US 11566277

Analysis of U.S. Patent 11,566,277

Report Date: May 12, 2026

This report provides a concise summary of United States Patent 11,566,277, including details of its prosecution history, an overview of its independent claims, and its current legal status.

I. Patent Overview

Title	Compositions and methods for analyte detection
Assignee	Harvard University
Inventors	George M. Church, Jehyuk Lee, Daniel Levner, Michael Super
Filing Date	February 15, 2022
Issue Date	January 31, 2023
Application No.	17/671,803

Abstract:
The patent describes methods and compositions for detecting multiple analytes in a sample. The core of the invention lies in using "detection reagents" which consist of a probe (like an antibody or nucleic acid) that binds to a specific target analyte, and a nucleic acid "label." This nucleic acid label contains a unique sequence of "subsequences" that acts as a barcode or identifier for the probe. By detecting these subsequences in a specific time-ordered sequence (temporally-sequential manner), the system can identify which probe has bound to its target, and thus identify the analyte present in the sample. The detection can be performed through methods like hybridization with fluorescently labeled "decoder probes" or by sequencing. This allows for multiplexed analysis, meaning many different analytes can be detected simultaneously in the same sample, such as a tissue section.

II. Independent Claims in Plain Language

U.S. Patent 11,566,277 has three independent claims: Claim 1, Claim 14, and Claim 21. Below is a plain-language explanation of each.

Independent Claim 1: This claim describes a method for identifying multiple different substances (analytes) within a sample. The process involves:

1. Contacting the sample with a collection of specialized "detection reagents." Each type of reagent is designed to stick to a specific substance you want to find.
2. Each detection reagent has a unique "barcode" made of a nucleic acid (like DNA). This barcode is composed of several pre-determined segments, or "subsequences."
3. Detecting these barcode segments one after another over time. Each segment, when detected, produces a specific signal (e.g., a color).
4. The specific order of signals you detect over time identifies the barcode, which in turn tells you which detection reagent is present, and therefore what substance has been found in the sample.

Independent Claim 14: This claim focuses on the "detection reagent" itself as a product. The reagent is a composite molecule that includes:

1. At least one "probe," which is the part that recognizes and binds to a specific substance (analyte) of interest. This probe could be an antibody, a nucleic acid, or other types of molecules.
2. At least one nucleic acid "label" attached to the probe. This label functions as a barcode.
3. This nucleic acid barcode is made up of at least one pre-determined segment ("subsequence") that is designed to be detected in a time-ordered fashion.
4. The sequence of these segments serves as a unique identifier for the probe it's attached to.

Independent Claim 21: This claim describes a kit for performing the detection method. The kit contains:

1. A collection of the detection reagents described in Claim 14 (or at least the nucleic acid label part of them).
2. At least one other reagent needed for the detection process, such as a set of "decoder probes." These decoder probes are complementary to the barcode segments and carry a detectable label (like a fluorescent dye), producing a unique signal when they bind.

III. Legal Status and Litigation

As of May 12, 2026, U.S. Patent 11,566,277 is active.

A search of court dockets and legal news indicates that this patent is the subject of recent litigation. In early May 2026, 10x Genomics Inc. and Harvard College filed a lawsuit against Element Biosciences Inc. in the U.S. District Court for the District of Delaware. The complaint alleges that Element Biosciences' AVITI24 platform and Teton chemistry infringe on U.S. Patent 11,566,277, along with three other patents. This case is in its early stages in district court, and there are no proceedings before the Court of Appeals for the Federal Circuit (CAFC) at this time.

IV. Known Litigation

As of May 12, 2026, U.S. Patent 11,566,277 is involved in one known litigation case. The details are as follows:

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Case 1: 10X Genomics, Inc. et al v. Element Biosciences, Inc.

• Plaintiff(s): 10X Genomics, Inc. and President and Fellows of Harvard College
• Defendant(s): Element Biosciences, Inc.
• Jurisdiction: U.S. District Court for the District of Delaware
• Case Number: 1:26-cv-00538
• Filing Date: May 8, 2026
• Status: Active/Pending. The complaint was filed on May 8, 2026. According to the docket, the summons was served on Element Biosciences, Inc. the same day, with an answer due by May 29, 2026.
• Details: The lawsuit alleges that Element Biosciences' AVITI24 multiomic analysis platform and Teton chemistry infringe on four patents, including U.S. Patent 11,566,277. The plaintiffs are seeking a permanent injunction or an ongoing royalty for future infringement, and lost profits or a reasonable royalty for past infringement. In a public statement, Element Biosciences has responded that it "strongly disagrees with the allegations" and "will vigorously defend its technology."

Proceedings Overview

There are no AIA trial proceedings on file at the PTAB for U.S. Patent 11,566,277. For a defendant, this means the patent's validity has not been tested or affirmed in a post-grant proceeding, leaving all defensive options, including arguments based on prior art, fully available.

Strategic Summary

Based on a review of the USPTO's Open Data Portal and public search records as of May 12, 2026, U.S. Patent 11,566,277 has not been the subject of any inter partes review (IPR), post-grant review (PGR), or covered business method (CBM) proceedings.

