US 11021737

Analysis of U.S. Patent 11,021,737

Washington, D.C. - A detailed analysis of U.S. Patent No. 11,021,737 reveals a significant invention in the field of molecular biology and diagnostics, focusing on methods for detecting multiple analytes within a sample. This patent, assigned to Harvard University, is at the center of recent litigation, underscoring its perceived commercial importance.

Key Patent Details:

• Title: Compositions and methods for analyte detection
• Assignee: President and Fellows of Harvard College
• Inventors: George M. Church, Je-Hyuk Lee, Daniel Levner, Michael Super
• Filing Date: July 29, 2020
• Issue Date: June 1, 2021
• Abstract: The patent describes compositions and methods for detecting and analyzing a plurality of analytes, such as proteins or nucleic acids, in a sample. The core of the invention lies in using detection reagents that are labeled with nucleic acid "barcodes." These barcodes allow for the identification of numerous analytes simultaneously through a process of sequential detection, which can be read, for example, by DNA sequencing or hybridization with fluorescent probes. This technology enables highly multiplexed analysis, meaning many different targets can be identified within a single sample at the same time, while preserving spatial information.

Plain-Language Overview of Independent Claims:

U.S. Patent 11,021,737 contains several independent claims that define the core scope of the invention. In essence, these claims protect methods for identifying multiple substances (analytes) in a biological sample.

• Claim 1: This claim outlines a method where a sample is treated with a variety of "detection reagents." Each reagent is designed to bind to a specific target molecule (like a particular protein). Crucially, each detection reagent is tagged with a unique DNA or RNA label. This label has a series of pre-determined "subsequences." The method involves reading these subsequences in a specific order over time. The sequence of signals generated from these subsequences acts like a barcode, uniquely identifying the detection reagent and, therefore, the target molecule it has attached to.

• Claim 12: This claim is similar to Claim 1 but specifies that the detection of the subsequences is done by repeatedly hybridizing (binding) and then removing sets of "decoder probes." Each decoder probe is a short nucleic acid that is complementary to one of the subsequences and carries a detectable label, such as a fluorescent dye. By using different sets of labeled probes in sequence, the unique barcode of each detection reagent can be read.

• Claim 20: This claim focuses on the detection reagent itself. It describes a construct that includes a "probe reagent" (the part that binds to the target molecule, such as an antibody) linked to a nucleic acid label. This nucleic acid label is the key feature, containing at least two different, pre-determined subsequences that can be detected in a specific order to identify the probe reagent. This claim protects the tool used in the methods described in the other claims.

Litigation Status:

As of April 2026, U.S. Patent 11,021,737 is the subject of patent infringement lawsuits. Notably, 10x Genomics, Inc., as an exclusive licensee of the patent from Harvard University, has filed suits against competitors Vizgen, Inc. and Element Biosciences, Inc. in the U.S. District Court for the District of Delaware. These cases suggest that the technology covered by this patent is considered foundational for certain commercial spatial biology and genomic analysis platforms. A search of the 2026 dockets for the U.S. Court of Appeals for the Federal Circuit (CAFC) did not reveal any appeals directly related to patent 11,021,737 at this time. However, it is common for district court patent decisions to be appealed to the CAFC.

Disclaimer: This analysis is for informational purposes only and does not constitute legal advice. The legal status and interpretation of patents can be complex and may change over time.

Known Litigation Involving U.S. Patent 11,021,737

As of May 12, 2026, U.S. Patent 11,021,737 is asserted in multiple patent infringement lawsuits, reflecting its significance in the competitive spatial biology market. The exclusive licensee, 10x Genomics, Inc., alongside patent owner President and Fellows of Harvard College, has actively enforced this patent against competitors.

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Case 1: Action Against Vizgen Inc.

• Plaintiff(s): 10x Genomics, Inc. and President and Fellows of Harvard College
• Defendant(s): Vizgen, Inc.
• Jurisdiction: U.S. District Court for the District of Delaware
• Case Number: 1:22-cv-00595
• Filing Date: May 4, 2022
• Outcome or Current Status: Settled. This complex case involved claims of infringement by 10x and Harvard, with Vizgen filing counterclaims for non-infringement, invalidity, and antitrust violations. After extensive legal proceedings, the parties reached a settlement in early February 2025, resolving the global patent dispute. The terms of the settlement were not publicly disclosed.

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Case 2: Action Against NanoString Technologies, Inc.

• Plaintiff(s): 10x Genomics, Inc. and President and Fellows of Harvard College
• Defendant(s): NanoString Technologies, Inc.
• Jurisdiction: U.S. District Court for the District of Delaware
• Case Number: 1:22-cv-00261
• Filing Date: February 28, 2022.
• Outcome or Current Status: Active. While this case involves a broader dispute over multiple spatial technology patents, US 11,021,737 is part of the litigation. The case is ongoing, with proceedings linked to the related action against Vizgen (1:22-cv-00595) before that case was settled. Detailed current status would require review of the most recent court filings on PACER.

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Case 3: Action Against 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: 1:2026cv00538
• Filing Date: May 8, 2026.
• Outcome or Current Status: Newly filed. The complaint alleges that Element's AVITI24 multiomic analysis platform infringes on patent 11,021,737 and three other related patents. Element Biosciences has stated its intent to "vigorously defend its technology." As a new filing, this case is in its earliest stages.

Proceedings Overview

Based on a thorough review of the USPTO's public databases and available records, there have been no Inter Partes Review (IPR), Post-Grant Review (PGR), or other AIA trial proceedings filed against U.S. Patent No. 11,021,737 as of May 12, 2026. This means the patent has not yet faced a validity challenge at the Patent Trial and Appeal Board (PTAB), giving a defendant the full range of available prior art arguments in a first-instance challenge.

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Strategic Summary

The absence of any PTAB proceedings for U.S. Patent 11,021,737 is a critical data point. Despite the patent being asserted in high-stakes litigation by an exclusive licensee, 10x Genomics, against multiple competitors since 2022, no defendant or third party has yet filed an IPR or PGR petition. This is unusual for a patent central to a competitive and litigious technology space.

• Claim Status: All claims of U.S. Patent 11,021,737 remain as originally granted. No claims have been canceled, sustained, or amended through a PTAB trial.
• Estoppel Landscape: For a defendant currently facing an assertion of this patent, the estoppel landscape is clear. Because no prior IPRs have been filed, there is no § 315(e)(2) estoppel in play. A defendant is free to petition for IPR based on any prior art patents or printed publications they identify, without restriction from a prior PTAB final decision. All grounds that could be raised in an IPR are available.
• Pattern Signals: The lack of PTAB challenges could suggest several strategic possibilities:
  • Potential challengers and their counsel may believe the patent is strong and an IPR would have a low probability of success.
  • Parties may be opting to litigate validity exclusively in district court, forgoing the parallel PTAB track.
  • Settlements in past litigation, such as the one between 10x Genomics and Vizgen, may have occurred before an IPR could be filed or completed.

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Recommended Next Steps

For a defendant, such as Element Biosciences, which was recently sued on May 8, 2026, the path to challenging patent validity at the PTAB is entirely open.

1. Conduct a Thorough Prior Art Search: Given that no prior art has been vetted by the PTAB for this patent, a comprehensive search for patents and printed publications that predate the December 22, 2011, priority date is the immediate first step. The focus should be on grounds of anticipation (§ 102) and obviousness (§ 103).
2. Evaluate IPR as a Strategic Option: An IPR offers a faster and often less expensive route to invalidate a patent compared to district court litigation. The standard of proof is lower ("preponderance of the evidence" vs. "clear and convincing evidence" in court), and the judges are technically expert Administrative Patent Judges.
3. Monitor Statutory Deadlines: A defendant has a one-year window to file an IPR petition from the date they are served with an infringement complaint. For Element Biosciences, served on or after May 8, 2026, this deadline would be in May 2027. Careful tracking of this date is essential.
4. No PTAB Activity on File: To be clear, there are no active proceedings to monitor. The primary recommendation is to initiate the first such proceeding if a meritorious invalidity case can be built. The absence of PTAB history means a petitioner has the advantage of making a first impression on the Board with their invalidity arguments.

Inventors

The four inventors named on the patent are:

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

All inventors were affiliated with Harvard University, the original assignee, at the time of the invention. This represents a standard inventor-to-employer assignment pathway for university-developed technology.

Original assignee

The original assignee of record is the President and Fellows of Harvard College (commonly known as Harvard University), located in Cambridge, Massachusetts.

As a major research university, Harvard's primary business is education and research. It does not commercially manufacture or sell products. Instead, its Office of Technology Development actively licenses its intellectual property to established companies and startups to commercialize inventions developed by its faculty. The litigation history confirms that this patent was exclusively licensed to 10x Genomics, Inc., a company that sells spatial biology and genomics analysis platforms that appear to practice the patent's claims. Harvard remains an active, world-renowned educational and research institution.

Assignment timeline

A search of the USPTO Patent Assignment Database shows that title for US 11,021,737 has never been transferred from the original assignee. The ownership has remained with Harvard University since its grant. The database does, however, record other transactions related to the patent's rights.

• 2021-03-25 (executed) / recorded 2021-03-25 — Reel 058379/0421

  • Conveyance: Assignment of Assignor's Interest
  • Assignor: George M. Church, Je-Hyuk Lee, Daniel Levner, Michael Super
  • Assignee: President and Fellows of Harvard College
  • Correspondent: David A. Bello, Esq.; Rosenberg, Klein & Lee; Ellicott City, MD
  • Context: Standard confirmatory assignment of invention rights from the named inventors to their employer, Harvard University.

