DETAILED ACTION
Notice of Pre-AIA or AIA Status
1. The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
2. Applicant’s election without traverse of Group II in the reply filed on September 29, 2025 is acknowledged.
Claims 1-35 are currently pending.
Claims 1-9 and 18-35 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on September 29, 2025.
Claim Objections
3. Claims 10-17 are objected to because of the following informalities: Claim 10 refers to a withdrawn claim. Appropriate correction by amending claim 10 to incorporate the limitations of claim 1 is required.
Claim Rejections - 35 USC § 103
4. 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.
5. Claims 10 and 14-15 are rejected under 35 U.S.C. 103 as being unpatentable over Shi (US 2019/0226024 Pub 7/25/2019) in view of Liu (ACS Nano 2022 16 pages 6916-6928), Portu (Reports of Practical Oncology and Radiotherapy 2016 Vol 21 pages 129-134), and Lubeck (Nature Methods Vol 9 No 7 July 2012).
Regarding Claim 10 Shi teaches a method comprising the steps of:
-providing the cells in a medium containing the probe;
-placing an array of functionalized nanoneedles on the surface of the medium;
- applying a centrifugal force to move the array of functionalized nanoneedles towards the cells for piercing the cells to allow the diffusion of the probe into the cells, and to form double-stranded sequences;
- allowing the functionalized nanoneedles (that are functionalized with a RNA binding protein such as P19) to bind with the double-stranded sequence;
-extracting the double stranded sequence out from the living cells via the temporarily formed holes.
Shi teaches that the bound double-stranded sequence remains on the functionalized nanoneedles after extraction. The application of the array of nanoneedles can extract the desired target substance from the cells without causing significant damages to the cells (paras 0062-0069, 0059). Shi further describes how the array of functionalization nanoneedles was prepared. Shi teaches that the nanoneedle patch was immersed in a NHS-Biotin solution for 1 h, a streptavidin solution containing streptavidin/alexa-488 modified streptavidin for 2 h, and a solution containing biotinylated p19 for 1 h (para 0101). Thus Shi teaches a method comprising: contacting an array of nanoprobes functionalized with a molecular target fishing molecule (p19) with cells at an interface where the one or more molecular target fishing molecules and one or more fluorescent dyes that are crosslinked with the nanoprobes (the alexa-488 is crosslinked with nanoneedles by biotin/streptavidin) will be in contact with a superficial layer of the cells; extracting the molecular targets by the one or more molecular target fishing molecules from the cells; removing the cells from the array and subjecting the array to fluorescent imaging.
Shi does not teach a method wherein the cells are part of an acute tissue slice (clm 10). Shi does not teach imprinting an outline of the acute tissue slice on the array (clm 10).
However Liu teaches a tissue imprinting technology. Liu teaches that this imprinting process can selectively transfer/adsorb analytes from a complex tissue surface onto the substrate, save pretreatment time, require lower laser energy, and improve anti-interference capability, compared with other LDI-MSI techniques (page 6917). Liu teaches that 50µm tissue section was transferred onto a precooled substrate, and then the underside of the substrate was carefully pressed on the back of a hand. After the warm substrate and tissue section were sufficiently warm, the imprinting process lasted for 1 min. Immediately after the tissue section was remove. The substrate was placed under room temperature and dried for mass spectrometry imaging (page 6925). Liu teaches that the tissue sections were from sliced mouse lung sections and the substrate was a hybrid NGQD@MoS2/SiNWs substrate. As shown in Fig 5D an outline of the tissue sample is clearly imprinted.
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In summary, Liu states that they have developed a TCSI-MSI platform based on 2D nanoflakes-capped SiNWs as a tissue imprinting substrate for lipidomic imaging and cancer diagnosis. Liu states that their method allows for authentical mapping of molecular spatial distribution in a sliced tissue sample (page 6924).
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Shi by imprinting an outline of the sample onto the array as suggested by Liu. One of skill in the art would have been motivated to performing the imprinting for the benefit of authentical mapping of molecular spatial distribution in a sliced tissue sample.
The combination of Shi and Liu do not teach a method wherein the irradiation is used to imprint the outline of the acute tissue slice onto the array (clm 10).
However Portu teaches a method to produce the imprint of cells cultivated on a polycarbonate detector by exposure of the detector to UV C radiation. Portu teaches that melanoma cells on Lexan films were exposed to a UV C lamp for 8 hours, stained with hematoxylin and explored with increasing magnification (1.25×, 10×, 20×, 40× and 100×) with a light microscope (Olympus DP70). Reference points were established on different regions in order to localize them after the chemical etching and correlate the cellular images with its corresponding autoradiographies (page 130). Figure 5 shows on the left, the stained culture images matched with the cell imprints of the same zones (obtained after the etching) At low magnification, the reference marks can be localized. At greater magnification, it can be observed how the imprints reproduce the cells morphology.
