Multiplex Protein Probes for Region-Specific Tissue Quantification
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Solution Overview
Problem
Existing immunohistochemical methods are limited to the simultaneous detection of six to ten protein targets, with three to four being typical, and there is a need for probes and methods to detect and quantify protein expression in user-defined regions of tissues, cells, and subcellular structures with higher multiplexing capabilities.
Innovation Solution
A method involving probes with target-binding domains and signal oligonucleotides that allow for the detection and quantification of multiple protein targets by applying a force to release signal oligonucleotides, which are then collected and identified, enabling the detection of up to 1000 targets in user-defined regions, with techniques like nCounter® systems for digital quantification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If standard immunohistochemical methods are used, then detection of protein targets is achieved, but the number of simultaneously detectable targets is limited to six to ten
Solution Approach 1:
The probe is segmented into distinct functional modules: a target-binding domain (e.g., antibody) and a signal oligonucleotide domain connected by a cleavable linker. This segmentation allows the binding function and signal generation function to operate independently and be optimized separately, enabling multiplexed detection of multiple protein targets simultaneously without increasing overall method complexity
Solution Approach 2:
A cleavable linker acts as an intermediary between the target-binding domain and the signal oligonucleotide. This intermediary element can be selectively cleaved under specific conditions (e.g., by proteases or chemical agents), enabling controlled release of signal oligonucleotides for detection while maintaining the integrity of the target-binding domain throughout the assay process
2Quantity of substance
If multiplexed detection of multiple protein targets is implemented, then the number of detectable targets increases, but spatial resolution and user-defined region detection become more difficult
Solution Approach 1:
The method enables local quality detection by allowing probes to bind to specific protein targets within user-defined regions of interest in tissue sections. The cleavable linker design ensures that signal oligonucleotides are released only from probes bound to targets in the specified region, maintaining spatial resolution even when detecting multiple protein targets simultaneously across different locations
Solution Approach 2:
The probe is pre-configured with the target-binding domain and signal oligonucleotide connected by a cleavable linker before application to the tissue sample. This preliminary assembly ensures that only probes specifically bound to target proteins in the user-defined region will release signal oligonucleotides upon cleavage, preventing background signal from unbound probes and maintaining precise spatial localization
3Measurement precision
If quantitative detection of protein expression is performed, then protein abundance measurement is achieved, but detection precision and reliability across multiple centers vary
Solution Approach 1:
The invention changes the detection parameter from direct optical signal intensity (which varies between laboratories) to digital counting of released signal oligonucleotides. This parameter change enables absolute quantification of protein expression levels based on the number of detected oligonucleotides, providing consistent and reliable measurements across multiple centers without requiring standardization of imaging conditions or subjective interpretation
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 simultaneous, multiplexed detection and quantification of up to 1000 protein targets in user-defined regions, providing higher multiplexing capabilities and digital quantification for reliable comparison across multiple centers.
Implementation Method 1
The target-binding domain comprises a protein-binding molecule, e.g., an antibody, a peptide, an aptamer, and a peptoid
Implementation Method 2
providing a force to a location of the tissue sample sufficient to release the signal oligonucleotide
Data Source
AI summary
The present invention relates to, among other things, probes, compositions, methods, and kits for simultaneous, multiplexed detection and quantification of protein expression in a user-defined region of a tissue, user-defined cell, and/or user-defined subcellular structure within a cell.