• 2012-09-06 (executed) / recorded 2023-10-13 — Reel 069695/0285

  • Conveyance: Confirmatory License
  • Assignor: President and Fellows of Harvard College
  • Assignee: United States of America as represented by the Secretary, Department of Health and Human Services
  • Correspondent: Richard A. Hertling; National Institutes of Health; Bethesda, MD
  • Context: A license granting the U.S. Government rights to the invention, a standard requirement for patents developed using federal research funding under the Bayh-Dole Act.

• 2023-10-06 (executed) / recorded 2023-10-16 — Reel 069726/0890 & 069726/0932

  • Conveyance: Security Agreement
  • Assignor: 10x Genomics, Inc.
  • Assignee: Royal Bank of Canada
  • Correspondent: Hylina E. Cannon; Dentons US LLP; San Diego, CA
  • Context: The exclusive licensee, 10x Genomics, pledged its license rights to this patent as part of a larger portfolio of collateral for financing. This securitization does not transfer ownership of the patent itself.

• 2024-04-30 (executed) / recorded 2024-05-02 — Reel 072554/0651

  • Conveyance: Release of Security Interest (Collateral)
  • Assignor: Royal Bank of Canada
  • Assignee: 10x Genomics, Inc.
  • Correspondent: Hylina E. Cannon; Dentons US LLP; San Diego, CA. The correspondent is the same as for the preceding Security Agreement.
  • Context: The security interest from the October 2023 financing was released, likely indicating the underlying loan was satisfied or refinanced.

Timeline diagram

timeline
    title Ownership of US 11021737
    2011 : Priority date
    2020 : Application filed
    2021 : Inventors assign rights to Harvard
         : Patent issues to Harvard
    2022 : First infringement suit filed by 10x and Harvard
    2023 : 10x Genomics pledges license as collateral
    2024 : Security interest is released

NPE / troll-pattern signals

1. Shell-entity transfer: Not present. The patent's title has remained with Harvard University, an operating educational and research institution, since its grant.

2. Known asserter in the chain: Not present. The owner is Harvard University. The party asserting the patent is 10x Genomics, the exclusive licensee, which is a major life sciences company that sells commercial products in the field of the invention.

3. Repeat correspondent across the chain: Not present. Different law firms and attorneys handled the distinct transaction types (inventor assignment, government license, commercial security agreement), which is typical. The recurrence of Hylina E. Cannon of Dentons is limited to the single securitization transaction (grant and release), which is not a signal of NPE activity.

4. Cascading transfers: Not present. There have been no transfers of title.

5. Pre-litigation transfer: Not present. No assignment of title occurred prior to the first infringement suit filed in February 2022.

6. Bankruptcy fire-sale: Not present.

7. Privateering: Not present. The patent was licensed by a university to an operating company (10x Genomics) which is now directly asserting the patent against its own competitors. This is a standard university tech-transfer and commercialization model, not a privateering arrangement.

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

Verdict

• Operating-company assertion

The ownership chain is clean and institutional. The patent originated at Harvard University and has remained there. It is being asserted by its exclusive licensee, 10x Genomics, a significant operating company, against direct competitors in the spatial biology market. This fact pattern is the definition of a standard operating-company assertion, intended to protect market position for a commercialized product, and displays no signals associated with NPE or patent troll behavior.

Verification Link: USPTO Patent Assignment Search for Pat. No. 11,021,737

Analysis of Cited Prior Art

This analysis focuses on the prior art references cited by the USPTO examiner during the prosecution of U.S. Patent 11,021,737. The patent's priority date is December 22, 2011. The following references were published before this critical date and were considered material to the patentability of the invention.

Key Prior Art References and Potential Anticipation

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1. U.S. Patent Application Publication No. US 2007/0231824 A1 ("Gunderson")

• Full Citation: US 2007/0231824 A1
• Title: Methods for decoding sensor arrays
• Filing Date: March 27, 2007
• Publication Date: October 4, 2007
• Brief Description: Gunderson discloses a method for identifying analytes using a sensor array, such as a fiber optic array with beads in wells. Each bead has a specific probe and is associated with a unique "address" sequence. The method involves sequentially hybridizing and stripping labeled "decoder probes" to these address sequences to identify the location of each bead type. After decoding the array, the sample is introduced, and analyte binding is detected.
• Potential Anticipation of Claims:
  • Claim 1 & 12 (Method Claims): Gunderson teaches a form of sequential detection using decoder probes to identify unique nucleic acid sequences, which generates a temporal series of signals. However, a key distinction exists. Gunderson's method first decodes the spatial positions of the probes on a fixed array before the sample is introduced. The detection of the analyte is a separate, subsequent step. In contrast, the '737 patent claims a method where the detection reagent (e.g., antibody + nucleic acid label) first binds to the analyte in the sample, and then the temporal decoding of the nucleic acid label reveals the identity of the bound analyte, preserving its location within the sample itself. The '737 patent is designed for in-situ analysis where the analyte's position is unknown beforehand, whereas Gunderson decodes a pre-fabricated sensor array. Therefore, Gunderson likely does not anticipate claims 1 or 12 because it does not teach detecting the temporal order of signals from a detection reagent already bound to an analyte within a biological sample.
  • Claim 20 (Composition Claim): Gunderson describes probes attached to beads, where the beads are associated with nucleic acid tags (address sequences). This is structurally similar to the "probe reagent" and "nucleic acid label" of claim 20. However, the '737 patent specifies that the detection reagent is designed for use in a solution phase to contact a sample, whereas Gunderson's probes are part of a fixed, solid-support array. The '737 patent itself explicitly argues this distinction, stating the '824 application's microspheres are "immobilized on a solid support ... rather than designed to be in a solution phase" and "cannot be used and detected directly on a sample (e.g., on a tissue sample) or in situ as described herein." This distinction likely prevents a finding of direct anticipation.

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2. U.S. Patent No. 7,473,767 B2 ("Dimitrov")

• Full Citation: US 7,473,767 B2
• Title: Polynucleotide probes for detecting and identifying analytes and methods of using the same
• Filing Date: May 19, 2005
• Publication Date: January 6, 2009
• Brief Description: This patent, foundational to NanoString's technology, describes a method using "nanoreporters" or "nanostrings." These are probes with a nucleic acid backbone containing a series of repeating units, each labeled with a different colored fluorescent molecule. A unique target is identified by the specific spatial order of these colors along the nucleic acid backbone, creating a "barcode." The probes are imaged after binding, and the sequence of colors along the probe's length identifies the analyte.
• Potential Anticipation of Claims:
  • Claim 1 & 12 (Method Claims): Dimitrov does not anticipate these claims because its detection method is fundamentally different. It relies on detecting a spatial code—the ordered sequence of different colors along a single probe, captured in a single image. The '737 patent claims a temporal code, where a series of signals is generated over time from the same location through sequential hybridization/detection/removal steps. The '737 patent's specification highlights this, stating that NanoString's technology is "based on determination of the 'spatial location of signals' ... rather than temporal detection of signals as described herein."
  • Claim 20 (Composition Claim): Dimitrov discloses a probe reagent linked to a nucleic acid label that acts as an identifier. However, the label described in Dimitrov is structured to present a spatially-ordered code of optical labels. The '737 patent's claim 20 requires a nucleic acid label comprising subsequences "to be detected in a temporally-sequential manner." Because Dimitrov's label is designed for spatial, not temporal, decoding, it would not anticipate this claim element.

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3. U.S. Patent No. 7,785,790 B1 ("Church")

• Full Citation: US 7,785,790 B1
• Title: In-situ sequencing
• Filing Date: January 24, 2007
• Publication Date: August 31, 2010
• Brief Description: This patent, which includes one of the same inventors (George M. Church), describes methods for determining nucleic acid sequences directly within a fixed sample (in situ). The core method involves amplifying nucleic acids (like mRNA) within a cell to create localized clusters of amplicons ("rolonies"). These rolony clusters are then sequenced in place, for example, by sequencing-by-ligation or sequencing-by-synthesis. This provides the sequence of the nucleic acid while preserving its original location in the cell or tissue.
• Potential Anticipation of Claims:
  • Claim 1 & 12 (Method Claims): Church '790 teaches in-situ analysis and sequential detection (as part of the sequencing process). However, it is focused on directly sequencing endogenous nucleic acids (like RNA) or synthetic DNA. It does not describe the key step of the '737 invention: using a pre-made detection reagent (e.g., an antibody) conjugated to a synthetic nucleic acid barcode to first target a non-nucleic acid analyte (like a protein), and then decoding that barcode to identify the protein. Church '790 is about identifying the nucleic acids that are already there, not using nucleic acids as labels to identify other types of molecules. This difference in purpose and method means it would not anticipate claims 1 or 12.
  • Claim 20 (Composition Claim): This patent does not describe or suggest the claimed detection reagent—a probe reagent like an antibody conjugated to a nucleic acid label with subsequences that form an identifier. The subject matter is the in-situ analysis of nucleic acids themselves, not a composition for detecting other analytes via a nucleic acid barcode. Therefore, it does not anticipate claim 20.

Obviousness Analysis Under 35 U.S.C. § 103

This analysis evaluates whether the claimed invention in U.S. Patent 11,021,737 would have been obvious to a Person Having Ordinary Skill in the Art (PHOSITA) as of the priority date of December 22, 2011. An invention is considered obvious if the differences between the invention and the prior art are such that the invention as a whole would have been obvious to a PHOSITA at the time the invention was made.