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Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Shi and Liu by imprinting an outline of the sample onto the array by using UV irradiation as suggested by Portu. One of skill in the art would have been motivated to perform imprint irradiation for the benefit of using a method known in the art that is capable of forming an imprint of biological material which reproduces cellular and nuclear contours of a sample (page 133).
The combined references do not teach a method further comprising a step of barcoding (clm 10).
However Lubeck discloses fluorophore based barcodes attached to molecular species. Lubeck teaches hybridization of the fluorophore based barcodes to a target nucleic acid. Lubeck teaches that the abundance of a target molecule in a cell can be quantified by counting the number of times that a corresponding barcode is observed in the super-resolution image of a cell (page 743).
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Shi, Liu, and Portu by subjecting the samples on the array to barcoding as suggested by Lubeck. One of skill in the art would have been motivated to perform barcoding for the benefit of being able to detect multiple distinct target nucleic acid.
Regarding Claim 14 Shi teaches using an array of functionalized nanoneedles to make holes on the cell membrane. Shi teaches applying a centrifugal force to move the array of functionalized nanoneedles towards the cells for piercing the cells (para 0062). The functionalized nanoneedles are capable of binding with the double-stranded sequence and extracting them out from the living cells via the temporarily formed holes (para 0063). Thus Shi teaches a method wherein after contacting the array of nanoprobes with the surface of the cell sample, a pressure (centrifugal force) is applied to the nanoprobes towards the cells in order to puncture the nanoprobes into the superficial layer of the cells for subsequent molecular target extraction.
Regarding Claim 15 the combined references do not teach a method wherein an image of the tissue and relative position of the nanoprobes are captured as a spatial registration of one or more cell types in the tissue associated with the nanoprobes prior to the removal of the tissue sample from the array. However the cited arts teaches methods of imaging the nanoprobes (Shi) and nanowires (Liu) after they were contacted with the tissue and it would have been obvious to also take an image of the tissue sample while it is still on the array of nanoprobes for the benefit of being able to use this image later to help with spatial orientation.
6. Claims 16-17 are rejected under 35 U.S.C. 103 as being unpatentable over Shi (US 2019/0226024 Pub 7/25/2019) in view of Liu (ACS Nano 2022 16 pages 6916-6928), Portu (Reports of Practical Oncology and Radiotherapy 2016 Vol 21 pages 129-134), and Lubeck (Nature Methods Vol 9 No 7 July 2012) as applied to claims 10 and 15 above and in further view of Carney (Annals of the American Thoracic Society 20156 Vol 12 Issue 3).
The teachings of Shi, Liu, Portu, and Lubeck are presented above.
The combined references do not teach a method wherein one or more cell types in the tissue associated with the nanoprobe are labelled by immunostaining with specific markers (clm 16).
However Carney teaches that there are many panels of immunostains currently available to help differentiate between common types of pulmonary malignancies. Carney teaches that there are four immunohistochemical stains routinely used for distinguishing primary lung ADC from primary lung SqCa and have become valuable ancillary tests for surgical pathologists. Thyroid transcription factor-1 (TTF-1), p63, cytokeratins (CK) 5/6, and CK 7 all aid in differentiating lung ADC and lung SqCa. More recently, p40 has become a valuable adjunct immunostain in the diagnosis of SqCa (page 430).
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Shi, Liu, Portu, and Lubeck by using immunostaining of specific cell markers to detect one or more cell types in a sample as suggested by Carney. The skilled artisan would have been motivated to identify specific cell types present by immunostaining in situations where it is desirable to distinguish between different types of lung cancer in a lung sample.
Regarding Claim 17 the combination of Shi and Lui do not teach a method wherein barcoding is performed by subjecting the array of nanoprobes to a plurality of reporter sequences complementary to the molecular targets extracted by the nanoprobes, wherein each of the reporter sequences is associated with a pre-determined mix ratio of multiple labelling agents for spectral analysis.
However this is taught Lubek. In particular Lubek teaches methods of spatial barcoding and spectral barcoding, both which uses multiple labeling agents for spectral analysis.
Accordingly, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Shi, Liu, and Portu by subjecting the samples on the array to the spatial or spectral barcoding methods of Lubek for the benefit of being able to detect multiple distinct target nucleic acid.
7. The closest prior art has been cited herein. The prior art does not teach or suggest a method wherein imprint irradiation results in the fluorescent dyes being cleaved from the nanoprobes (see claim 11). As such claims 11-13 are free from the prior art.
8. Any inquiry concerning this communication or earlier communications from the examiner should be directed to AMANDA HANEY whose telephone number is (571)272-8668. The examiner can normally be reached Monday-Friday, 8:15am-4:45pm EST.
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/AMANDA HANEY/Primary Examiner, Art Unit 1682