• Claim Status: All claims of U.S. Patent 11,566,277—including independent claims 1, 14, and 21—are UNTESTED before the PTAB. No claims have been canceled or sustained through an AIA trial.

• Estoppel Landscape: For a defendant, such as Element Biosciences in the current district court litigation (10X Genomics, Inc. et al v. Element Biosciences, Inc., D. Del. 1:26-cv-00538), there is no petitioner estoppel under 35 U.S.C. § 315(e)(2). This provides maximum flexibility, as any and all grounds of invalidity that can be raised in an IPR or PGR are still available. A future petitioner would not be barred from using prior art that it "reasonably could have raised" in a prior proceeding, because no prior proceeding exists.

• Pattern Signals: The absence of PTAB challenges is a significant signal. Patents that are frequently asserted by major commercial players, like 10x Genomics, often attract IPRs from competitors or defensive aggregators. The lack of any such challenge to date suggests that the patent may not have been widely asserted prior to the recently filed litigation against Element Biosciences.

Recommended Next Steps

For a defendant facing an assertion of U.S. Patent 11,566,277, the path is clear:

• No PTAB Activity: The primary takeaway is that there is no history of PTAB litigation for this patent. This is a "clean slate" scenario. The patent has not been "hardened" by surviving a previous validity challenge at the PTAB, which can be a significant advantage.
• IPR as a Viable Defense: A defendant should strongly consider filing an IPR as a key part of its defensive strategy. All potential prior art references are available for use in a petition. Given the statutory one-year timeline for an IPR from institution to a final written decision, this can provide a faster and often more cost-effective route to a validity determination than district court litigation.
• Monitor for New Filings: Given the active litigation, it is highly probable that the defendant, Element Biosciences, will file one or more IPR petitions against this patent in the near future. Any party with an interest in this technology space should monitor the PTAB docket for such filings, as they will provide the first indication of the prior art being used to challenge the claims and the patent owner's initial defensive arguments. The statutory deadline for filing an IPR is one year from the date of service of the infringement complaint. With a service date of May 8, 2026, a petition would be due by May 2027.

Inventors

The patent names four inventors:

• George M. Church: At the time of filing, Dr. Church was and remains a Professor of Genetics at Harvard Medical School and a Core Faculty Member of the Wyss Institute for Biologically Inspired Engineering at Harvard University.
• Jehyuk Lee: At the time of the invention, Dr. Lee was a Postdoctoral Fellow at the Wyss Institute at Harvard University.
• Daniel Levner: At the time of the invention, Dr. Levner was a Senior Staff Scientist at the Wyss Institute at Harvard University.
• Michael Super: At the time of the invention, Dr. Super was a Senior Staff Scientist at the Wyss Institute at Harvard University.

All inventors were employed by or affiliated with Harvard University at the time of the invention, which is a standard pattern for university-led research. There are no unusual patterns, such as mass departures, associated with this patent.

Original assignee

The original assignee is the President and Fellows of Harvard College (commonly known as Harvard University), located in Cambridge, Massachusetts. Harvard is a major private research university and does not itself manufacture or sell commercial products. Instead, it commercializes technology developed by its researchers through its Office of Technology Development, primarily by licensing patents to established companies and spin-out ventures.

In this instance, the litigation history indicates that Harvard has licensed the patent to 10x Genomics, Inc., a major life sciences technology company that sells instruments, reagents, and software that embody the types of technologies described in the patent. Harvard remains an operating university.

Assignment timeline

A search of the USPTO Patent Assignment Search system reveals a single recorded assignment for this patent.

• 2012-12-21 (executed) / recorded 2022-06-27 — Reel 064372/0839
  • 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. J. D'afonseca, Proskauer Rose LLP, One International Place, Boston, MA 02110
  • Context: Standard initial assignment of rights from inventors to their employer (Harvard University), executed shortly after the first priority application filing but recorded years later to perfect the chain of title.

No other assignments have been recorded. This indicates that the President and Fellows of Harvard College remains the current owner of record of US patent 11,566,277.

Timeline diagram

timeline
    title Ownership of US 11566277
    2012 : Inventors assign rights to Harvard
    2022 : Application filed
         : Inventor assignment recorded
    2023 : Patent issued to Harvard
    2026 : Harvard and 10x Genomics sue Element

NPE / troll-pattern signals

1. Shell-entity transfer — Not present. The only recorded transfer is from the inventors to their university employer, a major operating research institution.

2. Known asserter in the chain — Not present. The sole assignee of record is Harvard University, which is not considered an NPE.

3. Repeat correspondent across the chain — Not present. There is only one assignment, recorded by counsel at Proskauer Rose, a large, full-service law firm not primarily associated with NPEs.

4. Cascading transfers — Not present.

5. Pre-litigation transfer — Not present. No assignment was recorded in the six months preceding the May 8, 2026 litigation filing. Harvard University has owned the patent since its issuance.

6. Bankruptcy fire-sale — Not present.

7. Privateering — Not present. The litigation pattern, where the patent owner (Harvard) joins its exclusive licensee (10x Genomics) as a co-plaintiff against a competitor, is a standard operating procedure for patent enforcement and is not considered privateering. The patent was not transferred to a third-party assertion entity.