Definition of a Person Having Ordinary Skill in the Art (PHOSITA)

As of late 2011, a PHOSITA in this field would possess a Ph.D. in molecular biology, biochemistry, or a related discipline, coupled with several years of experience in academic or industrial research. This individual would be well-versed in nucleic acid biochemistry, including hybridization, amplification, and sequencing technologies (both Sanger and next-generation methods). They would have practical knowledge of protein detection methods, such as immunohistochemistry and immunofluorescence, and would be familiar with standard bioconjugation techniques for linking nucleic acids to proteins like antibodies. The PHOSITA would also understand the scientific goal of highly multiplexed biomolecule analysis and the challenges of achieving it while preserving spatial context within a biological sample.

Obviousness Combination 1: Gunderson (US 2007/0231824) in view of Church (US 7,785,790)

A strong argument can be made that the independent claims of the '737 patent are rendered obvious by the combination of Gunderson and Church '790.

• Primary Reference: Gunderson (US 2007/0231824)
  Gunderson teaches the core detection methodology claimed in the '737 patent: a temporal code generated by the sequential application, detection, and removal of labeled "decoder probes" to identify unique nucleic acid tag sequences. This directly teaches the method of claims 1 and 12, including generating a "temporal order of the signal signatures" (claim 1) through repeated hybridization and removal steps (claim 12). However, Gunderson applies this method to decode a pre-fabricated, fixed microarray of beads before introducing a sample. It does not teach applying this method to soluble detection reagents bound to analytes within a biological sample.

• Secondary Reference: Church (US 7,785,790)
  Church '790 teaches the art of performing complex, multi-step, sequential nucleic acid analysis (specifically, sequencing) directly in situ within a fixed cell or tissue sample. This established the principle that the necessary enzymes, buffers, and oligonucleotides could successfully diffuse into and function within a complex biological matrix to provide spatially resolved nucleic acid information.

• Motivation to Combine and Reasonable Expectation of Success:
  By 2011, a primary goal in biology was to move from analyzing single molecules to analyzing many simultaneously (multiplexing) within their native context. Church '790 provided a breakthrough for in situ analysis of nucleic acids. A PHOSITA, aware of the need to apply similar high-plex spatial analysis to other molecules like proteins, would look for robust barcoding and decoding schemes.

  Gunderson provided just such a scheme—a powerful temporal decoding method capable of identifying thousands of tags. The motivation to combine these references would have been to take Gunderson's superior temporal decoding method "off the chip" and apply it to the in situ environment demonstrated to be viable by Church '790. Instead of decoding a static array of beads, the PHOSITA would be motivated to use Gunderson's method to decode probes that were freely binding to targets within a tissue sample. This combination would directly address the well-understood need for highly multiplexed in situ proteomics and transcriptomics.

  The PHOSITA would have had a reasonable expectation of success. Church '790 demonstrated that the microenvironment of a fixed cell was permeable to the reagents needed for sequential nucleic acid chemistry. Since Gunderson's method relies on basic hybridization, which is a less complex process than the enzymatic ligation or synthesis used by Church, it would be reasonably expected that Gunderson's decoder probes and buffers would also function effectively in an in situ setting.

• Conclusion for this Combination:

  • Claims 1 and 12 (Method Claims): The combination of Gunderson's temporal decoding method with the in situ application context taught by Church renders the method claims obvious. It would have been an obvious step to apply a known decoding technique (Gunderson) to a known environment for sequential analysis (Church) to achieve a desired and predictable result: highly multiplexed spatial analyte detection.
  • Claim 20 (Composition Claim): To practice the obvious method derived from this combination, one would need a suitable tool. This tool is a probe reagent (e.g., an antibody known to bind a protein of interest) conjugated to one of Gunderson's nucleic acid tags. The creation of such antibody-DNA conjugates was a routine technique known to the PHOSITA. Therefore, the detection reagent of claim 20 would have been obvious as the necessary and readily created composition for carrying out the obvious method.

Obviousness Combination 2: Church (US 7,785,790) in view of General Knowledge and Dimitrov (US 7,473,767)

An alternative argument starts with the problem Church '790 was trying to solve—multiplexed spatial analysis—and combines it with other known labeling technologies.

• Primary Reference: Church (US 7,785,790)
  Church establishes the goal and feasibility of high-plex in situ analysis but is limited to sequencing endogenous nucleic acids. A PHOSITA would immediately be motivated to extend this powerful technique to proteins.

• Secondary References: General Knowledge of Immunoassays and Dimitrov (US 7,473,767)
  The most common tool for specific protein detection is an antibody. The general knowledge in the art was to attach a label to an antibody to make it detectable. To adapt the sequencing-based detection of Church to proteins, the obvious label would be a synthetic nucleic acid barcode.

  Dimitrov teaches the use of such a "nanostring" probe: a probe reagent linked to a nucleic acid backbone that serves as a barcode. While Dimitrov's barcode is read spatially, it teaches the fundamental concept of using a nucleic acid molecule as a high-capacity information carrier to identify a probe.

• Motivation to Combine and Reasonable Expectation of Success:
  A PHOSITA seeking to adapt Church's in situ method for proteins would obviously arrive at using an antibody conjugated to a nucleic acid barcode, a concept explicitly taught by Dimitrov. The final step would be to decide how to "read" the barcode in situ. While one could use the sequencing-by-ligation method from Church, it would also be obvious to consider other known methods for reading nucleic acid tags. As discussed in the first combination, Gunderson's temporal decoding is a well-documented and efficient method for this purpose. Therefore, it would have been obvious to a PHOSITA to:

  1. Start with the goal of in situ protein detection (an extension of Church).
  2. Select a DNA-barcoded antibody as the tool (taught by Dimitrov and general knowledge).
  3. Choose an efficient, known method for reading the DNA barcode (such as the temporal decoding from Gunderson).

  This logical progression of design choices, each step being well-supported by the prior art, would lead directly to the claimed invention with a reasonable expectation of success.

Patent Term Adjustments (PTA) and Patent Term Extensions (PTE)

The USPTO does not calculate patent expiration dates or provide official PTA/PTE figures directly in its publicly searchable databases. However, general principles can be applied:

• Patent Term Adjustment (PTA): PTA compensates applicants for certain administrative delays by the USPTO during patent prosecution (35 U.S.C. § 154(b)). These delays can include the USPTO failing to: issue a first Office Action or notice of allowance within 14 months of filing; respond to an applicant's reply within four months; or issue the patent within four months of the issue fee payment. PTA can also be granted if the patent is not issued within three years of its actual filing date. Applicant delays can reduce any awarded PTA.
• Patent Term Extension (PTE): PTE is distinct from PTA and is granted to compensate for delays incurred in obtaining regulatory approval for a patented product, particularly pharmaceuticals (35 U.S.C. § 156).

Given that US 11021737 relates to compositions and methods for analyte detection, which could be relevant to diagnostic products, a PTE might be sought if a product covered by the patent requires regulatory approval. Without access to the specific prosecution history and any potential regulatory approval details, the exact PTA and PTE for US 11021737 cannot be definitively stated.

Continuation, Divisional, and Related Family Members

To provide a comprehensive list of continuation and divisional applications, as well as related family members, a direct search of the USPTO Patent Center or other patent family databases is necessary. Based on the information available:

• Application Number: US16/941,585
• Publication Number (pre-grant): US20200354774A1
• Priority Date: December 22, 2011

The Google Patents entry for US 11021737 lists several "Other versions" and "Priority to" entries, which typically indicate related family members and continuations/divisionals. Based on this information:

• US20200354774A1: This is the pre-grant publication of US 11021737.
• Continuation Applications: The patent document itself indicates several "Priority to" entries which are US application numbers. These could represent continuation or divisional applications.
  • US17/122,168 (which issued as US11111521B2)
  • US17/238,642 (which issued as US11293051B2)
  • US17/238,682 (which issued as US11293052B2)
  • US17/366,151 (which issued as US11566276B2)
  • US17/366,127 (which issued as US11549136B2)
  • US17/498,829 (which published as US20220025448A1)
  • US17/584,959 (which issued as US11639518B2)
  • US17/671,803 (which issued as US11566277B2)
  • US17/664,095 (which published as US20220282301A1)
  • US18/064,956 (which issued as US11976318B2)
  • US18/146,483 (which published as US20230146985A1)
  • US18/459,068 (which published as US20240018569A1)
  • US18/614,970 (which published as US20240240235A1)
  • US18/936,042 (which published as US20250059590A1)
  • US18/989,119 (which published as US20250122556A1)
  • US18/989,169 (which published as US20250129408A1)

These applications generally represent continuations, continuations-in-part, or divisional applications, all of which claim priority back to an earlier application (in this case, likely US16/941,585 or its earliest priority application). A continuation application shares the same disclosure but pursues different claims, while a divisional application arises from a restriction requirement where a single application contains two or more independent inventions. A continuation-in-part (CIP) adds new subject matter while retaining some of the parent disclosure.

• International/PCT Applications:

  • PCT/US2012/071398 (Priority claimed from 2012-12-21, published as WO2013096851A1)
  • PCT/US2014/018580 (Priority claimed from 2014-02-26, published as WO2014163886A1)

• Foreign Equivalents:

  • ES23174395T (Priority to 2012-12-21, published as ES2991004T3)

Projected Expiration Date

The term of a U.S. utility patent (other than a design patent) is generally 20 years from the filing date of the earliest non-provisional application for which a benefit is claimed under 35 U.S.C. 120, 121, or 365(c).

US 11021737 claims priority from an application filed on December 22, 2011 (as indicated by the "Prior art date" and "Priority date" on the Google Patents page, which likely refers to an earlier application in the family). Assuming this date (December 22, 2011) is the earliest priority date from which the patent claims benefit, the base expiration date would be 20 years from this date.

