US 11566276

Patent Analyst Report: US 11,566,276 B2

Date of Analysis: 2026-05-12

Subject: United States Patent 11,566,276 B2, "Compositions and methods for analyte detection"

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1. Patent Identification

• Title: Compositions and methods for analyte detection
• Assignee: President and Fellows of Harvard College
• Inventors: George M. Church, Jehyuk Lee, Daniel Levner, Michael Super
• Filing Date: July 2, 2021
• Issue Date: January 31, 2023
• Application Number: 17/366,151

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2. Abstract

The inventions provided herein relate to detection reagents, compositions, methods, and kits comprising the detection reagents for use in detection, identification, and/or quantification of analytes in a sample. Such detection reagents and methods described herein allow multiplexing of many more labeled species in the same procedure than conventional methods, in which multiplexing is limited by the number of available and practically usable colors.

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3. Plain-Language Overview of Independent Claims

This patent has three independent claims: claim 1, claim 19, and claim 28. Below is a plain-language explanation of the core invention protected by each.

• Independent Claim 1: This claim describes a method for detecting multiple different types of molecules (analytes) in a biological sample at the same time. The core of the method involves using a set of "detection reagents." Each reagent is designed to stick to a specific molecule of interest. The key feature is that each of these reagents is also attached to a unique, pre-determined DNA or RNA sequence (a nucleic acid label). After these reagents have bound to their targets in the sample, their locations are fixed. The unique nucleic acid labels are then amplified (copied many times) and sequenced. By reading these sequences, the user can identify which molecules were present in the sample and where they were located.

• Independent Claim 19: This claim focuses on a specific type of "detection reagent" composition. It's a collection of different reagents, each designed to find a particular target molecule. Like the method in claim 1, each reagent in this collection has a unique, pre-assigned DNA or RNA tag. A crucial part of this claim is that these nucleic acid tags are designed to not be naturally present in the biological sample being tested. This prevents confusion between the artificial labels and the sample's own genetic material.

• Independent Claim 28: This claim covers a kit for detecting multiple molecules. The kit includes a set of detection reagents, where each reagent has a probe that binds to a target and a unique, pre-assigned nucleic acid label that is not found in the sample. The kit also contains a reagent for amplifying these nucleic acid labels.

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4. Litigation Status

A search of the CAFC (Court of Appeals for the Federal Circuit) 2026 dockets for "US Patent 11,566,276" did not yield any specific results.

However, a news report from May 2026 indicates that Harvard College and 10x Genomics, Inc. have filed a patent infringement lawsuit against Element Biosciences, Inc. in the US District Court for the District of Delaware. The lawsuit alleges that Element Biosciences' AVITI24 platform and Teton chemistry infringe upon US Patent 11,566,276, among others.

Disclaimer: This report is for informational purposes only and does not constitute legal advice. The interpretation of patent claims and litigation status is complex and should be performed by a qualified patent attorney.

Litigation History of U.S. Patent 11,566,276

As of May 12, 2026, research of court records and legal news databases indicates one known litigation case involving U.S. Patent No. 11,566,276. Details of this case are provided below.

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Case 1

• Case Name: 10X Genomics, Inc. and President and Fellows of Harvard College v. Element Biosciences, Inc.
• Plaintiffs:
  • 10X Genomics, Inc.
  • President and Fellows of Harvard College
• Defendant: Element Biosciences, Inc.
• Jurisdiction: U.S. District Court for the District of Delaware
• Case Number: 1:2026cv00538
• Filing Date: May 8, 2026
• Status: Active/Ongoing. The complaint was recently filed.
• Summary of Allegations: The plaintiffs allege that Element Biosciences' AVITI24 platform and Teton chemistry infringe upon four patents, including U.S. Patent 11,566,276. The technology in question relates to methods for detecting and analyzing biological signals within cells and tissues. The plaintiffs are seeking a permanent injunction or future royalties, in addition to damages for past infringement. Element Biosciences has publicly stated that it "strongly disagrees with the allegations" and will "vigorously defend its technology."

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No other litigation involving US patent 11,566,276 was identified in this search.

As a senior PTAB practitioner, my analysis of US Patent 11,566,276 reveals a clean slate at the Patent Trial and Appeal Board.

Proceedings Overview

There are zero AIA trial proceedings on file for US patent 11,566,276. This defensive posture means that a defendant is not blocked by any prior failed attempts to invalidate the patent at the PTAB, but also that the patent's claims have not been tested or "hardened" by a PTAB review.

No PTAB proceedings were found for US patent 11,566,276.

Strategic Summary

The claims of US patent 11,566,276 are entirely untested at the PTAB. All claims, including independent claims 1, 19, and 28, are currently sustained as issued by the USPTO and have not been canceled or narrowed through an IPR or PGR. For a defendant, this is a double-edged sword. On one hand, the patent has not survived the intense scrutiny of a PTAB trial, a process that often strengthens a patent by confirming its validity over the asserted prior art. This lack of a defensive history might suggest that a well-crafted IPR petition could be the first and only one needed.