8. Defensive aggregator (anti-NPE) — Not present.

Verdict

Operating-company assertion

The evidence strongly supports this verdict. The patent has a clean and simple ownership history, originating with researchers at Harvard University and assigned to the university itself, which remains the owner of record per the assignment at Reel 064372/0839. Harvard has licensed the patent to 10x Genomics, a significant operating company in the life sciences space. The subsequent litigation is a joint action by the patent owner (Harvard) and its licensee (10x Genomics) against a direct market competitor. All available evidence indicates this is a straightforward case of an operating company enforcing patent rights licensed from a university.

Verification link: USPTO Assignment Search for US 11,566,277

V. Prior Art Analysis

This section analyzes the most relevant prior art references cited during the prosecution of U.S. Patent 11,566,277. The analysis focuses on how each reference relates to the independent claims of the '277 patent and its potential to anticipate those claims under 35 U.S.C. § 102. The key inventive concept in the '277 patent is the use of a "temporally-sequential" detection of subsequences on a nucleic acid label to generate a "temporal order of the signal signatures," which acts as a unique identifier for a probe.

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1. U.S. Patent Application Publication No. 2007/0231824 ("the '824 application")

• Full Citation: US 2007/0231824 A1; Gunderson, et al.; "Methods for Decoding a Sensor Array"; Published Oct. 4, 2007; Filed Jan. 12, 2007.
• Brief Description: The '824 application describes methods for decoding sensor arrays, such as bead arrays, where each bead has a unique identifier. The decoding process involves sequentially applying different decoding probes to the array. In one embodiment, a first set of probes is hybridized, an image is taken, the probes are stripped, and then a second set of probes is hybridized, and a second image is taken. This cycle is repeated several times. The sequence of signals (e.g., colors) observed at a specific bead's location across the series of images reveals its unique code.
• Potential Anticipation Analysis:
  • This reference is highly relevant as it explicitly discloses a multi-step, sequential decoding process. The method of hybridizing, imaging, stripping, and re-hybridizing is a clear example of "detecting in a temporally-sequential manner" to generate a "temporal order of the signal signatures," as claimed in Claim 1.
  • The '824 application describes probes attached to beads, where the beads act as the substrate for the identifier nucleic acids. This maps closely to the "detection reagent" of Claim 14, which comprises a probe and a nucleic acid label with subsequences forming an identifier. The '824 disclosure of a decoding process over several cycles directly teaches using subsequences to be "detected in a temporally-sequential manner."
  • The '824 application also discloses kits containing the necessary reagents, such as the beads and the decoding probes, which is relevant to Claim 21.
  • Conclusion: The '824 application appears to disclose many key elements of the independent claims. The '277 patent itself attempts to distinguish this reference by stating the '824 application's methods are for immobilized microspheres on an array and "cannot be used and detected directly on a sample (e.g., on a tissue sample) or in situ" (Col. 14, ll. 5-11). However, whether this distinction is sufficient to overcome an anticipation rejection could be a central point of dispute. From a technical standpoint, the core concept of temporal decoding is present.

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2. U.S. Patent No. 7,473,767 ("the '767 patent")

• Full Citation: US 7,473,767 B2; Geiss, et al.; "Methods for Detection and Quantification of Analytes in Complex Mixtures"; Issued Jan. 6, 2009; Filed Jul. 3, 2001.
• Brief Description: The '767 patent, related to NanoString Technologies' core platform, describes using "nanoreporters" which consist of a target-specific probe attached to a nucleic acid backbone. This backbone contains a series of label attachment regions, where different fluorescent labels can bind to create a unique color code, or "barcode." The patent describes immobilizing and elongating these nanoreporters and then imaging the spatial pattern of the colors along the backbone to identify the analyte.
• Potential Anticipation Analysis:
  • This reference teaches the use of a probe conjugated to a nucleic acid label with multiple distinct regions that produce a signal to identify the probe, which is a core element of Claim 14.
  • However, the method of detection in the '767 patent is fundamentally different from that in the '277 patent. The '767 patent's method is based on reading the spatial arrangement of different colors along a single molecule at a single point in time. The '277 patent, in contrast, claims reading signals from the same location over a sequence of time steps.
  • Conclusion: The '767 patent does not appear to anticipate Claim 1 because it fails to teach the "temporally-sequential" detection and the generation of a "temporal order of signal signatures." It teaches a spatial order of signals. While it discloses a similar composition (probe + barcoded nucleic acid label), the method of using that composition is distinct. Therefore, anticipation under § 102 is unlikely.

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3. U.S. Patent No. 9,677,143 ("the '143 patent")

• Full Citation: US 9,677,143 B2; Church, et al.; "Compositions and Methods for Analyte Detection"; Issued Jun. 13, 2017; Filed Feb. 15, 2017.
• Brief Description: The '143 patent is an earlier patent from the same inventors and assignee as the '277 patent and shares a priority claim. It describes similar compositions and methods for detecting analytes using detection reagents with nucleic acid labels. It explicitly describes detecting subsequences in a "temporally-sequential manner" through repeated cycles of hybridization, imaging, and signal removal.
• Potential Anticipation Analysis:
  • This patent is part of the same patent family as the '277 patent and contains a very similar disclosure. However, as prior art, it must be analyzed for what it teaches. It discloses all the key features of the independent claims of the '277 patent: the detection reagent composition, the method of temporal-sequential detection, and kits for performing the method.
  • Conclusion: This reference would likely anticipate the claims of the '277 patent if it were not for their shared priority claim. Because they are related applications, the '143 patent is not prior art to the '277 patent under § 102. This reference is primarily of interest for understanding the prosecution history and the scope of the broader invention claimed by the applicants.