Therefore, the anticipated expiration date, before any PTA or PTE, would be December 22, 2031.

However, the Google Patents page for US 11021737 explicitly lists an "Anticipated expiration" date of 2032-12-21. This suggests that at least one year of Patent Term Adjustment (PTA) has been granted to the patent, pushing the expiration beyond the standard 20 years from the earliest priority date. Without direct access to the USPTO's Patent Center for a detailed calculation, the date provided by Google Patents will be used as the projected expiration.

This Defensive Disclosure document aims to define prior art that would render future incremental improvements on US Patent 11,021,737 "obvious" or "non-novel" to a Person Having Ordinary Skill in the Art (PHOSITA). The analysis focuses on deriving variations from the core independent claims (Claim 1, Claim 12, and Claim 20) of the patent.

Derivative Variations of US Patent 11,021,737

The following derivatives explore alternative materials, operational scales, cross-domain applications, integration with emerging technologies, and inverse/failure modes for the methods and compositions described in US Patent 11,021,737.

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Derivatives for Claim 1 (Method for detecting a plurality of analytes in a sample):

Claim 1 describes a method involving contacting a sample with detection reagents (probe + nucleic acid label with subsequences) and detecting these subsequences in a temporally-sequential manner to identify analytes.

Derivative 1.1: All-RNA Detection System

• Enabling Description: Instead of DNA nucleic acid labels, employ RNA oligonucleotides for the nucleic acid labels attached to probe reagents. The probe reagents (e.g., antibodies, aptamers) would be conjugated to single-stranded RNA sequences containing the predetermined subsequences. Detection would involve RNA-RNA hybridization with complementary decoder RNA probes, or RNA-DNA hybridization with DNA decoder probes. Ribonucleases (RNases) with specific cleavage sites could be engineered into the RNA labels or decoder probes for signal removal/displacement, replacing DNA-specific enzymes like USER. The matrix material for in-situ applications could be an alginate hydrogel optimized for RNA stability, perhaps with RNase inhibitors, allowing for the preservation of fragile native RNA analytes.
• Mermaid Diagram:

  sequenceDiagram
      participant Sample
      participant RNA_Probe_Reagent as Detection Reagent (Probe+RNA Label)
      participant RNA_Decoder_Probes as RNA Decoder Probes
      participant Imaging_System as Imaging System
      
      Note over Sample, RNA_Probe_Reagent: Contact sample with RNA detection reagents
      RNA_Probe_Reagent->>Sample: Bind to target analyte
      Note over RNA_Decoder_Probes: Apply Set 1 of RNA decoder probes (labeled)
      RNA_Decoder_Probes->>RNA_Probe_Reagent: Hybridize to subsequence 1
      Imaging_System->>RNA_Decoder_Probes: Detect Signal 1
      Note over RNA_Decoder_Probes: Remove Set 1 RNA decoder probes (e.g., RNase cleavage, heat)
      Note over RNA_Decoder_Probes: Apply Set 2 of RNA decoder probes (labeled)
      RNA_Decoder_Probes->>RNA_Probe_Reagent: Hybridize to subsequence 2
      Imaging_System->>RNA_Decoder_Probes: Detect Signal 2
      Note over Imaging_System: Continue sequentially for all subsequences
      Imaging_System->>Imaging_System: Generate temporal order of signals
      Imaging_System->>RNA_Probe_Reagent: Identify probe reagent / analyte
  

Derivative 1.2: High-Throughput Microfluidic System for Industrial-Scale Bioprocessing Monitoring

• Enabling Description: The method is scaled for real-time, high-throughput monitoring of bioprocessing vats (e.g., 10,000L fermenters). Samples (e.g., microbial cell suspensions, culture media) are continuously drawn into a microfluidic device, featuring channels approximately 50-100 µm wide. Detection reagents are introduced at high flow rates (e.g., 1-10 mL/min). The detection reagents are designed with highly stable (e.g., LNA-modified) nucleic acid labels to withstand harsh industrial conditions (e.g., pH 4-9, temperatures up to 60°C). Detection occurs within a dedicated "detection chamber" with multiple, spatially offset laser excitation and detection zones, allowing for rapid, pseudo-simultaneous temporal decoding across a continuous flow. The temporal detection sequence for each reagent must be completed within milliseconds as particles flow through the detection zone. The system uses high-frequency pulsed lasers (e.g., 10 kHz) and synchronized high-speed cameras (e.g., 10,000 frames/sec) to capture rapid signal changes. Data processing involves real-time algorithmic identification of temporal signal patterns from thousands of individual reagents per second.
• Mermaid Diagram:

  graph TD
      A[Bioprocess Vat] --> B(Microfluidic Sample Inlet)
      B --> C{Mixing Chamber: Sample + Detection Reagents}
      C --> D[Microfluidic Flow Channel]
      D -- High-Speed Flow --> E1(Detection Zone 1: Laser Excitation + Camera)
      E1 -- Milliseconds Later --> E2(Detection Zone 2: Wash + Next Decoder Probe Application)
      E2 -- Milliseconds Later --> E3(Detection Zone 3: Laser Excitation + Camera)
      E3 -- Sequential Operation --> E_N(Detection Zone N)
      E_N --> F[High-Speed Data Processing & Analysis]
      F --> G{Real-time Analyte Identification & Quantification}
      F --> H(Process Control System)
      H --> A
  

Derivative 1.3: Environmental Contaminant Monitoring in Water Treatment (Cross-Domain)

• Enabling Description: The method is applied to detect a plurality of specific environmental contaminants (analytes) in large-volume water samples (e.g., municipal water treatment plants, industrial effluent). Probe reagents would include: 1. Aptamers engineered to bind specific heavy metal ions (e.g., Pb2+, Hg2+). 2. Antibodies targeting residues of pesticides (e.g., glyphosate, atrazine) or pharmaceuticals (e.g., ibuprofen, diclofenac). 3. Bacteriophages/Antibodies targeting specific bacterial pathogens (e.g., E. coli O157:H7, Legionella pneumophila). These probe reagents are conjugated to unique nucleic acid labels. Water samples are continuously passed through a filtration system to concentrate particulate matter (e.g., bacteria, microplastics with adsorbed contaminants) or directly introduced for dissolved analytes. Detection reagents are added, allowed to bind, and unbound reagents are flushed. The remaining bound reagents are then subjected to temporal decoding using fluorescently-labeled decoder probes and automated fluidic systems. The generated temporal signatures indicate the presence and concentration of various contaminants.
• Mermaid Diagram:

  flowchart TD
      A[Water Sample Inlet] --> B{Filtration/Concentration (Optional)}
      B --> C[Mixing Chamber: Sample + Detection Reagents]
      C --> D{Incubation for Analyte Binding}
      D --> E{Wash: Remove Unbound Reagents}
      E --> F[Automated Fluidic System for Temporal Decoding]
      F -- Step 1: Add Decoder Probe Set 1 --> G1(Image Capture 1)
      G1 -- Remove Signal 1 --> F
      F -- Step 2: Add Decoder Probe Set 2 --> G2(Image Capture 2)
      G2 -- ... --> F
      F -- Final Step: Add Decoder Probe Set N --> GN(Image Capture N)
      GN --> H[Data Analysis: Temporal Signature Identification]
      H --> I{Contaminant Report: Type & Concentration}
      I --> J(Alert System for Water Quality)
  

Derivative 1.4: Livestock Disease Monitoring (AgriTech - Cross-Domain)

• Enabling Description: The method is used for rapid, multiplexed detection of pathogens and stress markers in livestock. Samples include blood, saliva, milk, or fecal matter from individual animals or pooled samples from herds. Probe reagents include antibodies or aptamers specific to: 1. Viral antigens, e.g., Foot-and-mouth disease virus (FMDV) proteins, Avian Influenza H5N1 proteins. 2. Bacterial toxins, e.g., E. coli enterotoxins, Salmonella LPS. 3. Hormones/Metabolites, e.g., cortisol as a stress indicator, specific inflammatory cytokines. Detection reagents with their unique nucleic acid labels are applied to prepared samples (e.g., on a microarray slide for pooled samples or directly in a microfluidic cartridge for individual animal diagnosis). After binding and washing, automated temporal decoding identifies the presence and relative abundance of disease agents or stress markers, enabling early intervention and herd management.
• Mermaid Diagram:

  flowchart LR
      A[Animal Sample (Blood/Saliva/Milk)] --> B{Sample Preparation (e.g., Centrifugation, Lysis)}
      B --> C[Microarray/Microfluidic Device]
      C --> D{Contact with Detection Reagents}
      D -- Bind to Analytes --> E{Wash Unbound Reagents}
      E --> F[Automated Temporal Decoding Module]
      F -- Cycle 1: Hybridize Decoder 1 --> G1(Capture Image 1)
      G1 -- Remove Signal 1 --> F
      F -- Cycle 2: Hybridize Decoder 2 --> G2(Capture Image 2)
      G2 -- ... --> F
      F --> H[Data Analysis: Disease/Stress Marker ID]
      H --> I{Veterinary Diagnostic Report}
      I --> J(Herd Management Decision Support)
  

Derivative 1.5: Authenticity Verification in Luxury Goods (Consumer Electronics/Supply Chain - Cross-Domain)

