Spatial Tissue Profiling With Cleavable Probes for High Multiplexing
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing methods for immunohistochemical and in situ hybridization are limited to the simultaneous detection of six to ten protein or nucleic acid targets, with three to four being typical, necessitating a need for improved probes and methods for multiplexed detection and quantification of protein and/or nucleic acid expression in user-defined regions of tissues, cells, and subcellular structures.
Innovation Solution
A method involving the use of probes with target-binding domains and signal oligonucleotides, where a force is applied to release signal oligonucleotides from specific locations in a tissue sample, allowing for the detection and quantification of multiple targets, including up to 1000 or more, through techniques like laser irradiation and collection via fluidic devices, enabling spatially-resolved DNA, RNA, and protein detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If standard immunohistochemical and in situ hybridization methods are used, then detection of protein and nucleic acid targets is achieved, but the number of simultaneous targets detected is limited to six to ten at most
Solution Approach 1:
The probe is segmented into distinct functional domains: a target-binding domain (antibody or nucleic acid) and a separate signal oligonucleotide domain connected by a cleavable linker. This segmentation allows the signal oligonucleotide to be released and detected separately after target binding, enabling multiplexed detection of numerous 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 component can be selectively cleaved (e.g., by UV light or enzymes) to release the signal oligonucleotide from the probe, enabling the detection system to distinguish between bound and unbound probes and thereby increase the number of simultaneously detectable targets
2Quantity of substance
If multiple targets are detected simultaneously, then comprehensive analysis of tissue expression is improved, but spatial resolution and quantification accuracy deteriorate
Solution Approach 1:
The signal oligonucleotide is extracted or released from the probe structure through cleavage of the linker after target binding. This extraction allows the signal component to be separated and quantified independently, maintaining measurement precision even when multiple targets are detected simultaneously, as each released signal oligonucleotide can be individually counted and localized
Solution Approach 2:
Different signal oligonucleotides can be labeled with distinct fluorescent labels or barcodes, enabling spatially-resolved multiplexed detection. Each target produces a detectable signal with unique identification, allowing simultaneous detection of multiple targets while maintaining the ability to distinguish and quantify each target's spatial distribution and abundance
3Area of stationary object
If user-defined region analysis is implemented, then spatial specificity is improved, but detection sensitivity deteriorates
Solution Approach 1:
The probe is pre-loaded with the signal oligonucleotide and target-binding domain in a single conjugated structure. This preliminary preparation ensures that when the probe binds to the target in the user-defined region, the signal is already attached and ready for detection, maintaining high detection sensitivity even when analyzing small or specific tissue regions
Solution Approach 2:
The mechanical or chemical binding between the target-binding domain and its target is replaced or supplemented by the cleavable linker mechanism. This allows for controlled release of the signal oligonucleotide upon application of a specific force (e.g., UV light, enzymatic cleavage), enhancing detection sensitivity in user-defined regions by providing a clear on/off signal mechanism that reduces background noise
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, high-multiplexed detection and quantification of proteins and nucleic acids in user-defined regions, providing a linear dynamic range of greater than 5 logs and reducing background noise through negative purification, thus enhancing the reliability and consistency of results across multiple centers.
Implementation Method 1
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 and/or nucleic acid expression in a user-defined region of a tissue, user-defined cell, and/or user-defined subcellular structure within a cell.