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4. U.S. Patent No. 9,447,468 ("the '468 patent")

• Full Citation: US 9,447,468 B2; Emily M. Leproust, et al.; "Multiplexed Selection and Detection of Nucleic Acids"; Issued Sep. 20, 2016; Filed Aug. 24, 2012.
• Brief Description: The '468 patent discloses methods for enriching and detecting target nucleic acids. The method involves using probes with unique barcode sequences. After hybridization and enrichment, the barcodes are typically identified by amplification and sequencing (e.g., next-generation sequencing).
• Potential Anticipation Analysis:
  • This reference describes a composition of a probe linked to a nucleic acid barcode for analyte identification, which is relevant to Claim 14.
  • The primary detection method disclosed is high-throughput sequencing. While sequencing is inherently a step-wise process, it is typically understood in the art as a method for determining a complete sequence, not for generating a "temporal order of signal signatures" in situ for probe identification in the manner of the '277 patent (i.e., multiple rounds of imaging). The '468 patent's method involves physically sequencing the barcode to read the code, not optically detecting a series of transient signals over time.
  • Conclusion: The '468 patent does not appear to anticipate Claim 1. It does not teach the repeated cycles of hybridization/imaging/stripping to generate a temporal code. The detection mechanism is fundamentally different. Therefore, anticipation under § 102 is unlikely.

Obviousness Analysis of U.S. Patent 11,566,277

This analysis assesses the validity of the claims of U.S. Patent 11,566,277 ("the '277 patent") under 35 U.S.C. § 103, focusing on whether the invention would have been obvious to a Person Having Ordinary Skill in the Art (PHOSITA) at the time of the invention.

A PHOSITA in this field circa 2011-2012 would typically have a Ph.D. in molecular biology, biochemistry, or a related field, with several years of postdoctoral or industry experience in areas such as nucleic acid chemistry, immunology, microscopy, and next-generation sequencing (NGS) technologies.

The central argument is that the claims of the '277 patent are rendered obvious by the combination of prior art teaching multiplexed analyte detection using nucleic acid barcodes with well-established methods for cyclic, sequential nucleic acid detection, such as sequencing-by-hybridization or sequencing-by-synthesis.

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Primary Obviousness Combination

A compelling case for obviousness can be made by combining the teachings of:

1. U.S. Pat. No. 7,473,767 to Geiss et al. ("Geiss" or "Nanostring"), which teaches the use of analyte-specific probes conjugated to nucleic acid barcodes for multiplexed detection.
2. The well-established principles and methods of cyclic sequencing-by-hybridization (SBH) or sequencing-by-synthesis (SBS), which were foundational to the field of next-generation sequencing long before the '277 patent's priority date.

1. Teachings of the Prior Art

• Geiss (Nanostring): Geiss discloses a system for detecting and quantifying numerous target molecules (analytes) simultaneously. The core of the Nanostring system is a "reporter probe" (a detection reagent) comprising a target-specific probe attached to a single-stranded DNA molecule. This DNA molecule is functionalized with a series of different fluorescent labels, creating a color-coded "barcode." The identity of the target analyte is determined by reading the spatial order of the colors along this barcode using high-resolution imaging. Geiss thus teaches the fundamental structure of the '277 patent's detection reagent: a probe conjugated to a nucleic acid label that serves as an identifier.

• Cyclic Sequencing (SBH/SBS): By 2011, cyclic sequencing methods were mature and widely practiced. These methods, which form the basis of platforms like those from Illumina and Applied Biosystems (SOLiD), operate on a core principle of sequential, iterative steps:

  1. Step 1 (Hybridization/Incorporation): A labeled oligonucleotide (in SBH) or a labeled nucleotide (in SBS) is introduced and binds to its complementary sequence.
  2. Step 2 (Imaging): The entire sample or flow cell is imaged to detect the signal (e.g., fluorescence) from the successfully bound molecule. This captures the identity of one position in the sequence.
  3. Step 3 (Signal Removal): The signal is removed, either by cleaving the fluorescent label or by stripping away the labeled oligonucleotide with a chemical wash or heat.
  4. Step 4 (Repeat): The cycle is repeated with a new set of labeled molecules to read the next position in the sequence.

  This process generates a temporal sequence of signals (e.g., Color 1 in cycle 1, Color 2 in cycle 2, etc.) for each nucleic acid molecule being sequenced. This is the exact process described in the '277 patent as "detecting in a temporally-sequential manner."

2. Motivation to Combine

A PHOSITA would have been motivated to combine the Nanostring reporter probe concept with the cyclic sequencing readout method to overcome a well-understood limitation of the Nanostring approach.

The primary motivation is to improve scalability and throughput while reducing instrument complexity.

The spatial decoding method of Geiss requires extremely high-magnification optics to resolve multiple distinct colors on a single molecule that is only nanometers long. This physical constraint limits the field of view, meaning only a small area of a sample can be analyzed at once, and it increases the cost and complexity of the imaging hardware.

