Single-Cell Secretome Analysis Using Barcoded Capture Probes
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Solution Overview
Problem
Current methods for detecting secreted proteins are limited in their ability to quantify the number of copies secreted by a single cell and simultaneously measure protein and gene expression, often relying on bulk measurements and lacking the necessary sensitivity and specificity for individual cell analysis.
Innovation Solution
A method involving the use of solid supports with capture probes and secreted factor-binding reagents, combined with oligonucleotide barcodes, to specifically bind and quantify secreted factors and nucleic acid targets from single cells, allowing for the determination of copy numbers through hybridization and sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional bulk measurement methods (bead-based assays, ELISA) are used to detect secreted proteins, then the measurement process is simple and robust, but the ability to quantify secreted factors at the single cell level is lost and measurement precision deteriorates
Solution Approach 1:
The patent segments the measurement process into distinct functional modules: capture probes for specific binding, secreted factor-binding reagents for detection, and oligonucleotide barcodes for identification. Each module performs a specific function, enabling single-cell resolution while maintaining operational simplicity through modular design
Solution Approach 2:
The patent introduces oligonucleotide barcodes as intermediary molecules that bridge the secreted factor detection and the sequencing-based quantification system. These barcodes are incorporated into the detection complex and allow the system to translate protein detection into a high-precision nucleic acid sequencing measurement
2Measurement precision
If fluorescence-based methods (flow cytometry, microscopy) are used to detect secreted proteins, then the system can provide visual detection capability, but the number of detectable proteins is limited by the number of fluorescence markers and measurement precision deteriorates due to fluorescence intensity limitations
Solution Approach 1:
The patent replaces the fluorescence-based detection system with a sequencing-based detection system. Instead of relying on fluorescence intensity measurements limited by optical detection capabilities, the system uses oligonucleotide barcodes that are identified through high-precision nucleic acid sequencing, enabling both superior quantification precision and expanded multiplexing capacity
Solution Approach 2:
The oligonucleotide barcode system serves multiple functions: it identifies the secreted factor type, quantifies the number of copies, and enables multiplexed detection of numerous different factors simultaneously. This universal barcode approach replaces multiple fluorescence markers with a single scalable nucleic acid-based identification system
3Measurement precision
If bulk measurement approaches are used to analyze secreted factors, then the analysis process is straightforward and device complexity is low, but the ability to correlate secretion activity with complex cell phenotype is lost and measurement precision at single cell level deteriorates
Solution Approach 1:
The patent merges single-cell secretion analysis with nucleic acid sequencing technology. By combining the capture of secreted factors from individual cells with the high-precision quantification capabilities of sequencing, the system achieves single-cell quantification precision while leveraging the operational simplicity and scalability of established sequencing workflows
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 quantitative analysis of secreted factors and nucleic acid targets at the single cell level, providing a more accurate and comprehensive understanding of cellular secretion and gene expression profiles.
Implementation Method 1
each first solid support comprises a plurality of capture probes capable of specifically binding to at least one of the plurality of secreted factors secreted by a single cell
Implementation Method 2
contacting a plurality of oligonucleotide barcodes with the secreted factor-binding reagent specific oligonucleotides for hybridization
Data Source
AI summary
Systems, methods, compositions, and kits for measuring secreted factors from cells are disclosed herein, including those capable of determining single cell secretion activity and protein expression and/or gene expression simultaneously. Disclosed herein include solid supports comprising a plurality of capture probes capable of specifically binding to at least one of the plurality of secreted factors secreted by a single cell. Also disclosed herein include secreted factor-binding reagents capable of specifically binding to a secreted factor bound by a capture probe. A secreted factor-binding reagent can comprise a secreted factor-binding reagent specific oligonucleotide comprising a unique factor identifier sequence for the secreted factor-binding reagent.