Derivative works

Defensive Disclosure and Prior Art Declaration

Publication Date: April 26, 2026
Subject: Derivative Methods and Applications for Spatially Resolved In Situ Nucleic Acid Sequencing
Reference Technology: The core inventive concept disclosed in U.S. Patent 12,264,358, pertaining to the formation of an in situ 3D nucleic acid matrix for spatially resolved amplification and sequencing.
Purpose: This document is intended to enter the public domain as prior art. It discloses a plurality of variations, extensions, and applications of the reference technology to render subsequent, incremental, and obvious improvements non-patentable.

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Axis 1: Material & Component Substitution

Derivative 1.1: Thermo-Reversible Hydrogel Matrix for Sample Recovery

Enabling Description: A method for in situ nucleic acid sequencing where the matrix is composed of a thermo-reversible polymer, such as poly(N-isopropylacrylamide) (PNIPAM) or a Poloxamer (e.g., Pluronic® F-127), co-polymerized with acrydite-modified nucleic acid primers and linkers. The gelation process is initiated by raising the temperature of the permeabilized biological sample above the polymer's lower critical solution temperature (LCST), typically to 32-37°C. All subsequent steps, including reverse transcription, rolling circle amplification, and iterative sequencing, are performed at this elevated temperature to maintain the gel state. Following data acquisition, the sample temperature is lowered below the LCST (e.g., to 4°C), causing the hydrogel to dissolve into a liquid phase. This enables the non-destructive recovery of specific cells or tissue regions, identified by their spatial coordinates from the sequencing map, for subsequent multi-omic analyses like proteomics or metabolomics.

flowchart TD
    A[Permeabilize Biological Sample at 4°C] --> B{Infuse with liquid PNIPAM/Poloxamer precursor mix containing Acrydite-cDNA primers};
    B --> C[Raise Temperature to 37°C];
    C --> D[Thermo-reversible Hydrogel Matrix Forms In Situ];
    D --> E[Perform In Situ Amplification & Sequencing Cycles at 37°C];
    E --> F[Acquire 3D Spatial Sequence Data];
    F --> G[Lower Temperature to 4°C];
    G --> H[Matrix Dissolves, Releasing Cellular Content];
    F --> I{Identify Target Cells based on Sequence Map};
    I --> J[Micro-dissect/Recover Target Cells from Liquid Phase for Proteomics];

Derivative 1.2: Electrically Conductive Polymer Matrix for Electronic Detection

Enabling Description: This variation replaces the optically transparent hydrogel with an electrically conductive polymer matrix, enabling a label-free electronic readout of sequencing events. A biocompatible conductive polymer, such as a poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) hydrogel, is formed in situ. The native nucleic acids are immobilized within this matrix. The sequencing-by-synthesis process is monitored electronically instead of optically. The incorporation of each nucleotide by a polymerase causes a transient, localized change in the ionic environment and pH, which is detected as a minute fluctuation in impedance or potential by a grid of micro-electrodes integrated at the base of the sample. An array of amplifiers and analog-to-digital converters translates these signals into a base call for each specific spatial coordinate, obviating the need for fluorescent labels and complex optical imaging systems.

sequenceDiagram
    participant P as Polymerase
    participant M as Micro-electrode Array
    participant A as Amplifier & ADC
    participant C as Base Caller

    loop For each sequencing cycle
        P->>M: Incorporates nucleotide at coordinate (x,y,z)
        Note over M: Local impedance change (ΔZ) occurs
        M->>A: Transmits analog signal for (x,y,z)
        A->>C: Sends digitized ΔZ signal
        C->>C: Identifies base ('G') from signal signature
    end