A PHOSITA, being intimately familiar with the massive scalability of cyclic sequencing, would have recognized that applying this temporal readout method to the Nanostring barcodes was an obvious solution. Instead of trying to resolve all the colors at once in space, it would be a predictable and logical step to treat the barcode as a short sequence to be read one "base" (or subsequence) at a time using the established cyclic SBH/SBS workflow.

This combination would allow for the use of lower-magnification, wider-field-of-view optics, as the system would only need to detect a single color at a specific location in each cycle. The identity is derived from the sequence of colors over time at that location, not the arrangement of colors in space. This would directly lead to higher throughput, lower cost, and the ability to analyze larger samples, such as entire tissue sections, more efficiently.

3. Application to the Claims

This combination of Geiss and the principles of cyclic sequencing renders the independent claims of the '277 patent obvious.

• Claim 1 (Method):

  • contacting the sample with a plurality of detection reagents...: Taught by Geiss.
  • each detection reagent comprises at least one probe reagent and at least one nucleic acid label: This is precisely Geiss's reporter probe structure.
  • said at least one nucleic acid label comprises a plurality of pre-determined subsequences: This is the barcode structure taught by Geiss, where the "subsequences" correspond to the binding sites for the different colored labels.
  • detecting in a temporally-sequential manner... wherein a temporal order of the signal signatures... identifies a subpopulation of the detection reagents: This is the key element not explicitly taught by Geiss, but it is the fundamental and well-known process of cyclic SBH/SBS. The motivation to apply this temporal readout to Geiss's barcodes, as explained above, makes this step obvious.

• Claim 14 (Composition):

  • A detection reagent comprising at least one probe reagent and at least one nucleic acid label...: This composition is taught by Geiss.
  • said at least one nucleic acid label comprises at least one pre-determined subsequence to be detected in a temporally-sequential manner: While Geiss teaches detecting the subsequences spatially, designing a nucleic acid barcode to be read by a known temporal method (cyclic sequencing) would have been an obvious design choice for a PHOSITA motivated to improve scalability. The structure of the molecule itself is not materially different; what changes is the intended method of reading it, which was a well-known alternative.

• Claim 21 (Kit):

  • A kit containing the obvious detection reagents from claim 14 and other reagents (like decoder probes used in SBH) would be obvious to a PHOSITA implementing the obvious method of claim 1. Assembling the necessary components for an obvious process into a kit is a routine and obvious practice in the art.

Conclusion

The core concept claimed in the '277 patent—using a temporal sequence of signals from a nucleic acid barcode to identify an analyte—is not an inventive leap but rather the logical and predictable convergence of two well-established technologies. The prior art taught the use of nucleic acid barcodes for multiplexed detection (Geiss) and separately taught the use of cyclic, temporal detection for reading nucleic acid sequences (SBH/SBS). A person of ordinary skill in the art would have been motivated to combine these teachings to create a more scalable and robust system for multiplexed analysis, directly arriving at the invention claimed in the '277 patent. Therefore, the claims of the '277 patent are invalid as obvious under 35 U.S.C. § 103.

Prosecution and Term Analysis

Patent Term Adjustment (PTA) and Patent Term Extension (PTE)

A review of the USPTO Patent Center file wrapper for Application No. 17/671,803, which matured into patent 11,566,277, shows the following:

• Patent Term Adjustment (PTA): The patent was granted 0 days of PTA. The application was prosecuted efficiently, with no delays attributable to the USPTO that would warrant an adjustment to the patent's term.
• Patent Term Extension (PTE): There is no record of a Patent Term Extension being sought or granted for this patent under 35 U.S.C. § 156. Such extensions are typically for delays associated with regulatory review of human drugs or medical devices and do not apply here.
• Terminal Disclaimer: A terminal disclaimer was filed on October 18, 2022, to obviate a non-statutory double patenting rejection over U.S. Patent No. 11,293,061. This disclaimer ties the enforceability of patent 11,566,277 to that of the '061 patent, ensuring it does not expire later. As both patents share the same priority date for term calculation, this disclaimer does not shorten the patent's term.

Application and Family History

U.S. Patent 11,566,277 is a continuation of a long line of applications, indicating a sustained research and development effort. The patent's 20-year term is calculated from the earliest non-provisional application in its priority chain.

The lineage is as follows:

• This patent, US 11,566,277, issued from Application No. 17/671,803, filed on February 15, 2022.
• It is a continuation of Application No. 16/797,015 (now US Patent 11,293,061).
• Which is a continuation of Application No. 15/951,349 (now US Patent 10,612,058).
• Which is a continuation of Application No. 14/419,053 (now US Patent 9,957,557).
• Which was the U.S. national stage of international application PCT/US2014/018580, filed on February 26, 2014.
• The '580 PCT application was a continuation-in-part of international application PCT/US2012/071398, filed on December 21, 2012.

The earliest non-provisional filing date from which the patent claims benefit is the December 21, 2012 filing date of PCT/US2012/071398. This date serves as the starting point for calculating the patent's 20-year term.

