Single Cell Multiplex Analysis via Nucleic Acid Tag Sequencing
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Solution Overview
Problem
Current methods for high multiplex analysis of proteins in single cells are limited by spectral overlap in flow cytometry and the number of available isotope tags in mass cytometry, and cannot combine protein detection with DNA readout, necessitating a method for high multiplex analysis of cellular constituents in single cells.
Innovation Solution
A method involving the use of nucleic acid tags linked to existing ligand binding and/or antibody technologies for proteomic or cellular constituent detection and relative quantification by next-generation sequencing (NGS), where cellular constituents are embedded in polymer matrices, labeled with oligonucleotide tags, and sequenced to identify and quantify cellular components.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If flow cytometry is used for protein detection, then single cell analysis is enabled, but spectral overlap limits the number of proteins that can be detected simultaneously
Solution Approach 1:
The patent replaces the optical detection system of flow cytometry with a sequencing-based detection system. Instead of using fluorescent tags that suffer from spectral overlap, the invention uses oligonucleotide tags that are detected through next-generation sequencing, eliminating the spectral overlap problem while enabling detection of many more proteins simultaneously
Solution Approach 2:
The patent introduces oligonucleotide tags as intermediaries between proteins and detection. These tags are attached to proteins via antibodies or other binding molecules, and the tags themselves are sequenced rather than directly detecting the proteins, allowing for high multiplexing without spectral interference
2Quantity of substance
If mass cytometry is used for protein detection, then isotope tags enable multiplexed detection, but the number of available isotope tags limits the analysis capacity
Solution Approach 1:
The patent changes the detection parameter from mass-to-charge ratio (in mass cytometry) to nucleotide sequence. This allows using the vast diversity of oligonucleotide sequences instead of the limited number of isotope tags, enabling detection of hundreds or thousands of proteins simultaneously without increasing physical device complexity
Solution Approach 2:
The patent transitions from detecting proteins in the mass spectrum dimension to detecting them in the sequence space dimension. By encoding protein identity in oligonucleotide sequences rather than mass tags, the system achieves much higher multiplexing capacity
3Adaptability or versatility
If conventional methods are used, then protein detection is possible, but combining protein detection with DNA readout in single cells is not achievable
Solution Approach 1:
The patent merges protein detection and DNA analysis into a single unified workflow. By using oligonucleotide tags that can be co-sequenced with genomic DNA from the same single cell, the method simultaneously provides protein expression profiles and genomic information without requiring separate experiments
Solution Approach 2:
The oligonucleotide tag system serves multiple functions: it identifies proteins through sequencing, enables quantification of protein abundance, and can be integrated with genomic DNA analysis from the same cell, creating a universal platform for multi-omics analysis
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
Enables high multiplex analysis of proteins and cellular constituents in single cells, allowing for comparison of protein data variation between cells and different biological conditions, and provides massively parallel profiling of circuit aspects from RNA to chromatin organization.
Implementation Method 1
admixing at least one isolated aggregation of cellular constituents with monomers of a polymerizable gel; polymerizing the gel, to embed the cellular constituents in discrete polymer matrices
Implementation Method 2
incubating the cellular constituents embedded in the polymer matrices with one or more labeling ligands with specific binding affinity for one or more target cellular constituents
Implementation Method 3
sequencing the oligonucleotide label, whereby detecting the UCI by sequencing indicates the presence of the target cellular constituent
Data Source
AI summary
The present invention relates to methods for high multiplex protein or cellular constituent analysis in single cells or single isolated aggregations of cellular constituents. The methods provide for embedding cells or isolated aggregations of cellular constituents in a hydrogel mesh and labeling of cellular constituents with labeling ligands linked to a nucleic acid tag. Cellular constituents can be determined using sequencing methods.