Single-Cell Secretome Quantification via Barcoded Capture Probes
Find Innovative SolutionsGenerate Solutions
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 sensitivity in fluorescence analysis.
Innovation Solution
A method involving solid supports with capture probes and secreted factor-binding reagents, combined with oligonucleotide barcodes for hybridization and sequencing, allows for the quantification of secreted factors and nucleic acid targets in individual cells, using unique identifier sequences for precise counting.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional bulk detection methods are used, then the measurement process is simple, but the ability to quantify secreted factors at the single-cell level is lost
Solution Approach 1:
The detection system is segmented into discrete solid supports (beads), each capable of independently capturing secreted factors from individual cells. This segmentation enables parallel processing of multiple single cells, achieving single-cell resolution while maintaining scalability through the modular bead-based architecture.
Solution Approach 2:
Oligonucleotide barcodes serve as intermediary molecules that bridge the connection between captured secreted factors and detectable signals. These barcodes are attached to secreted factors during capture and subsequently amplified and sequenced, converting the physical presence of secreted factors into quantifiable digital data without requiring direct fluorescence measurement.
2Measurement precision
If fluorescence-based methods are used, then real-time detection is possible, but quantitative accuracy is limited due to fluorescence intensity measurement limitations
Solution Approach 1:
The patent replaces the optical/fluorescence-based detection system with a sequencing-based detection system. Instead of measuring fluorescence intensity, the method uses oligonucleotide barcode amplification and sequencing, where the number of sequenced barcode reads directly corresponds to the number of secreted factor copies. This substitution eliminates the limitations of fluorescence quantification while providing digital, absolute counting capability.
Solution Approach 2:
The method creates multiple copies of oligonucleotide barcodes associated with each secreted factor through PCR amplification. These copied barcodes serve as proxies for the original secreted factors, allowing indirect quantification through sequencing depth. The copying process preserves the quantitative information while enabling scalable detection through molecular amplification.
3Adaptability or versatility
If the number of fluorescence markers is increased to detect more proteins, then the detection capability is improved, but the technical limitations of microscopy or flow cytometry are reached
Solution Approach 1:
The solid support system uses universal capture mechanisms (e.g., anti-tag antibodies) that can recognize diverse secreted factors through a common feature (such as a FLAG tag or other epitope). This universal approach allows the same platform to detect any secreted factor that has been engineered with the recognition tag, eliminating the need for factor-specific detection reagents and enabling scalable multiplexing limited only by the number of unique barcodes available.
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, overcoming limitations of bulk detection and enhancing the accuracy of protein and gene expression measurements.
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.