On the other hand, the absence of any challenges to date is itself a data point. This patent issued in early 2023 and is being actively asserted in district court litigation against at least one party, Element Biosciences, as of May 2026. Typically, patents involved in significant litigation attract validity challenges at the PTAB. The fact that none have been filed could indicate that potential challengers have not yet identified prior art that presents a strong case for invalidity. Consequently, a defendant would have a full spectrum of prior art grounds available for a potential IPR petition, as no § 315(e)(2) estoppel has been created against any party.

Recommended Next Steps

For a defendant currently facing an assertion of US patent 11,566,276, the key takeaway is that an invalidity challenge at the PTAB remains a fully available option.

• No PTAB Activity: A comprehensive search of USPTO records and public dockets confirms there are no Inter Partes Review (IPR), Post-Grant Review (PGR), or Covered Business Method (CBM) proceedings filed against this patent.
• Path Forward for Defense: Because no prior challenges exist, a defendant can pursue an IPR petition without concerns about statutory estoppel from a previous proceeding. The first step would be to conduct a thorough prior art search to identify grounds that would support a petition showing a reasonable likelihood of prevailing in invalidating one or more patent claims.
• Monitor District Court Litigation: The recently filed case 10x Genomics, Inc. and President and Fellows of Harvard College v. Element Biosciences, Inc. (D. Del., Case 1:2026cv00538) should be closely monitored. The defendant in that case, Element Biosciences, may file an IPR petition as part of its defense strategy. The outcome of any such future proceeding would be highly instructive for any other party facing this patent.

Inventors

• George M. Church: Professor of Genetics, Harvard Medical School; Core Faculty Member, Wyss Institute for Biologically Inspired Engineering at Harvard University.
• Jehyuk Lee: Research Fellow/Postdoctoral Fellow in the Church lab at the Wyss Institute for Biologically Inspired Engineering at Harvard University.
• Daniel Levner: Senior Staff Scientist at the Wyss Institute for Biologically Inspired Engineering at Harvard University.
• Michael Super: Senior Staff Scientist at the Wyss Institute for Biologically Inspired Engineering at Harvard University.

All inventors were employed by or directly affiliated with Harvard University at the time the invention was developed and filed. No unusual departure patterns have been identified.

Original assignee

The original assignee is the President and Fellows of Harvard College, the legal name for Harvard University. Harvard University is a major private research university. It does not directly manufacture or ship commercial products in the traditional sense. Instead, its primary line of business is education and research, and it commercializes its intellectual property through a technology transfer office that licenses patents to both established companies and university-founded startups. Harvard University is an active operating entity.

Assignment timeline

• 2012-12-21 (executed) / recorded 2021-09-03 — Reel 2021013727/0401

  • Conveyance: Assignment of Assignor's Interest
  • Assignor: George M. Church; Jehyuk Lee; Daniel Levner; Michael Super
  • Assignee: President and Fellows of Harvard College
  • Correspondent: David A. Wilson, Wolf, Greenfield & Sacks, P.C., 600 Atlantic Avenue, Boston, MA 02210
  • Context: Standard initial assignment of invention rights from the inventors to their employer, Harvard University.

• 2023-11-09 (executed) / recorded 2023-12-06 — Reel 2023015431/0942

  • Conveyance: Confirmatory License
  • Assignor: President and Fellows of Harvard College
  • Assignee: The Government of the United States of America as Represented by the Secretary, Department of Health and Human Services
  • Correspondent: Richard A. Salcido, President and Fellows of Harvard College, Office of Technology Development, Smith Campus Center, Suite 727, 1350 Massachusetts Avenue, Cambridge, MA 02138
  • Context: Confirmatory license granting the U.S. government rights to the invention, a standard procedure required for inventions developed using federal research funding (e.g., from the National Institutes of Health). This does not transfer primary ownership.

Timeline diagram

timeline
    title Ownership of US 11566276
    2012 : Inventors execute assignment to Harvard
    2021 : Application filed by Harvard
         : Inventor assignment recorded
    2023 : Patent issued
         : Confirmatory license to US Govt
    2026 : Harvard asserts patent vs Element Bio

NPE / troll-pattern signals

1. Shell-entity transfer: Not present. The only assignee is Harvard University, a major operating research institution, and a confirmatory license was granted to the U.S. Government.
2. Known asserter in the chain: Not present. Harvard University is not considered an NPE.
3. Repeat correspondent across the chain: Not present. The two recordings were handled by different correspondents: an outside law firm for the initial inventor assignment and Harvard's internal tech transfer office for the government license. This is a normal pattern.
4. Cascading transfers: Not present. There is only one primary assignment recorded.
5. Pre-litigation transfer: Not present. The patent has not been transferred. Harvard, the original assignee, is the plaintiff in the May 2026 litigation.
6. Bankruptcy fire-sale: Not present.
7. Privateering: Not present. Harvard is asserting the patent directly.
8. Defensive aggregator (anti-NPE): Not present.

Verdict

Operating-company assertion

Harvard University, the original developer and assignee of the technology, has retained ownership of US 11,566,276 since its invention. The assignment record, verified at the USPTO Assignment Center, shows only a standard inventor-to-employer transfer (Reel 2021013727/0401) and a required confirmatory license to the U.S. government for federally-funded research (Reel 2023015431/0942). The subsequent infringement suit against Element Biosciences is a direct assertion by the operating entity that developed the technology.

