Sequential Biomarker Detection Probe Sets Without Coverslip Removal
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Solution Overview
Problem
Existing multiplex immunohistochemistry methods require multiple rounds of imager strand application and coverslip removal for each detection round, increasing manipulation and time required for analyzing biomarkers in tissue samples.
Innovation Solution
A method involving probe-sets with nucleic acid barcodes and cleavage agents that allow simultaneous application of probes and sequential detection without coverslip removal, using complementary nucleic acid sequences and cleavage agents to release and activate labels for multiplex detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If multiple rounds of imager strand application and coverslip removal are used for each detection round, then sequential detection of multiple biomarkers is achieved, but manipulation time and handling steps increase
Solution Approach 1:
The probe is pre-designed with a quench moiety and cleavage site before the detection process. The quench moiety is attached to the probe in advance, and the cleavage site is prepared for specific cleavage agent action. This preliminary preparation allows the probe to remain attached to the target throughout multiple detection rounds without requiring coverslip removal, as the quenching function is already in place and activated only when needed through cleavage agent application.
Solution Approach 2:
The quench moiety is extracted as a separate functional component from the probe structure. By removing the quenching function as a distinct element that can be independently controlled through cleavage, the system allows the probe to maintain its binding function while the quenching is selectively removed only when detection is required, eliminating the need for repeated coverslip removal and manipulation.
2Adaptability or versatility
If coverslip removal is performed between detection rounds, then imager strand exchange is enabled, but handling complexity and time consumption increase
Solution Approach 1:
The probe system performs self-quenching through the integrated quench moiety that automatically deactivates the fluorescent signal after detection without requiring external intervention or coverslip removal. The cleavage agent selectively removes the quench moiety only when detection is needed, allowing the system to service itself through chemical modification rather than mechanical manipulation, thereby reducing handling complexity.
Solution Approach 2:
The mechanical operation of coverslip removal and imager strand exchange is replaced by a chemical mechanism using cleavage agents. Instead of physically removing the coverslip to access the sample for strand exchange, the system uses chemical cleavage to remove the quench moiety and activate detection, substituting mechanical handling with chemical transformation to achieve the same functional outcome.
3Productivity
If a single probe is used for multiple detection rounds, then manipulation time is reduced, but signal activation and specificity control become challenging
Solution Approach 1:
Different regions of the probe molecule are assigned different functional properties: the 5' end contains the fluorophore for signal generation, the middle region contains the quench moiety for signal control, and the 3' end contains the cleavage site for selective activation. This local differentiation of functional qualities within the single probe structure allows independent control of signal activation without requiring multiple probe types or complex handling procedures.
Solution Approach 2:
The probe's functional state is changed by altering the chemical environment at the cleavage site through application of specific cleavage agents. By changing the chemical parameter (introduction of cleavage agent), the system transitions the probe from a quenched inactive state to an activated detectable state, providing precise control over signal activation while maintaining the same physical probe structure throughout multiple detection rounds.
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
Reduces manipulation and time required for biomarker analysis by enabling simultaneous probe application and sequential detection of multiple biomarkers without coverslip removal, enhancing efficiency and reducing handling steps.
Implementation Method 1
a cleavage site for a first cleavage agent, wherein the first cleavage agent is capable of releasing the first label
Implementation Method 2
a cleavage site for the first cleavage agent, wherein the first cleavage agent is capable of releasing the quench moiety, whereby the second label is rendered detectable
Implementation Method 3
a nucleic acid sequence complementary to a nucleic acid barcode of a corresponding target-specific binding partner
Data Source
AI summary
Compositions, kits and methods for detecting a plurality of targets are provided herein. A probe-set composition is provided, including one or more first probes and one or more second probes. Each of the first probe includes a nucleic acid sequence complementary to a nucleic acid barcode of a corresponding target-specific binding partner, a first label, and a cleavage site for a first cleavage agent, wherein the first cleavage agent is capable of releasing the first label. Each of the second probes includes a nucleic acid sequence complementary to a nucleic acid barcode of a corresponding target-specific binding partner, a second label, a quench moiety that renders the second label undetectable, and a cleavage site for the first cleavage agent. The first cleavage agent is capable of releasing the quench moiety, whereby the second label is rendered detectable.