• Enabling Description: To combat counterfeiting, luxury goods (e.g., designer handbags, high-end electronics components) are invisibly marked with microscopic, unique "authenticity tags." These tags are composed of an inert polymer particle (e.g., nanodiamond, quantum dot embedded polymer bead) acting as the "probe reagent" (though not binding to an analyte, it is the item of interest) and are surface-conjugated with multiple identical, custom-synthesized nucleic acid labels. Each batch or individual item receives tags with a unique "temporal barcode" encoded in its nucleic acid labels. For verification, a minute sample is taken from the item (e.g., by swab, micro-abrasion) and introduced into a portable detection device. Detection reagents (here, the authenticity tags) are then analyzed via temporal decoding of their nucleic acid labels using fluorescent decoder probes. The detected temporal sequence is compared against a secure database of legitimate product codes. This allows for rapid, unambiguous authentication, even for high volumes of goods. The "analyte" is the unique authenticity tag itself.
• Mermaid Diagram:

  graph TD
      A[Luxury Good] --> B(Micro-sampling/Swab)
      B --> C[Portable Detection Device]
      C --> D{Isolate Authenticity Tags}
      D --> E[Automated Fluidics: Temporal Decoding]
      E -- Cycle 1: Add Decoder Probe Set 1 --> F1(Image Capture 1)
      F1 -- Remove Signal 1 --> E
      E -- Cycle 2: Add Decoder Probe Set 2 --> F2(Image Capture 2)
      F2 -- ... --> E
      E --> G[Decode Temporal Barcode]
      G --> H{Compare with Secure Blockchain Database}
      H -- Match --> I(Authentic Product)
      H -- No Match --> J(Counterfeit Detected)
  

Derivative 1.6: AI-Optimized Real-time Spatial Profiling (Integration with Emerging Tech)

• Enabling Description: The method is integrated with an AI-driven imaging and fluidics control system for advanced spatial biology. IoT sensors (e.g., embedded temperature, pH, fluid flow sensors) in a microfluidic chip provide real-time environmental data. An AI agent (e.g., deep reinforcement learning model) dynamically optimizes the experimental parameters during temporal detection: 1. Decoder Probe Incubation Times: Adjusted based on real-time binding kinetics and signal intensity observed. 2. Wash Stringency: Optimized based on unspecific binding levels and desired removal efficiency. 3. Fluorophore Excitation Power/Duration: Minimized to prevent photobleaching while ensuring adequate signal-to-noise. 4. Z-stack Imaging Depth/Frequency: Adjusted based on tissue heterogeneity and signal localization. The AI processes raw image data (e.g., 3D fluorescent images) in real-time, performing signal deconvolution, drift correction, and temporal pattern recognition. It then reconstructs a 3D spatial map of multiple analytes, presenting quantification and co-localization data. This autonomous system significantly improves detection speed, accuracy, and resolution, especially for complex or sparse analyte distributions.
• Mermaid Diagram:

  graph TD
      A[Biological Sample (Tissue Slice)] --> B{Microfluidic Chamber with IoT Sensors}
      B --> C[Detection Reagents]
      C -- Bind Analytes --> D[Automated Fluidics Control]
      D -- Apply/Wash Decoder Probes --> E[High-Res 3D Imaging System]
      E -- Real-time Image Data --> F(AI Optimization Engine)
      F -- Environmental Data --> F
      F -- Control Signals --> D
      F --> G[Signal Deconvolution & Temporal Pattern Recognition]
      G --> H[3D Spatial Map of Analytes]
      H --> I(Visualization & Quantification Interface)
  

Derivative 1.7: Blockchain for Supply Chain Provenance of Biomolecules (Integration with Emerging Tech)

• Enabling Description: The patent's method is adapted to verify the provenance and integrity of critical biomolecule reagents (e.g., antibodies, enzymes, synthetic oligonucleotides) throughout a cold chain supply. Each batch of a biomolecule is internally "barcoded" by conjugating a small, known fraction of the biomolecule to a specific nucleic acid label with a unique temporal signature. This signature is then recorded as a transaction on a private blockchain. At various checkpoints in the supply chain (e.g., manufacturing, shipping, distribution, end-user QC), a small sample of the reagent is taken, and its internal nucleic acid labels are temporally decoded. The resulting temporal signature is hashed and compared to the blockchain record. Any discrepancy (e.g., incorrect signature, missing signature, degradation leading to altered signal intensity) flags a potential counterfeit, contamination, or breach in the cold chain. This provides an immutable, verifiable audit trail.
• Mermaid Diagram:

  sequenceDiagram
      actor Manufacturer
      participant ReagentBatch as Biomolecule Reagent Batch
      participant DNA_Labeling as DNA Barcoding Module
      participant Blockchain as Blockchain Network
      participant QC_Check1 as QC Checkpoint 1
      participant QC_CheckN as QC Checkpoint N
      actor EndUser
      
      Manufacturer->>ReagentBatch: Produce Biomolecule
      DNA_Labeling->>ReagentBatch: Conjugate unique SeqTag to aliquot
      DNA_Labeling->>Blockchain: Record SeqTag Hash (Batch ID, Timestamp)
      ReagentBatch->>QC_Check1: Ship to Distributor
      Note over QC_Check1: Sample & Decode SeqTag (Claim 1 method)
      QC_Check1->>Blockchain: Verify SeqTag Hash
      alt Verification Fails
          QC_Check1->>Blockchain: Record Tamper Event
          QC_Check1->>Manufacturer: Alert: Integrity Compromised
      else Verification Succeeds
          QC_Check1->>ReagentBatch: Continue Shipment
      end
      ...
      ReagentBatch->>QC_CheckN: Ship to End User
      QC_CheckN->>Blockchain: Verify SeqTag Hash
      EndUser->>Blockchain: Final Verification
  

Derivative 1.8: Self-Deactivating/Limited-Functionality Diagnostic for Point-of-Care (The "Inverse" or Failure Mode)

• Enabling Description: Develop a point-of-care (POC) diagnostic kit using the temporal detection method, designed to operate in a limited-functionality mode after initial use or to self-deactivate for safe disposal and privacy. The nucleic acid labels on the detection reagents are engineered with multiple cleavable sites (e.g., UV-cleavable linkers, specific restriction enzyme sites) interspersed within the subsequences or at the probe-label conjugation point. After a predetermined number of detection cycles (e.g., sufficient for a single diagnostic readout, say 3 cycles), a "deactivation reagent" (e.g., a UV light pulse, a specific enzyme solution) is automatically introduced. This reagent cleaves the nucleic acid labels or the linker, irreversibly destroying the barcode information and preventing further or unauthorized detection. In a "low-power" mode, only the first 1-2 subsequences are decoded, providing a rapid "yes/no" or "high/low" qualitative result, consuming fewer reagents and less power, before self-deactivation. This ensures patient data privacy and prevents re-use of diagnostic components.
• Mermaid Diagram:

  stateDiagram-v2
      [*] --> Initialized: Kit Ready
      Initialized --> Sample_Added: Sample Contact
      Sample_Added --> Analyte_Binding: Detection Reagents Bind
      Analyte_Binding --> Temporal_Decoding_Cycle_1: Start Decoding
      Temporal_Decoding_Cycle_1 --> Signal_Capture_1: Detect Subsequence 1
      Signal_Capture_1 --> Decoder_Removal_1: Remove Decoder 1
      Decoder_Removal_1 --> Temporal_Decoding_Cycle_2: If More Cycles Needed
      Temporal_Decoding_Cycle_2 --> Signal_Capture_2: Detect Subsequence 2
      Signal_Capture_2 --> Decoder_Removal_2: Remove Decoder 2
      Decoder_Removal_2 --> Temporal_Decoding_Cycle_N: If More Cycles Needed (N <= Max Cycles)
      Temporal_Decoding_Cycle_N --> Final_Signal_Capture: Final Detection
      Final_Signal_Capture --> Interpretation: Analyze Results
      Interpretation --> Deactivation_Triggered: Max Cycles Reached OR Readout Complete
      Deactivation_Triggered --> Barcode_Irreversible_Cleavage: Apply Deactivation Reagent
      Barcode_Irreversible_Cleavage --> [*]: Self-Deactivated / Dispose
      
      Temporal_Decoding_Cycle_1 --> Low_Power_Mode: (Optional) Limit Cycles
      Low_Power_Mode --> Limited_Readout: Fast Qualitative Result
      Limited_Readout --> Deactivation_Triggered
  

Derivative 1.9: Quantum Dot-Encoded Decoder Probes with Hyperspectral Imaging (Material & Component Substitution)

• Enabling Description: Instead of organic fluorescent dyes, the decoder probes are labeled with quantum dots (QDs). Each set of decoder probes uses QDs emitting at distinct, narrow wavelengths when excited by a broad-spectrum light source, or uses QDs with varying excitation/emission characteristics that are distinguishable. For instance, a set of 4 decoder probes might use QDs emitting at 520 nm, 580 nm, 620 nm, and 680 nm. Detection is performed using a hyperspectral imaging system capable of resolving multiple distinct emission spectra simultaneously in each temporal step. This provides higher spectral resolution, greater photostability, and reduced bleed-through compared to traditional fluorophores, allowing for more multiplexing within a single temporal step and more robust signal detection over many cycles. Signal removal could involve UV degradation of the QD's surface passivation layer, or enzymatic cleavage of a linker attaching the QD to the probe.
• Mermaid Diagram:

  graph TD
      A[Detection Reagents Bound to Analytes] --> B{Apply QD Decoder Probes (Set 1)}
      B --> C[Hyperspectral Imaging System]
      C -- Capture Multiple Wavelengths --> D{Detect Temporal Signal 1 (Spectral Signature)}
      D --> E{Remove QD Decoder Probes 1}
      E --> F{Apply QD Decoder Probes (Set 2)}
      F --> C
      C -- Capture Multiple Wavelengths --> G{Detect Temporal Signal 2 (Spectral Signature)}
      G --> H[Temporal Sequence of Spectral Signatures]
      H --> I(Analyte Identification)
  

