Cleavable Fluorescent Streptavidin for Multiplexed Biomolecule Detection
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Solution Overview
Problem
Existing methods for multiplexed in situ protein and nucleic acid analysis in single cells suffer from low sensitivity, requiring long imaging exposure times and limited sample throughput, making it difficult to detect low-expression proteins and short RNA.
Innovation Solution
A method involving cleavable biotin-conjugated targeting agents and streptavidin with a detectable label, allowing for layer-by-layer signal amplification through reiterative cycles of staining, imaging, and chemical cleavage to enhance detection sensitivity and throughput.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional immunofluorescence and FISH methods are used for multiplexed analysis, then spectral overlap of fluorophores limits the number of biomolecules that can be profiled, but increasing the number of fluorophores does not resolve the sensitivity issue for low-expression proteins
Solution Approach 1:
The detection process is segmented into multiple cycles, each targeting a different biomolecule. After imaging one target, the fluorescent label is chemically cleaved and removed, allowing the same sample to be reused for detecting additional biomolecules. This segmentation enables multiplexed analysis without spectral overlap limitations.
Solution Approach 2:
The patent changes the detection parameter from simultaneous multi-color fluorescence detection to sequential detection with chemical cleavage. By using cleavable fluorescent labels and performing repeated imaging cycles with different targeting agents, the system achieves both high sensitivity for low-expression proteins and high throughput for multiple biomolecules.
2Measurement precision
If long imaging exposure time is used to improve detection sensitivity for low-expression proteins, then signal detection improves, but sample throughput decreases and assay time increases
Solution Approach 1:
The patent maintains continuous useful action by chemically cleaving and removing fluorescent labels after each imaging cycle, then immediately proceeding to the next cycle with a different targeting agent. This eliminates idle time and ensures that the sample is continuously utilized for detection purposes throughout the assay.
Solution Approach 2:
The detection process employs periodic action through repeated cycles of: (1) incubating with a new targeting agent, (2) imaging the fluorescent signal, and (3) chemically cleaving and removing the label. This periodic regimen allows multiple biomolecules to be detected sequentially with high sensitivity while maintaining overall assay efficiency.
3Ease of manufacture
If conventional direct conjugation of detection tags to affinity probes is used, then the method is simple, but detection sensitivity is limited and cannot detect low-expression proteins
Solution Approach 1:
The patent introduces an intermediary chemical cleavage step that temporarily attaches fluorescent labels to targeting agents during detection, then selectively removes them after imaging. This intermediary approach allows for signal amplification and repeated use of the same sample, dramatically improving detection sensitivity for low-expression proteins while maintaining procedural simplicity.
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
The method increases detection sensitivity by at least one order of magnitude, enabling the analysis of low-expression proteins and short nucleic acids with reduced assay times and without sample damage, allowing for comprehensive profiling in single cells.
Implementation Method 1
contacting a tissue with a plurality of targeting agents conjugated to biotin via a cleavable linker, wherein the targeting agents are configured to specifically bind or hybridize to a target biomolecule
Implementation Method 2
the targeting agents are configured to specifically bind or hybridize to a target biomolecule in the contacted tissue
Implementation Method 3
contacting a tissue comprising a plurality of biomolecules to streptavidin comprising a detectable label via a cleavable linker, wherein the second contacting step occurs under conditions that promote conjugation of the detectably-labeled streptavidin to biotin of the targeting agents
Implementation Method 4
imaging the cell after the second contacting step whereby a detectable signal generated from an interaction of biotin-conjugated targeting agents with the detectably-labeled streptavidin is detected
Implementation Method 5
removing the detectable label from the detectably-labeled streptavidin and unbound biotin
Data Source
AI summary
Provided herein are methods and systems for sensitive and multiplexed in situ analysis of samples such as biological samples using cleavable fluorescent streptavidin. In particular, provided herein are methods for multiplexed single-cell in situ biomolecule profiling in samples, including fixed or fresh tissues, and also 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.