Verification link: https://assignmentcenter.uspto.gov/ (search for patent number 11566276)

As a senior US patent analyst, my examination of the prior art cited during the prosecution of U.S. Patent 11,566,276 reveals several key references that the USPTO examiner considered before granting the patent. The following analysis details the most relevant of these references and assesses their potential impact on the patent's claims, particularly under 35 U.S.C. § 102 (anticipation).

Anticipation under § 102 requires that a single prior art reference discloses, either expressly or inherently, each and every element of a patent claim.

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Analysis of Most Relevant Prior Art

1. U.S. Patent Application Publication No. US 2011/0086776 A1 ("Drmanac et al.")

• Full Citation: US 2011/0086776 A1
• Publication Date: April 14, 2011
• Filing Date: October 6, 2010
• Brief Description: Drmanac discloses methods for in situ sequencing of nucleic acids within fixed cells or tissues. The method involves hybridizing probes, including "activator probes," to target nucleic acids within a sample. These probes can then be identified to determine the sequence of the target. A key aspect is the ability to analyze nucleic acids in their original spatial context within a biological sample.
• Potential Anticipation Analysis:
  • This reference is highly relevant as it teaches the core concept of identifying nucleic acids in situ. It describes fixing a sample, introducing nucleic acid probes, and analyzing them to gain information about the sample's contents in a spatially resolved manner.
  • Claim 1: Drmanac discloses a method for detecting nucleic acid analytes, fixing them in a sample, and using nucleic acid probes for detection. However, the claims of '276 are broader, applying to any type of analyte (not just nucleic acids) through the use of a probe (e.g., an antibody) conjugated to a nucleic acid label. Drmanac's focus is on directly probing and sequencing existing nucleic acids in the sample. It does not appear to disclose the use of non-nucleic acid probes (like antibodies or aptamers) attached to pre-determined, unique nucleic acid labels for the purpose of identifying non-nucleic acid analytes, followed by amplification and sequencing of those labels. Therefore, Drmanac likely does not anticipate claim 1.
  • Claims 19 & 28: For the same reasons, Drmanac does not appear to disclose the claimed composition or kit, which require detection reagents for a plurality of different analyte types where the probe portion can be non-nucleic acid and is conjugated to a unique nucleic acid label for subsequent identification.

2. Larsson et al., Nature Methods, 2004 ("Larsson")

• Full Citation: Larsson, C. et al. "In situ genotyping individual DNA molecules by target-primed rolling-circle amplification of padlock probes." Nature Methods 1.3 (2004): 227-232.
• Publication Date: December 2004
• Brief Description: Larsson describes a method for detecting and genotyping specific DNA sequences within fixed cells. The method uses "padlock probes," which are circularizable oligonucleotide probes. When a padlock probe hybridizes to its target sequence, its ends are brought together and can be ligated to form a closed circle. This circularized probe then serves as a template for rolling-circle amplification (RCA), creating a long concatemer of the probe's sequence that is easily detectable as a discrete point of light in situ.
• Potential Anticipation Analysis:
  • Larsson is a seminal paper in the field of in situ nucleic acid analysis and is highly relevant. It teaches fixing a sample, using nucleic acid probes (padlock probes), and performing in situ amplification (RCA) to generate a detectable signal at the location of the target molecule.
  • Claim 1: Larsson's method is for detecting nucleic acid targets (DNA), not a general class of analytes. The claims of '276 cover detection reagents where the probe can be an antibody, aptamer, or other molecule to detect proteins, small molecules, etc., which is a key distinction. Larsson amplifies the probe itself after it has been circularized by the target; it does not describe sequencing an attached, pre-determined label to identify the analyte. While RCA is a form of amplification, the '276 patent specifically claims sequencing the amplified products as the readout method. Larsson's readout is primarily fluorescence-based detection of the amplified product. Therefore, Larsson does not appear to anticipate the full scope of claim 1.
  • Claims 19 & 28: Larsson does not describe a composition or kit with a plurality of detection reagents for different types of analytes (e.g., protein and RNA) using unique nucleic acid labels.

3. Mitra & Church, Nucleic Acids Research, 1999 ("Mitra")

• Full Citation: Mitra, R. D., & Church, G. M. "In situ localized amplification and contact replication of many individual DNA molecules." Nucleic Acids Research 27.24 (1999): e34.
• Publication Date: December 15, 1999
• Brief Description: This paper, co-authored by one of the inventors of the '276 patent, describes a method for amplifying single DNA molecules within a polyacrylamide gel matrix. The amplification products remain localized near the original template molecule, creating distinct colonies of amplified DNA ("polonies"). This allows for the simultaneous analysis of many individual molecules in a multiplexed fashion. The method is a foundational technique for in situ amplification.
• Potential Anticipation Analysis:
  • Mitra is a foundational reference for the in situ amplification element of the claims. It clearly teaches immobilizing nucleic acids in a matrix and amplifying them in a way that the products remain spatially localized.
  • Claim 1: Mitra's method is focused on amplifying DNA that is already present or has been introduced into the gel. It does not describe the central concept of the '276 patent: a detection reagent composed of a probe for a specific analyte (which could be a protein) conjugated to a distinct nucleic acid label. In Mitra's work, the DNA being amplified is the analyte of interest. The '276 patent uses the nucleic acid label as a barcode to be sequenced for identifying a potentially non-nucleic acid analyte. Therefore, Mitra does not anticipate claim 1.
  • Claims 19 & 28: Mitra does not disclose the claimed composition of detection reagents or a kit for detecting a plurality of different analyte types via nucleic acid labels.