Derivative 1.10: Ultra-low Temperature/Cryogenic Sample Processing (Operational Parameter Expansion)

• Enabling Description: The method is adapted for detecting analytes in cryogenically preserved samples, or performing detection steps at ultra-low temperatures (e.g., -80°C to -196°C, within a cryostat). This would be beneficial for preserving delicate analytes, minimizing diffusion, or studying cellular processes at extremely low metabolic rates. The detection reagents and decoder probes would need to be synthesized with cryo-compatible modifications (e.g., increased GC content for nucleic acids to maintain hybridization strength at lower temperatures, specific cryoprotectant formulations). The matrix material for in-situ applications would be a cryogel or cryo-resistant polymer (e.g., highly cross-linked polyacrylamide with glycerol). Fluidics systems would need to handle cryogenic liquids (e.g., ethanol, specialized buffers). Imaging systems would be integrated into the cryostat, using specialized optics for low-temperature environments. Signal removal (e.g., displacement hybridization) would be optimized for slow diffusion kinetics at these temperatures.
• Mermaid Diagram:

  flowchart TD
      A[Cryo-preserved Sample] --> B{Cryostat Integration & Sample Thawing/Preparation (if needed)}
      B --> C[Cryo-compatible Detection Reagents]
      C -- Bind Analytes at Low Temp --> D{Cryo-Fluidics: Wash Unbound Reagents}
      D --> E[Cryo-Imaging Module (Integrated within Cryostat)]
      E -- Cycle 1: Apply Cryo-Decoder 1 --> F1(Cryo-Image 1)
      F1 -- Remove Signal 1 (optimized for cryo) --> E
      E -- Cycle N: Apply Cryo-Decoder N --> FN(Cryo-Image N)
      FN --> G[Low-Temperature Data Analysis]
      G --> H(Analyte Detection Report)
  

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Derivatives for Claim 12 (Method for detecting via hybridization and removal):

Claim 12 further specifies the detection method of Claim 1, detailing the sequential hybridization, detection, and optional removal of decoder probes.

Derivative 12.1: Self-Assembling DNA-Origami Decoder Probes (Material & Component Substitution)

• Enabling Description: Instead of linear oligonucleotide decoder probes, utilize complex DNA-origami structures as decoder probes. Each DNA-origami structure is precisely engineered to present multiple copies of a specific hybridization sequence complementary to a subsequence on the detection reagent's nucleic acid label. The origami structures also integrate multiple copies of specific, spatially arranged detectable labels (e.g., a cluster of 10 fluorescent molecules or a single, large quantum dot). This multivalency significantly increases binding affinity and signal intensity. Signal removal could involve inducing a structural change in the DNA origami (e.g., via a "trigger" oligonucleotide that causes dissociation) or enzymatic degradation specifically targeting the origami structure, leaving the detection reagent intact.
• Mermaid Diagram:

  classDiagram
      class DetectionReagent {
          +ProbeReagent
          +NucleicAcidLabel (w/ Subsequences)
      }
      
      class DNA_Origami_Decoder {
          +HybridizationSequence[]
          +DetectableLabel[]
          +StructuralTriggerSite
      }
      
      DetectionReagent "1" -- "1" NucleicAcidLabel
      NucleicAcidLabel "1" -- "*" Subsequence
      DNA_Origami_Decoder "1" -- "*" HybridizationSequence
      DNA_Origami_Decoder "1" -- "*" DetectableLabel
      
      Note for DNA_Origami_Decoder "Increased signal strength and binding affinity due to multivalency."
  

Derivative 12.2: Millimeter-Wave Spectroscopy for Label Detection in Opaque Samples (Operational Parameter Expansion)

• Enabling Description: For detection in optically opaque or highly scattering samples (e.g., dense tissue, whole blood, turbid fermentation broth), replace optical labels and fluorescence imaging with labels detectable by millimeter-wave (MMW) spectroscopy. Decoder probes are conjugated to specific MMW-resonant tags (e.g., metamaterial resonators, specially engineered nanoparticles with distinct dielectric properties, or molecular tags exhibiting unique rotational spectra in the MMW range). The detectable "signal signature" is a unique MMW absorption or reflection spectrum. A MMW transceiver system scans the sample, and the temporal order of detected MMW signatures identifies the analytes. Signal removal could involve chemical degradation of the MMW tags or altering their resonant properties via a pH/redox shift. This allows for deep penetration and detection without optical transparency requirements.
• Mermaid Diagram:

  graph LR
      A[Opaque Sample] --> B(Detection Reagents Bound)
      B --> C{MMW Transceiver System}
      C -- Emit MMW Signal --> D[MMW-Resonant Decoder Probes (Set 1)]
      D -- Absorb/Reflect MMW --> C
      C --> E{Detect MMW Signal 1 (Spectral Signature)}
      E --> F{Remove MMW Decoder Probes 1}
      F --> G{Apply MMW-Resonant Decoder Probes (Set 2)}
      G --> C
      C --> H{Detect MMW Signal 2 (Spectral Signature)}
      H --> I[Temporal Sequence of MMW Signatures]
      I --> J(Analyte Identification in Opaque Medium)
  

Derivative 12.3: Environmental Soil Contaminant Mapping (Geospatial/Environmental - Cross-Domain)

• Enabling Description: The method is used to create high-resolution 3D maps of contaminants within soil or sediment cores. Soil cores are collected, stabilized with a porous polymer (e.g., modified polyacrylamide gel to maintain structure), and then sectioned. Detection reagents (e.g., antibodies/aptamers against heavy metals, pesticides, petroleum hydrocarbons, radionuclides) are diffused into the soil sections. After binding and washing, sequential hybridization and removal of colorimetric or chemiluminescent decoder probes are performed. An automated imaging system captures the temporal signal signatures across the section, allowing for a detailed 3D reconstruction of contaminant distribution and concentration. This enables targeted remediation efforts.
• Mermaid Diagram:

  flowchart TD
      A[Soil Core Sample] --> B{Porous Polymer Stabilization & Sectioning}
      B --> C[Diffusion Chamber: Add Detection Reagents]
      C --> D{Incubation & Wash}
      D --> E[Automated Planar Imaging System]
      E -- Cycle 1: Apply Colorimetric Decoder 1 --> F1(Color Image 1)
      F1 -- Chem. Remove Signal 1 --> E
      E -- Cycle N: Apply Colorimetric Decoder N --> FN(Color Image N)
      FN --> G[3D Reconstruction & Contaminant Map]
      G --> H(Targeted Remediation Planning)
  

Derivative 12.4: Robotic Automation and Digital Twin Modeling (Integration with Emerging Tech)

• Enabling Description: The entire temporal detection process (fluidics, imaging, signal removal) is performed by a fully autonomous robotic platform. This robot operates within a sealed environment, handling delicate biological samples (e.g., brain slices, organoids). A digital twin of the biological sample and the experimental setup is created in real-time. This digital twin precisely models fluid dynamics, reagent diffusion, and light interaction within the sample. AI algorithms use this digital twin to predict optimal decoder probe concentrations, incubation times, and wash cycles for each specific tissue region, adapting to local variations. The temporal sequence of images is captured, processed, and immediately integrated into the digital twin, allowing for interactive, multi-dimensional analysis and simulation of experimental outcomes.
• Mermaid Diagram:

  sequenceDiagram
      actor Robotic_Platform
      participant Sample_Handling as Robotic Sample Handler
      participant Fluidics_Module as Automated Fluidics
      participant Imaging_Module as High-Res Imaging
      participant Digital_Twin_AI as Digital Twin & AI Optimizer
      
      Robotic_Platform->>Sample_Handling: Load Sample
      Sample_Handling->>Fluidics_Module: Deliver Detection Reagents
      Fluidics_Module->>Sample_Handling: Incubate & Wash
      loop Temporal Decoding Cycles
          Fluidics_Module->>Imaging_Module: Apply Decoder Probe Set (N)
          Imaging_Module->>Digital_Twin_AI: Stream Real-time Images
          Digital_Twin_AI->>Digital_Twin_AI: Update Digital Twin State
          Digital_Twin_AI->>Fluidics_Module: Adjust Fluidics/Imaging Parameters (AI Optimization)
          Fluidics_Module->>Sample_Handling: Remove Decoder Probe Set (N)
      end
      Digital_Twin_AI->>Robotic_Platform: Output 3D Analyte Map
  

Derivative 12.5: "Sentinel" Detection Reagents with Early Warning Degradation (The "Inverse" or Failure Mode)

• Enabling Description: Incorporate "sentinel" detection reagents into a multiplex assay. These sentinel reagents are designed to bind a common, stable cellular component (e.g., actin, ribosomal RNA) but have nucleic acid labels that are intentionally fragile or contain known degradation sites. For example, specific phosphodiester bonds within the subsequences are engineered to be hyper-sensitive to nucleases, or the detectable labels on the decoder probes are unusually prone to photobleaching. If the assay conditions (e.g., storage, washing, reagent quality) become suboptimal, these sentinel reagents will show a disproportionately rapid degradation of their temporal signal, or a distorted temporal sequence (e.g., certain steps fail prematurely). This provides an early warning indicator that the overall assay integrity is compromised, allowing for re-running the assay or flagging potentially unreliable results.
• Mermaid Diagram:

  stateDiagram-v2
      [*] --> Assay_Initialized
      Assay_Initialized --> Normal_Operation: Sentinel Signal Stable
      Normal_Operation --> Decoding_InProgress: Perform Multiplex Detection
      Decoding_InProgress --> Sentinel_Monitor_Active
      Sentinel_Monitor_Active --> Degradation_Threshold_Exceeded: Sentinel Signal Fails/Distorts
      Degradation_Threshold_Exceeded --> Alert_System: Integrity Compromised!
      Alert_System --> Rerun_Assay: Action Required
      Rerun_Assay --> Assay_Initialized
      Normal_Operation --> Results_Generated: All Signals Normal
      Results_Generated --> [*]: Report Valid Results
  

---

Derivatives for Claim 20 (Detection reagent):

Claim 20 defines a detection reagent comprising a probe reagent conjugated to a nucleic acid label with at least two different pre-determined subsequences for temporal detection.

