, because the dispenser can dispense capture antibodies and control antibodies uniformly as taught by Han.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the activating treatment step of Yun, by using PBS solution in dispersion step, e.g., by ultrasonicating, to obtain an activated RENPs solution or to obtain an antibody conjugated RENPs as taught by Zhong because the hydrophilic functionalized RENPs showed remarkable stability in aqueous buffers (1xPBS) without aggregation and exhibited zero photo-bleaching (see Zhong page 1325 col.1 and page 1332 col.1-3). Life Science network states that the 1xPBS comprises 10mM PBS.
Yun, Zhong and Delport do not teach the same amounts of reagents in the preparation steps, or the same mixing and centrifuging time as claimed. However, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to define the optimal concentrations of each chemical used in the activating treatment step to maximize the efficiency of the NPs as taught by Delport. Moreover, mixing time and centrifuging time are parameters that those skilled in the art can optimize from the known techniques according to practical needs. For example, Yun teaches that washing and collecting RENPs is done by centrifuging the solution at 12000~14000rpm for 5~15min (see page 6 lines 22), or Zhong teaches the solution of nanoparticles is washed by centrifugal filter (100 kDa) (see page 1332 col.2 par.1-2). It is obvious that the varying amounts of reagents used for activating the carboxyl groups on the surface of NP or for conjugating protein on the NP taught by Yun, Zhong and Delport is the consequence of the routine optimization process based on the material, shape or size of the NP and every type of biomolecule (see Delport Introduction). Absent unexpected results, it would have been obvious to one of ordinary skill to have arrived at the claimed concentration, mixing time and centrifuging by routine optimization in order to uncover the optimum workable ranges of the method of preparing conjugated RENPs. One having an ordinary skill in the art would have had a reasonable expectation of success in combining Yun, Zhong and Delport because they are directed to prepare nanoparticle probes for biosensing assay, where the nanoparticle probes need to be activated to conjugate with the biomolecule on their surface and the probes are used in immunoassay.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to replace the antibody in the test strip preparation method of Yun with the anti-CEA antibody to provide a useful and cost-effective test strip for monitoring colon cancer as taught by Hasanzadeh (see page 185 col.1 par.1, see page 189 col.1 par.2, and page 192 col.1 par.3). One having an ordinary skill in the art would have had a reasonable expectation of success in using anti-CEA antibody conjugated nanoparticles in CEA detection because Hasanzadeh states that nano-material based immunosensors can be used as quantitative analytical tools for detection of CEA in different cancers (see page 203 col.2 par.2).
Response to Arguments
Applicant's arguments filed 04/10/2026 with respect to claim(s) 1 have been fully considered but are moot because the new ground of rejection is made in view of the amendment of the claim. Yun and Zhang disclose the limitations of the claim.
Applicant’s arguments with respect to claim(s) 2-13 have been considered but they are not persuasive because Zhong teaches that the carboxylated RENPs are dispersed in 1xPBS solution at 4 °C for long-term storage to obtain an activated RENPs solution (page 1332 col.2 par.1). This teaching discloses the application of a PBS solution in the activating treatment of RENPs. Zhong also teaches the process of conjugating antibody onto the surface of activated RENPs (see page 1332 col.2 par.2-3), wherein all the steps from dispersing, mixing, to washing are done with PBS solution (see page 1332 col.2 par.3). This teaching discloses the application of a PBS solution to RENPs dispersion during the conjugation of antibodies on the surface of RENPs. Zhong teaches that the hydrophilic functionalized RENPs showed remarkable stability in aqueous buffers (1xPBS) and serum without aggregation and exhibited zero photo-bleaching (see page 1325 col.1).
Conclusion
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). 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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/CHAU N.B. TRAN/ Examiner, Art Unit 1677
/BAO-THUY L NGUYEN/ Supervisory Patent Examiner, Art Unit 1677 September 3, 2026
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