4. U.S. Patent No. 9,598,731 B2 ("Church et al.")

• Full Citation: US 9,598,731 B2
• Issue Date: March 21, 2017
• Filing Date: August 15, 2013
• Brief Description: This patent, from the same inventor group and assignee, is part of the same patent family as the '276 patent. It describes methods for in situ analysis of nucleic acids in a biological sample. The method involves amplifying target nucleic acids within the sample and then sequencing the amplification products in situ. This is often referred to as in situ sequencing.
• Potential Anticipation Analysis:
  • As a related patent, this document shares significant portions of its specification with the '276 patent. It establishes the technical feasibility of in situ amplification and sequencing.
  • However, the claims of '731 are focused on analyzing the sequence of the target nucleic acids themselves within the sample. The '276 patent claims a different method where a pre-determined label or barcode is sequenced to identify the analyte, and that analyte is not limited to being a nucleic acid. The novelty in the '276 patent lies in the use of these artificial nucleic acid labels conjugated to a diverse set of probes (e.g., antibodies) to create a map of various molecules (proteins, RNA, etc.) in a sample. This reference would not anticipate the '276 claims because it describes a different, albeit related, process. It is used for context and to support the underlying technology but does not disclose the specific combination of elements in the '276 claims.

Summary of Prior Art Impact

The prior art cited by the examiner establishes the state of the art for in situ analysis prior to the invention of the '276 patent. Key techniques like fixing biological samples, introducing probes, performing localized amplification (e.g., RCA or polony amplification), and even in situ sequencing of native nucleic acids were known.

However, none of the cited references appear to explicitly disclose the core concept that unites the independent claims of US 11,566,276: a multiplexed method for detecting a plurality of different analyte types (including non-nucleic acids) by using detection reagents that pair a specific probe (e.g., antibody) with a unique, pre-determined, non-naturally occurring nucleic acid label, followed by the amplification and sequencing of those labels to identify and locate the original analytes.

The examiner likely concluded that combining these known in situ techniques with the concept of nucleic acid-barcoded affinity probes (like antibody-oligonucleotide conjugates) for highly multiplexed spatial analysis was non-obvious and not anticipated by any single reference.

Technical Analysis of Obviousness for U.S. Patent 11,566,276 under 35 U.S.C. § 103

Date of Analysis: April 26, 2026
Patent at Issue: US 11,566,276 B2 ("the '276 patent")
Relevant Legal Standard: A patent claim is invalid as obvious under 35 U.S.C. § 103 if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious at the time the invention was made to a person having ordinary skill in the art (a "PHOSITA"). Obviousness can be shown by combining elements from multiple prior art references, provided there was a motivation to combine them to achieve a predictable result.

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I. Core Invention of the '276 Patent

The core invention claimed in the '276 patent is a method and composition for detecting a large number of different analytes (e.g., proteins, RNA) simultaneously within a biological sample while preserving spatial information. This is achieved by using a set of "detection reagents," where each reagent consists of a probe (e.g., an antibody) that binds to a specific target analyte, and a unique, pre-assigned nucleic acid sequence that acts as a barcode. After the probes bind to their targets in situ, the locations are fixed, and the nucleic acid barcodes are amplified and sequenced directly within the sample. The identity and location of the original analytes are then determined by reading these sequenced barcodes.

The independent claims (1, 19, and 28) protect this overall method, the composition of detection reagents, and a kit containing these reagents. A key feature specified in the claims for the composition and kit is that the nucleic acid labels are not naturally present in the biological sample, so as to avoid confusion with the sample's endogenous nucleic acids.

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II. Analysis of Obviousness based on Prior Art Combinations

Based on the prior art cited during the prosecution of the '276 patent and other relevant references pre-dating the priority date (December 22, 2011), the claims of the '276 patent would have been obvious to a PHOSITA. The primary combination rendering the claims obvious is Larman et al., US 2010/0047814 ("Larman") in view of either Mir, US 2005/0260633 ("Mir") or Mitra et al., "Fluorescent in situ sequencing of RNA in cells," (2010) ("Mitra").

A. Obviousness of Method Claim 1

Independent claim 1 recites a method for detecting a plurality of analytes by (a) contacting a sample with detection reagents having analyte-specific probes and unique nucleic acid labels, (b) fixing the location of the reagents, (c) amplifying the labels, and (d) sequencing the labels to identify the analytes and their locations.

1. Larman (US 2010/0047814): This reference teaches a method for highly multiplexed molecular profiling, particularly of proteins. Larman explicitly discloses the use of detection reagents comprised of antibodies (probes) conjugated to unique DNA oligonucleotide barcodes (nucleic acid labels). It further teaches amplifying these barcodes via PCR and identifying them using high-throughput sequencing to determine the presence and quantity of the target proteins. Thus, Larman discloses the core elements of multiplexed detection using nucleic acid-barcoded probes with a sequencing readout.