Projected Expiration Date

The expiration date of U.S. Patent 11,566,277 is calculated as follows:

Calculation Component	Date/Days
Earliest Effective Filing Date:	December 21, 2012
Add 20-Year Statutory Term:	+ 20 years
Base Expiration Date:	December 21, 2032
Add Patent Term Adjustment (PTA):	+ 0 days
Add Patent Term Extension (PTE):	+ 0 days
Projected Expiration Date:	December 21, 2032

Barring any changes due to terminal disclaimers tied to maintenance fee payments on parent patents, the patent is projected to expire on December 21, 2032.

Defensive Disclosure: Derivative Innovations for Analyte Detection by Temporal Barcoding

Publication Date: May 12, 2026
Reference Technology: U.S. Patent 11,566,277 ("Compositions and methods for analyte detection")
Objective: This document discloses a plurality of derivative inventions, alternative embodiments, and cross-domain applications of the core technology described in US 11,566,277. The purpose is to place these concepts in the public domain, thereby establishing them as prior art for patentability purposes.

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Derivative Set 1: Material & Component Substitution

1.1. Electrochemical Temporal Barcoding (ETB)

• Enabling Description: This variation replaces optical detection with an electrochemical readout. The nucleic acid label of the detection reagent is synthesized to include specific sites for binding decoder probes, as in the reference patent. However, each decoder probe is conjugated not to a fluorophore, but to a unique redox-active molecule (e.g., ferrocene, methylene blue, or a metal nanoparticle with a distinct redox potential). The sample is placed on a microelectrode array. In each cycle, a set of redox-labeled decoder probes is hybridized. A cyclic voltammetry (CV) or square wave voltammetry (SWV) scan is performed, generating a signal (a current peak at a specific voltage) for each redox molecule present. This "voltammogram" constitutes the signal signature for that cycle. The probes are then chemically or thermally stripped, and the next cycle begins. The temporal sequence of voltammograms provides the analyte's identifier.

• Mermaid Diagram:

  sequenceDiagram
      participant MEA as Microelectrode Array
      participant Sample as Sample Surface
      participant Reagent as Detection Reagent
      participant Decoder as Redox Decoder Probe
      participant Reader as Potentiostat
  
      MEA->>Sample: Provides detection surface
      Reagent->>Sample: Binds to target analyte
      loop Cycle 1
          Decoder->>Reagent: Hybridize (Ferrocene-labeled)
          Reader->>MEA: Apply Voltage Ramp (CV)
          MEA-->>Reader: Measure Current (Peak at V1)
          Decoder->>Reagent: Strip probe
      end
      loop Cycle 2
          Decoder->>Reagent: Hybridize (Methylene Blue-labeled)
          Reader->>MEA: Apply Voltage Ramp (CV)
          MEA-->>Reader: Measure Current (Peak at V2)
          Decoder->>Reagent: Strip probe
      end
      Reader->>System: Compile Temporal Voltammogram [V1, V2, ...]
  

1.2. Molecularly Imprinted Polymer (MIP) Probes with PNA Labels

• Enabling Description: This derivative replaces biological antibodies or aptamers with robust, synthetic molecularly imprinted polymers (MIPs). MIPs are created by polymerizing functional monomers around a template molecule (the analyte). After polymerization, the template is removed, leaving a cavity with high specificity for the analyte. These MIP nanoparticles serve as the probe reagent. The MIPs are conjugated to Peptide Nucleic Acid (PNA) labels. PNA has a neutral backbone, making its hybridization highly stable and resistant to enzymatic degradation and extreme pH, thus suitable for industrial or environmental samples. The temporal readout proceeds via hybridization with fluorescently-labeled DNA or PNA decoder probes as described in the reference patent, but leverages the superior stability of the PNA label backbone.

• Mermaid Diagram:

  flowchart TD
      subgraph Synthesis
          A[Analyte Template] --> B{Mix with Functional Monomers};
          B --> C{Polymerization};
          C --> D[Remove Template];
          D --> E(MIP Nanoparticle);
      end
      subgraph Conjugation
          E --> F{Activate Surface};
          G[PNA Barcode Label] --> H{Activate Terminus};
          F & H --> I[Conjugate MIP to PNA];
      end
      subgraph Assay
          I --> J(MIP-PNA Detection Reagent);
          J --> K{Apply to Sample};
          K --> L[Temporal Readout via Hybridization];
      end
  

Derivative Set 2: Operational Parameter Expansion

2.1. In-Situ Intracellular Transcriptome Mapping

• Enabling Description: This method scales the technology down to the nanoscale for use inside living or fixed cells. The detection reagents, targeting specific mRNA sequences, are encapsulated in lipid nanoparticles (LNPs) functionalized with cell-penetrating peptides. Upon entering a cell, the LNPs release the reagents. Each reagent's probe is a locked nucleic acid (LNA) oligonucleotide for high-affinity binding to its target mRNA. The attached DNA label is then detected temporally. The readout cycles are performed on a microscope stage with automated microfluidics. Decoder probes are modified to include a cell-permeable trans-activating transcriptional activator (TAT) peptide. This allows for repeated interrogation of the spatial location of individual mRNA molecules within the subcellular architecture, creating a 3D temporal map of the transcriptome.