Derivative 1.3: Quantum Dot (QD) Labels for Enhanced Multiplexing and Stability

Enabling Description: This method substitutes conventional organic fluorophores used in sequencing-by-synthesis with semiconductor quantum dots (QDs). Four populations of QDs, each with a distinct emission wavelength (e.g., 525 nm, 565 nm, 605 nm, 655 nm), are respectively conjugated to the four nucleotide triphosphates (A, C, G, T). Due to the QDs' high quantum yield and extreme photostability, the signal-to-noise ratio is significantly enhanced, and the number of sequencing cycles can be extended beyond 100 rounds without significant photobleaching. The narrow, symmetric emission peaks of QDs also allow for the potential of hyperspectral imaging to deconvolve signals from more than four labels simultaneously, enabling interrogation of multiple sequences or modifications at each location per cycle.

classDiagram
    class Nucleotide {
        +base: char
    }
    class QuantumDot {
        +emissionWavelength: int
        +coreMaterial: string
        +shellMaterial: string
    }
    class QD_Nucleotide {
        +conjugationLinker: string
    }

    Nucleotide <|-- QD_Nucleotide
    QuantumDot <|-- QD_Nucleotide
    QD_Nucleotide "1" -- "1" Nucleotide : conjugatedTo
    QD_Nucleotide "1" -- "1" QuantumDot : hasLabel

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Axis 2: Operational Parameter Expansion

Derivative 2.1: Cryo-Spatial Transcriptomics (Cryo-ST)

Enabling Description: A method to perform spatial sequencing on vitrified, non-fixed biological samples. The sample is flash-frozen in liquid ethane, preserving cellular ultrastructure with minimal ice crystal damage. A cryo-compatible matrix precursor (e.g., a low-concentration polyacrylamide solution with cryoprotectants like glycerol) is infused into the sample via cryo-focused ion beam (FIB) milling channels. Polymerization is initiated via UV light at cryogenic temperatures (e.g., -150°C). All subsequent enzymatic reactions (reverse transcription, amplification, ligation) are performed using cold-adapted enzymes (e.g., psychrophilic polymerases and ligases sourced from arctic microorganisms) in cryo-compatible buffers. Imaging is performed on a cryo-confocal microscope. This process provides a near-native-state spatial map of gene expression, completely avoiding chemical fixation artifacts.

stateDiagram-v2
    [*] --> Vitrification
    Vitrification --> FIB_Milling: Sample Flash-Frozen
    FIB_Milling --> Infusion: Create channels
    Infusion --> Cryo_Polymerization: Infuse matrix precursors at -150°C
    Cryo_Polymerization --> Cryo_Enzymatics: UV-initiated gelation
    Cryo_Enzymatics --> Cryo_Imaging: Use cold-adapted enzymes
    Cryo_Imaging --> Data_Analysis: Acquire sequence data
    Data_Analysis --> [*]

Derivative 2.2: Macroscopic Whole-Organ Spatial Sequencing

Enabling Description: Scaling the in situ sequencing methodology to intact macroscopic samples, such as an entire mouse brain or plant seedling. The organ is first decellularized and permeabilized via vascular perfusion with a detergent solution, followed by perfusion with the hydrogel precursor mix. After gelation, the resulting organ-hydrogel hybrid is optically cleared using techniques like CLARITY. The sequencing reagents are then cyclically flowed through the remnant vascular system of the organ. Data acquisition is performed using a light-sheet fluorescence microscope (LSFM), which allows for rapid optical sectioning and imaging of the entire cubic-centimeter-scale sample. The resulting dataset is a complete, 3D molecular atlas of the organ at cellular resolution.

flowchart LR
    A[Mouse Brain] --> B(Vascular Perfusion with Detergent);
    B --> C(Perfusion with Hydrogel Precursors);
    C --> D{Gelation & Optical Clearing (CLARITY)};
    D --> E(Mount in Light-Sheet Microscope);
    E --> F(Cyclic Reagent Flow via Cannulated Arteries);
    F --> E;
    E --> G[High-speed 3D Imaging of Sequencing];
    G --> H[Reconstruction of Whole-Brain Transcriptome];

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Axis 3: Cross-Domain Application