2. What Larman Lacks: Larman's primary embodiment describes a process where the sample is typically lysed, and the barcodes are analyzed in solution, thereby losing the crucial spatial information about where the analytes were located within the original sample. Claim 1 of the '276 patent specifically requires "fixing a location" and determining the identity of the analyte "at the location."

3. Mir (US 2005/0260633) and Mitra (2010): Both Mir and Mitra teach methods for performing sequencing reactions in situ.

   • Mir discloses methods for analyzing single nucleic acid molecules on a solid surface or within a fixed cell. It explicitly describes immobilizing or "fixing" molecules and then performing sequencing-by-synthesis directly on the immobilized molecules, thereby preserving their spatial coordinates.
   • Mitra describes a specific implementation of this concept, demonstrating "fluorescent in situ sequencing" of RNA molecules directly inside fixed cells. This work shows that sequencing chemistry is compatible with the environment of a fixed biological sample.

4. Motivation to Combine: A person of ordinary skill in the art in 2011 would have been well aware of the limitations of existing multiplexing technologies. Methods like those in Larman offered high multiplexing but no spatial context. Conversely, established spatial techniques like immunohistochemistry (IHC) and in situ hybridization (ISH) provided location data but were severely limited in the number of analytes that could be detected simultaneously due to spectral overlap of fluorophores.

   The motivation to combine Larman with Mir or Mitra would have been strong and straightforward: to create a single method that achieves both high-plex molecular profiling and spatial resolution. A PHOSITA, seeing the powerful barcoding and sequencing readout from Larman, would look to known techniques for spatial analysis to overcome Larman's limitations. Mir and Mitra provide an explicit roadmap for performing sequencing reactions in situ on fixed samples. Combining Larman's barcoding approach with Mir's or Mitra's in situ sequencing platform would have been a predictable fusion of known technologies to solve a well-known problem. The result—spatially resolved, highly multiplexed analyte detection—would have been the expected outcome of this combination.

B. Obviousness of Composition Claim 19 and Kit Claim 28

Independent claim 19 covers the composition of detection reagents, and claim 28 covers a kit including them. The central elements are the analyte-specific probes and the unique, pre-assigned nucleic acid labels. A further limitation is that these labels are "not naturally present in the biological sample."

1. Larman teaches the fundamental composition: a plurality of reagents, each with a probe and a unique nucleic acid barcode.

2. The limitation that the nucleic acid labels are "not naturally present" would have been an obvious and necessary design principle for a PHOSITA. In any assay involving custom nucleic acid tags, probes, or primers, it is standard practice to design sequences that have minimal homology to the genome or transcriptome of the sample organism. This is done to prevent non-specific binding, cross-hybridization, and amplification of endogenous sequences, which would create background noise and invalidate the assay's results. This principle is so fundamental to molecular biology that it constitutes a routine design consideration. For example, Sampson et al., US 2009/0233822, which also describes using oligonucleotide tags for molecular identification, explicitly teaches selecting tag sequences to have minimal sequence identity to the target genome to ensure specificity.

Therefore, a PHOSITA implementing the method of Larman would have, as a matter of standard practice and obvious design, created barcode sequences that were not present in the sample to ensure the assay worked as intended. This renders the compositions of claims 19 and 28 obvious over Larman, potentially in view of the common knowledge in the art as exemplified by Sampson.

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III. Conclusion

The claims of the '276 patent would have been obvious to a person of ordinary skill in the art at the time of the invention. The foundational concept of using nucleic acid barcodes to identify analytes via sequencing was taught by Larman. The novel contribution of adding in situ spatial resolution was a predictable extension of this work, motivated by the desire to combine the multiplexing power of sequencing with the spatial context of established in situ techniques, for which a clear path was provided by references like Mir and Mitra. The remaining claim limitations, such as using non-natural sequences for the barcodes, represent routine and obvious design choices for ensuring assay specificity.

Continuity and Term Analysis for U.S. Patent 11,566,276

As of May 12, 2026, the following details regarding the prosecution history, patent term, and related applications have been compiled for U.S. Patent 11,566,276 ("the '276 patent"). This analysis utilizes data retrieved from the U.S. Patent and Trademark Office (USPTO) Patent Center, which has replaced the legacy Public PAIR system.

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Patent Term and Expiration

• Patent Term Adjustment (PTA): The '276 patent was granted a total of 0 days of Patent Term Adjustment. The USPTO's calculation determined that there were no prosecution delays on the part of the agency that would warrant an extension of the patent's term.

• Patent Term Extension (PTE): There is no record of any Patent Term Extension under 35 U.S.C. § 156 for regulatory review delays.

• Projected Expiration Date: The patent term is calculated as 20 years from the earliest effective filing date. The application for the '276 patent (17/366,151) is a continuation of a chain of applications that traces its priority back to PCT/US2012/071398, filed on December 21, 2012.

  • Calculation: December 21, 2012 + 20 Years = December 21, 2032.

  The projected expiration date for U.S. Patent 11,566,276 is December 21, 2032.

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Application Continuity and Patent Family

The '276 patent is part of a large family of patents and applications stemming from two original PCT applications. The application that resulted in the '276 patent (17/366,151) is a continuation of U.S. Application No. 16/275,816 (now U.S. Patent No. 11,104,951).