• Mermaid Diagram:

  graph TD
      A[LNP Encapsulated Reagents] --> B{Cellular Uptake};
      B --> C[Reagent Release in Cytoplasm];
      C --> D{Probe binds target mRNA};
      subgraph Automated Microscopy Stage
          E[Cycle 1: Add TAT-Decoder Probes 1];
          E --> F[Hybridization at mRNA locations];
          F --> G[Image Acquisition (Signal 1)];
          G --> H[Photobleach or Displace Probes];
          H --> I[Cycle 2: Add TAT-Decoder Probes 2];
          I --> J[Hybridization at mRNA locations];
          J --> K[Image Acquisition (Signal 2)];
          K --> L[...]
      end
      D --> E;
      L --> M[Reconstruct 3D Temporal Transcriptome Map];
  

2.2. Continuous Industrial Bioreactor Monitoring

• Enabling Description: The technology is scaled up for monitoring a 10,000L industrial bioreactor producing monoclonal antibodies. A sidestream from the reactor flows through a detection cell with a transparent window. The detection reagents use probes targeting critical process parameters, such as host cell proteins, leaked protein A, or specific glycosylation patterns. The reagents are PNA-based for chemical robustness. The readout is automated. In each cycle, decoder probes are injected, allowed to hybridize, and detected via an integrated spectroscopic reader. The signal is removed by a high-pH wash cycle that the PNA-DNA duplex can withstand. The temporal signature identifies contaminants or product quality issues in near real-time, enabling dynamic process control.

• Mermaid Diagram:

  stateDiagram-v2
      [*] --> Idle
      Idle --> Cycle_Start: New measurement triggered
      Cycle_Start --> Hybridization: Inject Decoder Probe Set N
      Hybridization --> Detection: Flow paused, acquire signal
      Detection --> Signal_Removal: Inject high-pH wash buffer
      Signal_Removal --> Idle: If last cycle
      Signal_Removal --> Cycle_Start: If more cycles
      Detection --> Alert: If analyte level exceeds threshold
      Alert --> [*]
  

Derivative Set 3: Cross-Domain Application

3.1. Aerospace: Composite Material Fatigue Sensing

• Enabling Description: This system monitors the health of carbon fiber reinforced polymer (CFRP) structures in aircraft. The detection probes are aptamers selected to bind specifically to chemical markers of matrix degradation or delamination (e.g., specific epoxy breakdown products). These probe-reagents are infused into a sensor patch applied to a critical structural component. A portable, handheld device performs the temporal readout. It first applies a small amount of buffer to mobilize any bound reagents, then cycles through hybridization with decoder probes and imaging via a fiber-optic coupled camera. A "red-green-blue" temporal signature might indicate severe fatigue, while "green-null-red" indicates early-stage micro-cracking.

• Mermaid Diagram:

  flowchart LR
      subgraph Aircraft Wing
          A[CFRP Structure] -- contains --> B(Embedded Sensor Patch);
      end
      subgraph Handheld Reader
          C[Fluidics Module] -- injects --> B;
          D[Optics Module] -- images --> B;
          C <--> D;
      end
      subgraph Process
          E{Attach Reader to Patch};
          E --> F[Inject Decoder Set 1];
          F --> G[Image Signal 1];
          G --> H[Strip Signal];
          H --> I[Inject Decoder Set 2];
          I --> J[Image Signal 2];
          J --> K[...];
          K --> L(Analyze Temporal Signature);
          L --> M{Assess Structural Health};
      end
  

#### **3.2. AgTech: In-Field Plant Pathogen Detection**

*   **Enabling Description:** A ruggedized, battery-powered handheld device for farmers to diagnose crop diseases on-site. The farmer takes a leaf punch, places it in a disposable cartridge containing lysis buffer and a plurality of detection reagents. Each reagent's probe is a DNA oligonucleotide complementary to a specific pathogen's genomic RNA/DNA (e.g., for Tobacco Mosaic Virus, Wheat Rust Fungus). The cartridge is inserted into the device, which automates the temporal readout. Microfluidic pumps cycle decoder probes labeled with quantum dots (for high photostability in sunlight) over the sample. A CMOS sensor detects the signals. The device displays the identified pathogen and its concentration within minutes.

*   **Mermaid Diagram:**
    ```mermaid
    sequenceDiagram
        actor Farmer
        participant Cartridge
        participant Device

        Farmer->>Cartridge: Insert Leaf Sample
        Farmer->>Device: Insert Cartridge
        Device->>Cartridge: Lyse sample, release reagents
        Device->>Device: Start Temporal Readout Protocol
        loop For N cycles
            Device->>Cartridge: Pump Decoder Probes (Set N)
            Device->>Cartridge: Incubate
            Device->>Cartridge: Image with CMOS sensor
            Device->>Cartridge: Pump Wash Buffer
        end
        Device->>Device: Decode Temporal Barcodes
        Device->>Farmer: Display "Wheat Rust: High"
    ```

### **Derivative Set 4: Integration with Emerging Tech**

#### **4.1. AI-Powered Signal Deconvolution and Barcode Design**

*   **Enabling Description:** The temporal detection process is integrated with a deep learning model. A convolutional neural network (CNN) is trained to analyze the image stacks from each cycle. The AI can deconvolve signals from spatially overlapping detection reagents in dense samples, correcting for bleed-through between fluorescent channels and imperfect probe stripping. Furthermore, a generative adversarial network (GAN) is used for *in silico* design of the nucleic acid label subsequences. The generator proposes barcode sets, and the discriminator, trained on thermodynamic and kinetic simulation data, predicts their cross-hybridization potential and overall decoding error rate. The system evolves optimal barcode libraries for specific numbers of analytes and imaging conditions.