Derivative 3.1 (Aerospace): In-Situ Mapping of Material Fatigue via Engineered Biosensors

Enabling Description: Composite materials used in aerospace structures are embedded with engineered, dormant bacterial spores (Bacillus subtilis). The spores contain a DNA plasmid with a "reporter cassette" flanked by sequences recognized by a stress-activated recombinase. Upon experiencing specific mechanical stress thresholds (e.g., >200 MPa), the spores germinate and the recombinase is expressed, which inverts or excises the DNA reporter cassette. For maintenance checks, a handheld device drills a micro-core from the material, perfuses it in situ with the hydrogel matrix and sequencing reagents. The spatial sequencing map reveals the 3D locations of bacteria with altered DNA, providing a high-resolution, cumulative record of material fatigue and micro-fracture propagation.

graph TD
    subgraph Aircraft Wing Composite
        A(Spores with Intact DNA)
        B(Spores with Flipped DNA)
    end
    subgraph Maintenance Check
        C[Micro-core Extraction]
        D[In-situ Gelation & Sequencing]
        E{3D Fatigue Map}
    end
    A -- Mechanical Stress --> B
    A & B -- Core Sample --> C
    C --> D
    D --> E

Derivative 3.2 (AgTech): Spatially Resolved Soil Metatranscriptomics

Enabling Description: A hollow, porous probe is inserted directly into an agricultural soil bed. A solution of hydrogel precursors is injected through the probe, permeating a small volume of soil and encapsulating the native soil microbiome in situ. The probe then delivers a sequence of reagents to: (1) lyse the microorganisms, (2) immobilize the released RNA into the gel matrix, (3) perform reverse transcription, amplification, and sequencing. The probe contains an integrated fiber optic imaging bundle connected to a portable sequencer. The resulting data provides a 3D map of gene expression across different microbial species in their undisturbed microenvironment, revealing metabolic activity and symbiotic/competitive interactions around plant roots.

sequenceDiagram
    participant Probe
    participant Soil_Microbiome
    participant Sequencer

    Probe->>Soil_Microbiome: Injects hydrogel precursors
    Note right of Probe: Matrix forms, encapsulating microbiome
    Probe->>Soil_Microbiome: Injects lysis & RT reagents
    Probe->>Soil_Microbiome: Injects amplification & sequencing reagents
    loop Sequencing Cycles
        Probe->>Sequencer: Transmits optical data via fiber bundle
    end
    Sequencer-->>Probe: Generates 3D metatranscriptomic map

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Axis 4: Integration with Emerging Tech

Derivative 4.1 (AI-driven Optimization): Adaptive, Interest-Driven Sequencing

Enabling Description: The in situ sequencing process is controlled by a convolutional neural network (CNN) in a closed loop. After an initial, low-resolution sequencing pass (e.g., 2-3 cycles) across the entire sample, the CNN analyzes the nascent spatial gene expression patterns. It identifies "regions of interest" (ROIs) based on pre-trained models for features like tumor heterogeneity, immune cell infiltration, or neural activity boundaries. The system's controller then automatically modifies the data acquisition plan: it increases the imaging magnification, decreases the pixel binning, and allocates more sequencing cycles specifically to the ROIs, while continuing sparse sampling elsewhere. This AI-driven approach maximizes the information density acquired from biologically significant regions while minimizing instrument time and data storage costs.

flowchart TD
    A[Start] --> B(Perform Low-Res Global Sequencing: Cycles 1-3);
    B --> C{Send 3D Map to CNN for Analysis};
    C --> D{CNN Identifies Regions of Interest (ROIs)};
    D --> E(Update Instrument Protocol);
    subgraph High-Res Targeted Sequencing
        F[Increase Magnification on ROIs]
        G[Increase Cycle Count on ROIs]
    end
    E --> F & G
    G --> H(Acquire Deep Data from ROIs);
    H --> C;