Derivative 20.1: Universal Protein Scaffold for Nucleic Acid Label Attachment (Material & Component Substitution)

• Enabling Description: Instead of directly conjugating nucleic acid labels to diverse probe reagents (e.g., antibodies), a universal protein scaffold (e.g., SpyCatcher/SpyTag system, Affibody, Designed Ankyrin Repeat Protein (DARPin) based) is employed. This scaffold is engineered to have a high affinity binding site for a broad range of biotinylated probe reagents (e.g., via streptavidin fusion) on one end, and multiple highly specific and addressable conjugation sites (e.g., orthogonal click chemistry sites, unnatural amino acid incorporation for site-specific reactions) for the nucleic acid labels on the other end. The nucleic acid label itself could be a PNA (Peptide Nucleic Acid) for enhanced stability and binding. This modular design allows for rapid assembly of various detection reagents and simplifies manufacturing.
• Mermaid Diagram:

  classDiagram
      class UniversalScaffold {
          +ProbeBindingSite
          +NucleicAcidLabelConjugationSite[]
      }
      
      class ProbeReagent {
          +TargetBindingDomain
          +BiotinTag
      }
      
      class NucleicAcidLabel {
          +PNA_Backbone
          +Subsequence[]
          +ClickChemistryHandle
      }
      
      ProbeReagent --o UniversalScaffold : Binds via Biotin/Streptavidin
      NucleicAcidLabel --o UniversalScaffold : Covalently conjugated via Click Chemistry
      UniversalScaffold "1" -- "*" NucleicAcidLabel : Multiple labels per scaffold
      UniversalScaffold "1" -- "*" ProbeReagent : Multiple probes per scaffold (if multivalent)
  

Derivative 20.2: High-Density Nanoparticle Core with Encapsulated Nucleic Acid Labels (Operational Parameter Expansion)

• Enabling Description: The "detection reagent" is designed around a high-density, sub-50 nm nanoparticle core (e.g., gold nanoparticle, silica nanoparticle, upconverting nanoparticle). This core provides a large surface area for conjugating multiple probe reagents (e.g., 100s of antibodies). Crucially, the nucleic acid labels are not surface-conjugated but are encapsulated within the porous matrix of the nanoparticle itself. These encapsulated nucleic acid labels are protected from enzymatic degradation and harsh environmental conditions. They are released only upon specific triggering (e.g., pH change, light activation, enzymatic degradation of a sacrificial shell) into the local microenvironment for subsequent temporal decoding. This allows for high payload, robust reagents, and controlled release kinetics.
• Mermaid Diagram:

  graph TD
      A[Nanoparticle Core (e.g., Silica)] -- Porous Matrix --> B(Encapsulated Nucleic Acid Labels)
      A -- Surface Conjugation --> C[Multiple Probe Reagents (e.g., Antibodies)]
      B -- Triggered Release (e.g., pH, UV) --> D{Local Environment}
      D -- Diffuse --> E[Decoder Probes]
      E -- Hybridize --> D
      D -- Detect Temporal Sequence --> F(Analyte Identification)
      
      subgraph Detection Reagent
          A
          C
      end
  

Derivative 20.3: Biofouling Detection in Marine Sensors (Oceanography - Cross-Domain)

• Enabling Description: Detection reagents are integrated into anti-biofouling coatings for marine sensors. The "probe reagent" is a synthetic peptide or polymer embedded within the coating matrix, designed to specifically bind to early-stage biofouling organisms (e.g., bacterial adhesion proteins, algal extracellular polymeric substances) or their unique metabolic byproducts. These peptides/polymers are conjugated to nucleic acid labels. When biofouling occurs, the detection reagents bind to the target. A miniaturized, submersible temporal decoding unit periodically passes over the coated surface, applies decoder probes, and detects the temporal signatures. This allows for early, specific detection of biofouling types, triggering targeted, minimal intervention (e.g., localized UV burst, low-frequency sound pulse) rather than widespread, environmentally harmful antifoulants.
• Mermaid Diagram:

  flowchart LR
      A[Marine Sensor Surface] --> B{Anti-Biofouling Coating}
      B --> C[Embedded Detection Reagents (Probe+SeqTag)]
      C -- Bind to Biofouling Analytes --> D{Submersible Decoding Unit}
      D -- Periodically Scan --> E[Automated Fluidics (Decoder Probes)]
      E -- Temporal Decoding (Claim 12) --> F[Onboard Data Analysis]
      F --> G{Biofouling Alert & Characterization}
      G --> H(Targeted Anti-Fouling Intervention)
  

Derivative 20.4: Bio-Synthetic Hybrid Reagents for Adaptive Sensing (Integration with Emerging Tech)

• Enabling Description: The detection reagent is a bio-synthetic hybrid. The "probe reagent" is a live, engineered bacterium or yeast cell (a "sensing microbe") that expresses a surface receptor specific for a target analyte (e.g., a heavy metal ion, a specific pollutant). Upon binding the analyte, this microbe initiates a genetic circuit that results in the surface expression or secretion of multiple copies of a synthetic nucleic acid label. This nucleic acid label, containing the pre-determined subsequences, then becomes accessible for temporal decoding. The system could be further enhanced with AI optimization, where the AI dynamically adjusts the temporal decoding parameters based on the sensing microbe's metabolic activity, reported by IoT sensors integrated with the culture. This creates a living, adaptive biosensor with a synthetic readout mechanism.
• Mermaid Diagram:

  graph LR
      A[Target Analyte] --> B(Engineered Sensing Microbe)
      B -- Surface Receptor Binding --> C{Genetic Circuit Activation}
      C -- Express/Secrete --> D[Nucleic Acid Label (on Microbe Surface)]
      D --> E[Temporal Decoding System (Claim 12)]
      E --> F(Analyte Detection & Quantification)
      
      subgraph Bio-Synthetic Hybrid Detection Reagent
          B
          D
      end
      
      subgraph AI_Integration
          G[IoT Sensors (Microbe Metabolism)] --> H(AI Optimization)
          H --> E
      end
  

Derivative 20.5: Reagent with Built-in Quality Control (QC) Barcode (The "Inverse" or Failure Mode)

• Enabling Description: Each detection reagent is designed with an additional, short "QC subsequence" embedded within its nucleic acid label, distinct from the analyte-identifying subsequences. This QC subsequence is designed to be highly sensitive to common reagent degradation pathways (e.g., oxidation, nuclease contamination, pH extremes). During the first temporal decoding cycle, a specific QC decoder probe is always hybridized and detected. A robust signal from this QC subsequence confirms the integrity and functionality of the detection reagent itself (i.e., its nucleic acid label is intact and capable of hybridization). If the QC signal is absent or below a threshold, the entire batch of detection reagents is flagged as compromised, preventing false positives or negatives from faulty reagents. This provides a built-in quality control check for every individual detection reagent used in the assay.
• Mermaid Diagram:

  flowchart LR
      A[Detection Reagent (Probe + Nucleic Acid Label)]
      Nucleic_Acid_Label_Structure(Nucleic Acid Label)
      Nucleic_Acid_Label_Structure -- Contains --> B(Analyte_ID_Subsequence_1)
      Nucleic_Acid_Label_Structure -- Contains --> C(Analyte_ID_Subsequence_2)
      Nucleic_Acid_Label_Structure -- Contains --> D(QC_Subsequence)
      
      A --> E{Temporal Decoding (First Step)}
      E -- Hybridize QC Decoder Probe --> F(Detect QC Signal)
      F -- Signal > Threshold? --> G{Reagent OK}
      F -- Signal < Threshold? --> H{Reagent Failed QC}
      
      G -- Proceed --> I(Continue Analyte ID Decoding)
      H -- Discard --> J(Invalid Assay Result)
  

Derivative 20.6: Thermoplastic Polymer Matrix for Reversible Immobilization (Material & Component Substitution)

• Enabling Description: For in-situ applications, instead of a thermoset hydrogel, utilize a thermoplastic polymer matrix (e.g., polymethyl methacrylate (PMMA), polycarbonate) that is micro-perforated or has precisely engineered pores. Nucleic acid labels are derivatized with a UV-cleavable crosslinker to functional groups within this thermoplastic matrix. The advantage is that the matrix can be reversibly softened or dissolved (e.g., by mild heating or specific solvent washing) after detection, allowing for retrieval of the sample or embedded cellular components, or for re-analysis under different conditions. The "detectable label" on the decoder probes could be a surface-enhanced Raman scattering (SERS) tag, providing narrow, photostable spectral signals.
• Mermaid Diagram:

  graph TD
      A[Biological Sample] --> B(Embed in Thermoplastic Matrix)
      B -- UV-cleavable Crosslinker --> C[Detection Reagents w/ Nucleic Acid Labels]
      C --> D{Temporal Detection (SERS Labels)}
      D --> E[Data Analysis & Analyte Map]
      E --> F{Reversible Matrix Dissolution/Softening}
      F --> G(Sample Retrieval / Further Analysis)
  