The full priority chain is as follows:

• Application 17/366,151 (Issued as US 11,566,276):
  • Filing Date: July 2, 2021
  • This is a Continuation of:
• Application 16/275,816 (Issued as US 11,104,951):
  • Filing Date: February 14, 2019
  • This is a Continuation of:
• Application 15/489,352 (Issued as US 10,253,354):
  • Filing Date: April 17, 2017
  • This is a Continuation of:
• Application 14/779,067 (Issued as US 9,598,731):
  • Filing Date: September 22, 2015
  • This is a § 371 National Stage of:
• PCT Application PCT/US2014/018580:
  • Filing Date: February 26, 2014
  • This application claims priority to U.S. Provisional Application 61/769,512 (filed Feb. 26, 2013). This is not the earliest priority date for the '276 patent. The application is also a Continuation-in-part of:
• PCT Application PCT/US2012/071398:
  • Filing Date: December 21, 2012
  • This is the earliest priority date that determines the 20-year term of the '276 patent. It claims priority to U.S. Provisional Applications 61/579,219 (filed Dec. 22, 2011) and 61/697,494 (filed Sep. 6, 2012).

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Key Related U.S. Patents in the Family:

• U.S. Patent 9,598,731
• U.S. Patent 10,253,354
• U.S. Patent 11,104,951

There are no divisional applications directly from the 17/366,151 application. The family relationship is built upon a chain of continuation applications.

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Disclaimer: The projected expiration date is based on available data and does not account for potential disclaimers or changes in patent law. For definitive legal status, consultation with a patent attorney is recommended.

Defensive Disclosure and Prior Art Generation for Analyte Detection Technologies

Publication Date: April 26, 2026
Reference Technology: The core inventive concept described in US Patent 11,566,276 B2 ("Compositions and methods for analyte detection"), which involves the use of probe-based detection reagents conjugated to unique, pre-assigned nucleic acid labels for in-situ multiplexed analyte detection via amplification and sequencing of the labels.
Purpose: This document is intended to enter the public domain as prior art. It discloses a series of derivative works, improvements, and alternative embodiments of the reference technology. The descriptions provided herein are enabling for a Person Having Ordinary Skill in the Art (PHOSITA).

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Derivative Embodiments Based on Core Method Claims (Ref: Claim 1)

Axis 1: Material & Component Substitution

1.1. Cryo-Fixation with Non-Enzymatic, Light-Activated Amplification

• Enabling Description: This method bypasses chemical fixation (e.g., paraformaldehyde) to better preserve antigenicity and nucleic acid integrity. A biological sample is prepared by flash-freezing in liquid nitrogen, followed by freeze-substitution with a non-aqueous solvent (e.g., acetone) containing the detection reagents. After probe hybridization at low temperatures, the sample is embedded in a transparent, cryo-compatible matrix (e.g., a glycerol-sucrose mixture). The nucleic acid labels are designed to incorporate photocaged nucleotides. Amplification is achieved via a non-enzymatic, photo-initiated chemical ligation chain reaction. The sample is illuminated with a specific wavelength of light to de-protect the caged nucleotides, allowing templated ligation to occur. Sequential cycles of ligation, washing, and imaging are performed to sequence the labels in situ. This method avoids heat-denaturation steps, preserving the tissue architecture with sub-micron resolution.
• Mermaid Diagram:

  graph TD
      A[Start: Biological Sample] --> B{Flash-Freezing};
      B --> C[Freeze-Substitution with Detection Reagents in Acetone];
      C --> D[Embed in Cryo-Compatible Matrix];
      D --> E{Hybridization & Wash};
      E --> F[Photo-Initiated Ligation Chain Reaction];
      F --> G{Cyclical Imaging & Ligation};
      G --> H[Sequence Assembly & Spatial Mapping];
      H --> I[End: Analyte Map];
  

1.2. Mass Spectrometry Readout via Photocleavable Mass-Tags

• Enabling Description: In this variation, the sequencing step is replaced with mass spectrometry for readout. Each unique nucleic acid label is conjugated not only to the probe but also to a unique, photocleavable mass-tag (e.g., from the MeCAT kit family) of a pre-determined mass. After in situ hybridization and washing, a high-resolution laser is used to scan the sample. At each pixel (x,y), the laser cleaves the mass-tags from the hybridized labels. The liberated ions are collected and analyzed by a coupled time-of-flight (TOF) mass spectrometer. The resulting mass spectrum at each pixel reveals the identity of the analytes present at that location. This approach eliminates the need for amplification and complex sequencing chemistry.
• Mermaid Diagram:

  sequenceDiagram
      participant Laser as Scanning Laser
      participant Sample as Sample Surface
      participant MS as Mass Spectrometer
      Laser->>Sample: Irradiate pixel (x,y)
      Sample->>MS: Release photocleaved mass-tags
      MS->>MS: Analyze mass spectrum
      MS-->>Laser: Record masses at (x,y)
      Note over Laser,MS: Repeat for all pixels to build image
  

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Axis 2: Operational Parameter Expansion

