Cleavable Tyramide Detection for Sensitive Multiplexed Tissue Profiling
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Solution Overview
Problem
Existing methods for in situ analysis of proteins and nucleic acids in tissues are limited in their ability to detect a small number of molecules and often miss low-copy transcripts, lacking sensitivity and multiplexing capabilities.
Innovation Solution
A cleavable detectably-labeled tyramide (CLT) is used, comprising a detectable marker tethered via a chemically cleavable linker, allowing for multiplexed in situ analysis through cycles of target biomolecule binding, imaging, and signal removal using 1,3,5-Triaza-7-phosphaadamantane (PTA) and tris(2-carboxyethyl)phosphine (TCEP).
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If conventional immunohistochemistry and immunofluorescence methods are used, then the detection process is simple and straightforward, but only a handful of proteins can be detected in one tissue sample, limiting multiplexing capability
Solution Approach 1:
The patent implements cyclic detection where the same tissue section is sequentially stained with different antibody-enzyme conjugates over multiple days. Each cycle detects a new target protein while previous signals are amplified and preserved. This periodic action enables detection of many more proteins (50+) than conventional single-step methods, directly resolving the contradiction between multiplexing capability and process complexity.
Solution Approach 2:
The detection process is segmented into discrete cycles, each targeting a specific protein with a unique antibody-enzyme conjugate. The tyramide signal amplification system is also segmented, separating the amplification step from the detection step. This segmentation allows systematic multiplexing while maintaining manageable complexity through standardized repeated protocols.
2Measurement precision
If conventional immunofluorescence is used, then the methodology is straightforward, but transcripts present at low copy numbers are missed, lacking sensitivity
Solution Approach 1:
The patent fundamentally changes the detection parameter from direct fluorophore signal to enzyme-catalyzed tyramide deposition. The enzyme-tyramide reaction amplifies the signal by depositing multiple tyramide molecules per enzyme molecule, dramatically increasing detection sensitivity for low-copy transcripts. This parameter change enables detection of previously undetectable low-abundance targets while maintaining a relatively simple procedural framework.
3Adaptability or versatility
If high multiplexing is achieved through multiple tissue sections, then more biomolecules can be analyzed, but the cost and resource requirements increase significantly
Solution Approach 1:
The patent merges multiple detection objectives into a single tissue section by implementing sequential staining cycles on the same sample. Instead of requiring separate sections for each target protein, the system combines detection of 50+ different biomolecules in one section through repeated cycles with different antibody conjugates. This merging dramatically reduces tissue consumption and enables analysis of precious or limited samples.
4Productivity
If conventional methods are used for protein imaging, then the process is simple and quick, but only a small number of different molecules can be quantified, limiting comprehensive profiling
Solution Approach 1:
The patent performs preliminary signal amplification by incorporating enzyme-tyramide complexes during each staining cycle. This preliminary action ensures that sufficient signal is generated and preserved before the next cycle begins, enabling comprehensive profiling of many proteins without requiring excessive time for each individual detection step. The accumulated signals from multiple cycles provide comprehensive profiling capability.
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-throughput, sensitive, and comprehensive analysis of multiple biomolecules in intact tissues with single-molecule sensitivity, allowing for precise quantification and spatial organization understanding.
Implementation Method 1
Exemplary detectable makers include, without limitation, fluorophores
Implementation Method 2
luminescent agents (e.g., chemiluminescent agents)
Data Source
AI summary
Provided herein are methods for multiplexed in situ analysis of biomolecules in a tissue. In particular, provided herein are methods for multiplexed single-cell in situ protein and nucleic acid profiling in fixed or fresh tissues, that allows the investigation of the different cell compositions and their spatial organizations in intact tissues through consecutive cycles of probe hybridization, fluorescence imaging, and signal removal.