Derivative 20.7: High-Pressure/High-Temperature Sterilization for Biohazard Detection (Operational Parameter Expansion)

• Enabling Description: The detection reagent is engineered for applications in extreme environments requiring robust sterilization, such as biosafety level 4 (BSL-4) facilities or extraterrestrial sample analysis. The "probe reagent" (e.g., heat-stable aptamer, synthetic peptide) and the nucleic acid label (e.g., PNA, LNA) are designed to withstand high-pressure (e.g., >200 MPa) and high-temperature (e.g., >121°C for autoclave sterilization) conditions without degradation. The conjugation method (e.g., robust covalent linkages like amide bonds, silane chemistry) must also be resistant. The detectable labels on the decoder probes are also selected for thermal and pressure stability. This ensures the reagents remain functional even after rigorous sterilization protocols, crucial for handling highly infectious agents or sensitive extraterrestrial samples.
• Mermaid Diagram:

  stateDiagram-v2
      [*] --> Reagent_Synthesis
      Reagent_Synthesis --> High_Pressure_High_Temp_Sterilization
      High_Pressure_High_Temp_Sterilization --> Sterile_Storage
      Sterile_Storage --> Contact_with_Biohazard_Sample: In BSL-4 / Space Environment
      Contact_with_Biohazard_Sample --> Analyte_Binding
      Analyte_Binding --> Temporal_Decoding: Robust reagents maintain function
      Temporal_Decoding --> Results_Generated
      Results_Generated --> [*]
  

Derivative 20.8: Time-Limited Reagent Functionality for Controlled Diagnostics (The "Inverse" or Failure Mode)

• Enabling Description: The detection reagent is engineered with an inherent, controlled degradation mechanism that limits its functional lifetime. This is useful for diagnostics where a precise window of activity is desired or to prevent prolonged environmental presence. The nucleic acid label, or its linker to the probe reagent, is designed with a bio-degradable polymer segment (e.g., polylactic acid, polycaprolactone) whose degradation rate is precisely calibrated to ambient environmental factors (e.g., temperature, humidity, light exposure). After a specific period (e.g., 24-48 hours), the nucleic acid label detaches or degrades, rendering the detection reagent non-functional and preventing further signal generation. This ensures that the diagnostic information is only available for a defined period, preventing misinterpretation of aged results or environmental contamination.
• Mermaid Diagram:

  graph TD
      A[Detection Reagent Synthesized] --> B(Degradable Linker/Component)
      B -- Initial State --> C[Active Analyte Binding]
      C --> D[Active Temporal Decoding]
      D -- Time Progresses --> E{Degradable Component Breakdown}
      E -- Leads to --> F(Nucleic Acid Label Loss/Degradation)
      F --> G[Reagent Non-Functional]
      G --> H(Dispose Safely)
  

---

Combination Prior Art Scenarios with Open-Source Standards

These scenarios illustrate how the core concepts of US 11,021,737 could be rendered obvious when combined with existing open-source standards and practices known to a PHOSITA prior to the patent's priority date (December 22, 2011).

Combination Prior Art Scenario 1: '737 Patent + Open-Source Image Processing Libraries (e.g., OpenCV, scikit-image)

• Enabling Description: The core of the '737 patent involves capturing a temporal sequence of images (e.g., fluorescent images, as mentioned in the patent) from a sample. An obvious improvement or application would be to process these images using widely available and well-documented open-source image processing libraries. For example, a PHOSITA would use OpenCV or scikit-image to perform:
  1. Image Registration: Aligning sequential images to correct for sample drift (explicitly mentioned as a challenge in the '737 patent: "position shift, such as less than 1 µm per amplicon"). Standard algorithms like phase correlation, SIFT, or ORB feature matching are readily available in these libraries.
  2. Noise Reduction: Applying Gaussian filters, median filters, or more advanced denoising algorithms to improve signal-to-noise ratio in low-light fluorescence images.
  3. Spot Detection and Segmentation: Identifying individual "spots" or clusters of signals (amplicons/detection reagents) using blob detection algorithms (e.g., Laplacian of Gaussian, Difference of Gaussians) and watershed segmentation.
  4. Signal Quantification: Extracting intensity values for each detected spot over the temporal sequence.
     This combination would render the act of computationally enhancing or correcting the detected temporal signals using standard image processing techniques as obvious.
• Mermaid Diagram:

  graph LR
      A[Raw Temporal Image Sequence] --> B(OpenCV/scikit-image)
      B -- Image Registration --> C[Drift-Corrected Image Sequence]
      C -- Noise Reduction --> D[Denoised Image Sequence]
      D -- Spot Detection & Segmentation --> E[Identified Signal Spots (X, Y, Z, Time)]
      E -- Signal Quantification --> F[Temporal Signal Profiles per Spot]
      F --> G(Analyte Identification Algorithm)
      G --> H(Output: Analyte Map)
  

Combination Prior Art Scenario 2: '737 Patent + Open-Source Microfluidics Design Platforms (e.g., LPKF CircuitCam/BoardMaster, various academic CAD tools for microfluidics)

• Enabling Description: The '737 patent frequently mentions microfluidic devices, flow cells, and automated fluidics for applying reagents and performing washes. Combining the '737 method with existing open-source design principles and platforms for microfluidics would be obvious for implementing the described methods in a practical, automated format. A PHOSITA skilled in microfluidics would use design tools (e.g., freely available CAD software, or open-source libraries for generating designs compatible with standard fabrication techniques like soft lithography, laser cutting) to create microfluidic chips that:
  1. Integrate Sample Inlet/Outlet: Standard ports for sample introduction and waste removal.
  2. Reagent Chambers and Mixing Channels: Efficiently mix detection reagents and decoder probes with the sample.
  3. Wash Channels: Perform rapid and efficient removal of unbound reagents, as required by the '737 method.
  4. Detection Zones: Optically transparent regions designed for imaging, compatible with the specified imaging systems.
  5. Temperature Control: Integrated micro-heaters or coolers for controlling hybridization/wash stringency.
     The creation of a microfluidic device to implement the fluidic steps of the '737 patent using commonly known design principles and open-source or freely available CAD tools would be obvious to a PHOSITA.
• Mermaid Diagram:

  flowchart TD
      A[Sample Input] --> M1(Microfluidic Inlet Port)
      B[Detection Reagent Input] --> M2(Microfluidic Reagent Port)
      C[Decoder Probe Input (Set 1..N)] --> M3(Microfluidic Decoder Port)
      D[Wash Buffer Input] --> M4(Microfluidic Wash Port)
      
      M1 & M2 & M3 & M4 --> MIX_CHANNELS[Mixing & Reaction Channels]
      MIX_CHANNELS --> DETECTION_ZONE[Optical Detection Zone]
      DETECTION_ZONE --> WASH_CHANNELS[Wash & Waste Channels]
      WASH_CHANNELS --> E[Waste Output]
      
      subgraph Microfluidic Device (Open-Source Design)
          M1 --- MIX_CHANNELS
          M2 --- MIX_CHANNELS
          M3 --- MIX_CHANNELS
          M4 --- WASH_CHANNELS
          MIX_CHANNELS --- DETECTION_ZONE
          DETECTION_ZONE --- WASH_CHANNELS
          WASH_CHANNELS --- E
      end
  

Combination Prior Art Scenario 3: '737 Patent + Open-Source Bioinformatics Tools for Sequence Design (e.g., Primer-BLAST, Biopython, ViennaRNA Package)

• Enabling Description: The '737 patent specifies that nucleic acid labels comprise "pre-determined subsequences" and that "the nucleic acid label can be designed for minimal cross-hybridization of bases with each other." It also describes designing decoder probes to be complementary. This process of designing specific nucleic acid sequences to avoid undesired secondary structures, minimize cross-hybridization, and optimize melting temperatures is a standard bioinformatics task. A PHOSITA would routinely use open-source bioinformatics tools for:
  1. Subsequence Design: Generating random or constrained sequences for the nucleic acid labels and decoder probes.
  2. Cross-Hybridization Prediction: Using tools like Primer-BLAST or similar algorithms to check for potential off-target binding between subsequences, decoder probes, and other nucleic acids in the sample.
  3. Secondary Structure Prediction: Employing software (e.g., ViennaRNA Package) to predict potential hairpin loops or other secondary structures within the nucleic acid labels or decoder probes that could interfere with hybridization.
  4. Melting Temperature (Tm) Calculation: Optimizing sequence length and GC content to achieve desired Tm for efficient hybridization and removal, as mentioned in the patent ("thermal denaturing can be reduced or avoided by using a sequencing-by-ligation approach and by setting the nucleic acid label base that immediately follows the sequencing primer...").
     The application of these well-known, open-source computational tools to design the specific nucleic acid sequences required by the '737 patent is a routine and obvious practice for anyone skilled in the art of molecular biology and oligonucleotide design.
• Mermaid Diagram:

  graph TD
      A[Design Requirements (e.g., length, multiplex level)] --> B(Generate Candidate SeqTag Subsequences)
      B --> C{Biopython: Sequence Manipulation}
      C --> D{Primer-BLAST: Cross-Hybridization Check}
      C --> E{ViennaRNA: Secondary Structure Prediction}
      C --> F{Custom Script: Tm Optimization}
      D -- Fail --> B
      E -- Fail --> B
      F -- Fail --> B
      D & E & F -- Pass --> G[Validated SeqTag & Decoder Probe Sequences]
      G --> H(Synthesize Nucleic Acid Labels & Decoder Probes)
      H --> I(Fabricate Detection Reagents)
      I --> J(Perform '737 Method)