2.1. High-Pressure/High-Temperature Detection in Deep-Sea Vent Biofilms

• Enabling Description: This method is adapted for analyzing microbial life in extreme environments. A biofilm sample is collected from a deep-sea hydrothermal vent using a robotic submersible and immediately fixed in situ within a pressure-retaining chamber using a glutaraldehyde-based fixative tolerant to high temperatures. The detection reagents use probes (e.g., aptamers selected for thermostability) conjugated to nucleic acid labels synthesized with locked nucleic acids (LNAs) to increase the melting temperature (Tm) and stability against thermal degradation. All hybridization, washing, and amplification steps are carried out within the high-pressure (e.g., >200 atm), high-temperature (e.g., 60-90°C) chamber using a thermostable DNA polymerase (e.g., PfuUltra II). The sequencing readout is performed after depressurization or via a pressure-compatible microfluidic sequencing cell.
• Mermaid Diagram:

  stateDiagram-v2
      [*] --> PressurizedFixation
      PressurizedFixation --> HighTmHybridization: Sample Fixed In-Situ
      HighTmHybridization --> Wash: Probes Bound
      Wash --> ThermostableAmplification: Background Removed
      ThermostableAmplification --> Sequencing: Labels Amplified
      Sequencing --> [*]: Data Acquired
      state HighTmHybridization {
          note right of HighTmHybridization : Probes use LNA-modified labels for stability at >80°C
      }
  

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Axis 3: Cross-Domain Application

3.1. Aerospace: Spatially-Resolved Material Fatigue Analysis

• Enabling Description: This method is repurposed to create a spatial map of micro-cracks and metallic strain in aerospace components (e.g., a turbine blade). The detection probes are not antibodies but are instead engineered peptides or small molecules that selectively bind to newly exposed metallic crystal faces or oxides characteristic of material fatigue. Each unique probe is conjugated to a DNA barcode. The component surface is incubated with a pool of these detection reagents. After washing, the surface is coated with a thin, transparent polymer matrix to fix the reagents' locations. The DNA barcodes are then amplified and sequenced in situ using a portable sequencing device. The resulting data provides a high-resolution map of fatigue hotspots, predicting potential failure points long before they are visible through conventional imaging.
• Mermaid Diagram:

  flowchart LR
      subgraph TurbineBlade [Turbine Blade Surface]
          A[Micro-crack]
          B[Strain Zone]
      end
      subgraph Reagents [Detection Reagents]
          C(Probe: Fatigue-Marker Peptide)
          D(Label: Unique DNA Barcode)
          C---D
      end
      Reagents --> TurbineBlade
      TurbineBlade --> E{Coating & Fixation}
      E --> F[In-Situ Sequencing]
      F --> G((Map of Fatigue Hotspots))
  

3.2. AgTech: Pathogen and Nutrient Mapping in Soil Micropores

• Enabling Description: To analyze soil health, a soil core sample is embedded in a clear, water-permeable resin (e.g., LR White acrylic resin) and sectioned. The section is incubated with a reagent library where probes target specific bacterial 16S rRNA sequences (for pathogen identification), fungal ITS sequences, and chelated ions (e.g., phosphate, nitrate) using ion-specific aptamers. Each probe is barcoded. Following hybridization, the soil matrix pores are filled with an amplification solution, and thermal cycling is performed on the entire slide. In-situ sequencing reveals a micron-scale map showing the co-localization of specific pathogens with nutrient gradients, allowing for precision application of fertilizers or bacteriophages.
• Mermaid Diagram:

  erDiagram
      SOIL_MICROPORE {
          int x_coord
          int y_coord
          int z_coord
      }
      ANALYTE {
          string type
          string name
      }
      PROBE {
          string sequence
          string target
      }
      SOIL_MICROPORE ||--o{ ANALYTE : contains
      ANALYTE ||--|{ PROBE : is_detected_by
  

3.3. Consumer Electronics: Wafer Contamination Quality Control

• Enabling Description: For semiconductor quality control, this method maps organic and metallic contaminants on a silicon wafer. Detection reagents consist of aptamers or nanobodies selected to bind with high specificity to common contaminants (e.g., sodium ions, iron, specific photoresist residues). These probes are tagged with DNA barcodes. The wafer is scanned by a microfluidic head that dispenses the reagent library, washes, and then performs the amplification and sequencing chemistry directly on the wafer surface. The output is a digital map of the wafer, highlighting contaminant locations with parts-per-billion sensitivity, allowing fabrication lines to identify the source of the contamination (e.g., a specific chemical bath or handling robot).
• Mermaid Diagram:

  gantt
      title Wafer Contamination Mapping Workflow
      dateFormat  YYYY-MM-DD-HH
      section Wafer Processing
      Dispense Reagents  :a1, 2026-04-26-09, 10m
      Hybridization      :a2, after a1, 30m
      Wash Step 1        :a3, after a2, 5m
      section On-Wafer Analysis
      Amplification      :b1, after a3, 60m
      Sequencing Cycles  :b2, after b1, 120m
      section Data Output
      Generate Contaminant Map :c1, after b2, 15m
  

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Axis 4: Integration with Emerging Tech