Derivative 4.3 (Blockchain Verification): Immutable Provenance for Clinical Diagnostics

Enabling Description: For clinical applications, each step of the spatial sequencing workflow is tied to a private blockchain to ensure an immutable audit trail. When a patient biopsy is received, its metadata (patient ID, time, location) is used to generate a cryptographic hash, creating the genesis block. Subsequent steps, such as matrix formation, reagent lot number application, instrument ID for the sequencing run, and the raw image data hash, are added as sequential transactions to the chain. The final diagnostic report, containing the spatial gene expression map, is also hashed and linked. Any user, from pathologist to patient, can cryptographically verify the entire provenance of the result, ensuring the data has not been tampered with and is linked to the correct original sample.

erDiagram
    PATIENT ||--o{ BIOPSY : has
    BIOPSY ||--|{ BLOCKCHAIN_RECORD : "is recorded in" {
        string tx_hash PK
        string block_id
        timestamp ts
    }
    BLOCKCHAIN_RECORD ||--|{ HASHED_DATA : "contains" {
        string data_type
        string data_hash
    }
    INSTRUMENT ||--|{ BLOCKCHAIN_RECORD : "generates"
    DIAGNOSTIC_REPORT ||--|{ BLOCKCHAIN_RECORD : "finalizes"

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Axis 5: The "Inverse" or Failure Mode

Derivative 5.1: Enzymatically Dissolvable Matrix for "Read-then-Recover" Workflow

Enabling Description: The hydrogel matrix is formulated with cross-linkers containing a substrate for a specific, non-endogenous enzyme. For example, the bis-acrylamide cross-linker is replaced with a peptide linker containing a Tobacco Etch Virus (TEV) protease cleavage site. The entire spatial sequencing protocol is executed to generate the 3D gene expression map. Based on this map, specific cell populations are identified for further study (e.g., a rare cancer stem cell clone). A solution containing TEV protease is then introduced, which selectively digests the peptide cross-linkers and dissolves the hydrogel. The targeted cells are then recovered from the liquified sample using micromanipulation or FACS, enabling a direct link from spatial transcriptome to single-cell proteomics or functional assays.

graph TD
    A[In-Situ Sequencing in TEV-Cleavable Matrix] --> B(Generate 3D Transcriptome Map);
    B --> C{Identify Rare Cell at Coords (x,y,z)};
    C --> D(Perfuse Sample with TEV Protease);
    D --> E[Matrix Dissolves];
    E --> F(Recover Cell from (x,y,z) via Micropipette);
    F --> G[Perform Downstream Single-Cell Proteomics];

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Combination Prior Art Scenarios

1. Combination with OpenFlexure Microscope and Micro-Manager: The sequencing imaging system is built using the OpenFlexure Microscope, an open-source, 3D-printed microscopy platform. Instrument control for the multi-day, cyclic sequencing process is automated using Micro-Manager, an open-source software package for microscope control. Custom scripts written in Python or Beanshell within Micro-Manager handle the fluidics control, autofocus, and multi-channel image acquisition sequences.
2. Combination with FAIR Data Principles and Common Workflow Language (CWL): The entire data analysis pipeline, from raw image stacks to a spatially resolved gene expression matrix, is containerized (e.g., using Docker) and described using the Common Workflow Language (CWL), an open standard for portable, reproducible data analysis workflows. The final data and metadata are published according to FAIR (Findable, Accessible, Interoperable, Reusable) data principles, ensuring the results can be easily discovered and integrated with other public spatial omics datasets.
3. Combination with ONNX AI Model Format: The AI model used for adaptive sequencing (Derivative 4.1) is trained using a standard framework (e.g., PyTorch) and then exported to the Open Neural Network Exchange (ONNX) format. This open-source format allows the inferencing model to be deployed on diverse hardware platforms (e.g., NVIDIA Jetson, FPGA) embedded in the microscope controller, ensuring interoperability and decoupling the model's execution from the original training environment.