MS-Tagged Probe Linkers for In Vivo Multiplex Biomarker Quantification
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Solution Overview
Problem
Current methods for quantifying protein receptors in cancer cells are limited by interference and degradation of biological material, and cannot be used in vivo to detect or quantify biomarkers in their natural environment, allowing for only non-quantitative detection of up to 4 biomarkers per cycle.
Innovation Solution
The use of MS-tagged probes with a cleavable iminosydnone linker through a click-and-release bioorthogonal reaction, allowing for multiplexed analysis of biomarkers in vivo by administering the probes, which are released in physiological fluids for quantification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If cyclic immunofluorescence with fluorescent antibodies is used, then multiple biomarkers can be detected through repeated cycles, but the biological material degrades and interference increases with each cycle
Solution Approach 1:
The fluorescent label is extracted and removed from the antibody after detection, allowing the antibody to be reused for additional detection cycles without degradation from repeated fluorescent staining and bleaching processes
Solution Approach 2:
The method replaces the optical detection system (fluorescence microscopy) with a mass spectrometry-based detection system that quantifies antibodies bound to biomarkers through MS signal intensity, eliminating the need for fluorescent labeling and its associated degradation issues
2Quantity of substance
If immunofluorescence devices are used for biomarker detection, then up to 4 biomarkers can be detected per cycle, but the results are not quantitative and tissue removal is required
Solution Approach 1:
The patent replaces qualitative immunofluorescence imaging with quantitative mass spectrometry detection, where the MS signal intensity directly correlates with the amount of antibody-bound biomarker, enabling precise quantification of multiple biomarkers simultaneously
Solution Approach 2:
The mass spectrometry-based platform can detect and quantify any number of biomarkers limited only by the panel of antibodies used, rather than being restricted to 4 biomarkers per cycle as in immunofluorescence, while also providing quantitative measurement capability
3Adaptability or versatility
If harsh chemical agents or photobleaching are used to inactivate fluorophores, then fluorescence can be erased for new cycles, but biological material degradation and interference increase
Solution Approach 1:
Instead of using harsh chemicals or photobleaching to inactivate fluorophores, the method extracts and removes the fluorescent label from the antibody through chemical cleavage, allowing the antibody to be recovered and reused without exposing the biological material to degrading conditions
Solution Approach 2:
A cleavable linker acts as an intermediary between the fluorescent label and the antibody, allowing controlled release of the fluorophore without requiring harsh treatment of the antibody or biological material, thus preserving material integrity across multiple cycles
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 the characterization of tumors in their native environment with multiplexed analysis of multiple biomarkers, allowing for infinite cycles of detection and quantification without tissue removal, and provides a valuable tool for tumor characterization and therapy selection.
Implementation Method 1
click-and-release bioorthogonal reaction involving an iminosydnone linker
Implementation Method 2
click-and-release bioorthogonal reaction involving an iminosydnone linker
Implementation Method 3
Detecting, by mass spectrometry, the nature and optionally the amount of the released MS-tags
Data Source
Figure 1A~1B
Figure 2~3
Figure 4~5A
AI summary
The accurate quantification of biomarkers is paramount in modern medicine, particularly in cancer where precise diagnosis is imperative for targeted therapy selection. There is therefore a long felt need for techniques using cell surface specific agents that can be used for identifying cancerous tissue and therefore the need for cell surface specific biomarkers. In this context, the present invention proposes performant multiplexed analysis methods using probes linked to cleavable MS-tag isotopologues by a click-and-release bioorthogonal reaction involving an iminosydnone linker. The great potential of this approach is herein demonstrated in culture cells, in tissues as well as in vivo, thereby unveiling promising diagnostic avenues, especially for cancer cell immunoprofiling.