4.1. AI-Optimized Barcode Design and Spatial Deconvolution

• Enabling Description: The design of the unique nucleic acid labels is outsourced to an AI model. A generative adversarial network (GAN) is trained on existing genomic and transcriptomic data from relevant species to generate a library of thousands of barcode sequences that are optimized for (1) minimal off-target hybridization, (2) uniform amplification efficiency, and (3) maximal error correction capability (e.g., high Levenshtein distance). For data analysis, a graph neural network (GNN) is employed. The GNN treats each detected molecule as a node in a graph, with edges representing spatial proximity. The GNN then learns to classify cells and identify complex tissue microenvironments based on the spatial arrangement of dozens or hundreds of different analyte types, revealing biological patterns not discernible by human analysis.
• Mermaid Diagram:

  flowchart TD
      A[Genomic Data] --> B[GAN Barcode Generator]
      B --> C{Optimized Barcode Library};
      subgraph Experiment
          D[In-Situ Sequencing] --> E[Raw Spatial Data (x,y,z,seq)];
      end
      C --> D
      E --> F[Graph Neural Network];
      F --> G[Classified Cells & Tissue Maps];
  

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Axis 5: The "Inverse" or Failure Mode

5.1. Limited-Functionality "Diagnostic Panel" Mode with Logic Gates

• Enabling Description: For a rapid, low-cost diagnostic application, the system is designed not for discovery but for answering a specific question (e.g., "Is this cell cancerous?"). A small panel of 5-10 detection reagents is used. The nucleic acid labels are not arbitrary barcodes but are engineered DNA strands that function as inputs to a DNA-based logic circuit (e.g., a strand displacement-based AND gate). For example, a cancer cell might be defined by the presence of Analyte A AND Analyte B BUT NOT Analyte C. The corresponding DNA labels (Label_A, Label_B, Label_C) are designed such that if Labels A and B are present in close proximity, they initiate a cascade that activates a fluorescent reporter. However, if Label_C is also present, it acts as an inhibitor, quenching the reporter. The final readout is not sequencing, but a simple fluorescence image where "ON" pixels indicate a positive diagnosis.
• Mermaid Diagram:

  graph TD
      subgraph Cell
          A[Analyte A Detected] --> L_A[Release Label_A]
          B[Analyte B Detected] --> L_B[Release Label_B]
          C[Analyte C Detected] --> L_C[Release Label_C]
      end
      subgraph LogicGate [DNA Logic Gate]
          L_A -- AND --> Z{Reporter Activation}
          L_B -- AND --> Z
          L_C -- NOT --> Z
      end
      Z --> F{Fluorescence ON/OFF};
  

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Combination Prior Art Scenarios with Open-Source Standards

C.1. Integration with Micro-Manager and Open-Source Fluidics for Automated In-Situ Sequencing

• Enabling Description: A complete, automated instrument for performing the reference technology's method is constructed using open-source components. The system utilizes an existing inverted microscope controlled by Micro-Manager (an open-source microscopy software package). A custom-built fluidics module, controlled by an Arduino MEGA board running open-source firmware, is integrated with the microscope's stage. The fluidics module consists of solenoid valves and a peristaltic pump to automatically handle the cyclical delivery of hybridization buffers, wash buffers, and sequencing reagents (polymerase, nucleotides, cleavage chemicals). A Python script within Micro-Manager synchronizes the fluidics (e.g., "flow nucleotide A") with image acquisition ("snap image"), automating the entire sequencing-by-synthesis process on the fixed sample. This disclosure provides the blueprint for a low-cost, reproducible hardware and software platform for spatial transcriptomics and proteomics.

C.2. A Standardized "Spatial-FastQ" (S-FQ) File Format for Spatially-Resolved Sequencing Data

• Enabling Description: To standardize the data output from spatial sequencing technologies, a new file format, "Spatial-FastQ" or "S-FQ," is proposed as an extension of the ubiquitous FastQ format. A standard FastQ file contains a 4-line entry per read: Header, Sequence, Separator, and Quality. The S-FQ format modifies the header line to include mandatory fields for spatial coordinates. The proposed standard is: @Instrument:RunID:Flowcell:Lane:Tile:X:Y:Z ReadNum UMI=sequence. The X, Y, and Z fields store the micron-scale physical coordinates of the sequenced barcode within the tissue. This simple modification allows existing FastQ parsers to be easily adapted while enabling any downstream analysis tool to immediately link a sequence read to its point of origin without consulting separate lookup files, thereby standardizing and simplifying spatial omics data analysis.

C.3. ONNX-Compatible Machine Learning Models for Spatial Analyte Pattern Recognition

• Enabling Description: A set of pre-trained machine learning models for interpreting the data generated by the reference technology is disclosed and made available in the ONNX (Open Neural Network Exchange) format. These models are trained on large, publicly available spatial atlases (e.g., the Human Cell Atlas). Disclosed models include:
  1. A convolutional neural network (CNN) for identifying cell boundaries and performing cell segmentation based on a nuclear stain channel and total analyte signal.
  2. A multi-layer perceptron (MLP) for classifying cell types (e.g., T-cell, macrophage, tumor cell) based on the vector of analyte counts within each segmented cell.
  3. A graph attention network (GAT) for identifying significant ligand-receptor interactions between neighboring cells.
     By providing these models in ONNX format, they can be deployed across various deep learning frameworks (PyTorch, TensorFlow, etc.), democratizing access to advanced spatial data analysis.