Nucleic Acid Barcoding for Single-Cell Multiplexed Analyte Detection
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Solution Overview
Problem
Current methods for analyzing secreted proteins and molecules from cells are limited in their ability to multiplex analytes beyond tens of proteins and cannot simultaneously measure nucleic acids from the same cell, hindering the understanding of cellular interactions and communication mechanisms.
Innovation Solution
A method involving the use of reporter nucleic acid molecules and barcode nucleic acid molecules to label cells, allowing for the coupling of secreted analytes and cell surface molecules, followed by partitioning into droplets or microwells with barcode nucleic acid molecules, and subsequent sequencing to obtain nucleic acid sequences, enabling the multiplexing and simultaneous measurement of various analytes from a single cell.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If FACS method is used to measure secreted proteins, then single cell resolution is achieved, but the ability to multiplex analytes is limited to tens of proteins
Solution Approach 1:
The patent introduces nucleic acid barcodes as an intermediary between the secreted analyte and the detection system. Capture agents (antibodies) bind to secreted proteins and are brought into proximity with the cell, where they can be labeled with unique nucleic acid barcodes. These barcodes serve as mediators that can be amplified and sequenced, enabling high-throughput multiplexing while maintaining single-cell resolution through the original capture agent-analyte-cell association.
Solution Approach 2:
The patent replaces the mechanical fluorescence-based detection system of FACS with a nucleic acid-based detection system. Instead of relying on fluorescent labels and flow cytometry machinery, the invention uses nucleic acid barcodes that can be amplified by PCR and detected by sequencing technologies, thereby overcoming the multiplexing limitations of FACS while preserving single-cell analysis capability.
2Measurement precision
If FACS method is used to measure secreted proteins, then detection is achieved, but simultaneous measurement of nucleic acids from the same cell is impossible
Solution Approach 1:
The patent merges the measurement of secreted proteins and nucleic acids into a single unified workflow. By using nucleic acid barcodes that are introduced at the cell surface through capture agent labeling, the system enables simultaneous recovery of both protein information (through barcode identification) and cellular nucleic acid information (through extraction and sequencing from the same cell), thereby achieving correlated multi-analyte measurement from individual cells.
Solution Approach 2:
The nucleic acid barcode serves as a mediator that bridges protein detection and nucleic acid analysis. The barcode is introduced via the capture agent that bound to the secreted protein, creating a permanent molecular link between the protein identity and the cell's nucleic acid content. This mediator enables the subsequent simultaneous measurement of both analyte types from the same cellular source.
3Quantity of substance
If antibody-antibody complex is used to catch secreted molecules, then secreted proteins can be isolated, but limited multiplexing beyond tens of proteins is achieved
Solution Approach 1:
The patent uses nucleic acid barcodes as intermediaries attached to capture agents. Each capture agent (antibody) that binds to a secreted protein can be associated with a unique nucleic acid barcode sequence. This intermediary system allows for the isolation and identification of numerous different secreted proteins simultaneously, as each protein-capture agent-barcode complex can be distinguished through nucleic acid sequencing, vastly expanding multiplexing capacity beyond the tens of proteins achievable with traditional fluorescent antibody panels.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach allows for the detailed analysis of secreted analytes, cell surface proteins, and nucleic acids from a single cell, enhancing the understanding of cellular interactions and communication mechanisms, and increasing the throughput of sample analysis.
Implementation Method 1
the capture agent is configured to couple to both a cell surface molecule and the secreted analyte
Implementation Method 2
the reporter agent is configured to couple to the secreted analyte
Implementation Method 3
generating a barcoded nucleic acid molecule from a barcode nucleic acid molecule of the plurality of barcode nucleic acid molecules and the reporter nucleic acid molecule
Data Source
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AI summary
Provided herein are methods and systems for measuring secreted cytokines or other analytes from single cells. The methods disclosed herein include the use of analyte-specific and/or barcoded binding agents (e.g., antibodies) and beads in partitions (e.g., droplets or wells) to measure such analytes on a single cell basis. Further described herein are methods comprising the use of hydrogel-encapsulated cells (e.g., cell beads) to measure secreted and/or cellular analytes from single cells.