*   **Mermaid Diagram:**
    ```mermaid
    graph TD
        subgraph Barcode Design (GAN)
            A[Generator] -- Proposes Barcode Set --> B(Discriminator);
            B -- Predicts Error Rate --> A;
            B --> C{Optimal Barcode Library};
        end
        subgraph Assay Execution
            D[Sample] --> E{Temporal Imaging};
            E --> F[Raw Image Stack];
        end
        subgraph Signal Deconvolution (CNN)
            F --> G[AI Model];
            G --> H{Decoded Analytes & Locations};
        end
        C --> D;
        H --> I[Final Report];

4.2. Blockchain for Clinical Diagnostic Data Integrity

• Enabling Description: In a clinical pathology workflow, a tissue biopsy is processed using the temporal barcoding method to detect a panel of 50 cancer biomarkers. The unique temporal barcode for each of the 50 detection reagents is pre-registered. After the automated microscope completes the N cycles of readout, the raw image data is processed. For each detected analyte, the system generates a data packet containing: (1) the spatial (X,Y,Z) coordinates, (2) the detected temporal signature (e.g., R-G-B-N), and (3) a confidence score. This data packet is cryptographically hashed, and the hash is submitted as a transaction to a private, permissioned blockchain (e.g., Hyperledger Fabric). This creates an immutable, timestamped, and auditable record of the diagnostic result, ensuring data integrity from the instrument to the patient's record.

• Mermaid Diagram:

  sequenceDiagram
      participant Microscope
      participant AnalysisServer as Analysis Server
      participant Blockchain
      participant EMR as Electronic Medical Record
  
      Microscope->>AnalysisServer: Transfer Raw Image Stack
      AnalysisServer->>AnalysisServer: Decode Temporal Signatures
      AnalysisServer->>Blockchain: Submit Hash(Result Data) as Transaction
      Blockchain-->>AnalysisServer: Confirm Transaction
      AnalysisServer->>EMR: Push Verified Diagnostic Report
  

Derivative Set 5: The "Inverse" or Failure Mode

5.1. Limited-Use Reagents with Photolabile Linkers

• Enabling Description: This derivative is designed for single-use diagnostic kits or applications requiring data security. The nucleic acid label is synthesized with one or more internal photolabile linkers (e.g., a nitrobenzyl-based photocleavable group) placed between subsequences. The readout process proceeds normally for a pre-determined number of cycles. However, the final step in the protocol involves exposing the sample to a specific wavelength of UV light (e.g., 365 nm). This exposure cleaves the photolabile linkers, fragmenting the nucleic acid label and rendering it incapable of further hybridization or re-analysis. This ensures single-use compliance and prevents reverse engineering of proprietary probe sequences.

• Mermaid Diagram:

  flowchart TD
      A[Full-Length Label: Sub1-PL-Sub2-PL-Sub3] --> B{Bind to Analyte};
      B --> C{Perform N Readout Cycles};
      C --> D{Expose to 365nm UV Light};
      D --> E[Photocleavage at PL sites];
      E --> F[Fragmented Label: Sub1 + Sub2 + Sub3];
      F --> G(Assay Inactivated);
  

Combination Prior Art Scenarios

1. Combination with Micro-Manager and ImageJ: The entire temporal detection method is automated using the open-source Micro-Manager microscopy control software. A BeanShell script controls a fluidic pump (via serial port) to deliver decoder and wash buffers, triggers image acquisition using the system's camera at each time point, and calls an ImageJ/Fiji macro. The macro performs image registration to correct for sample drift between cycles and then extracts intensity values at specific ROIs, outputting a CSV file of the temporal signal signatures. This combination makes the core method obvious to implement with standard, freely available laboratory software.

2. Combination with FASTQ Data Standard: The output of a temporal barcoding experiment is converted into the standard FASTQ format used for next-generation sequencing. Each detected spatial location becomes a "read." The "sequence" line consists of characters representing the signals (e.g., 'R' for red, 'G' for green, 'N' for null). The "quality" line consists of ASCII characters encoding the signal-to-noise ratio or intensity of the signal at each corresponding cycle. This allows bioinformaticians to use existing NGS alignment tools like BWA or Bowtie to "align" the temporal codes against a reference file of known barcodes, leveraging a massive existing software ecosystem for decoding.

3. Combination with OME-Zarr Format: For large-scale spatial transcriptomics applications, the output data is stored in the OME-Zarr format, an open-source standard for large, multi-dimensional bioimaging data. The 5D image data (X, Y, Z, channel, time-point) is stored in a chunked, cloud-friendly Zarr array. The decoded temporal barcode identifying the analyte at each (X,Y,Z) coordinate is stored as an associated label mask or in an accompanying metadata table, directly linking the spatial and temporal information in a standardized, interoperable format.