Photo-cleavable Probe Multiplexing in FFPE Tissue Analysis
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Solution Overview
Problem
Current methods for detecting proteins in formalin-fixed, paraffin-embedded (FFPE) tissue samples face challenges due to low sensitivities, narrow dynamic ranges, and limited multiplexing capabilities, as well as the destruction of tissue context during isolation processes, making it difficult to analyze multiple targets simultaneously.
Innovation Solution
A method involving probes with a photo-reactive moiety, specific binding moiety, and detection tag is used, where the probe binds to targets in the sample, and upon selective irradiation, the detection tag is released and extended by a polymerase, allowing for contextual molecular diagnostics and multiplexed analysis without disrupting the tissue sample.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If sample isolation methods (macroscopic dissection or laser capture microdissection) are used to remove testable analytes from tissue, then analytes can be analyzed, but tissue context is disrupted and tissue destruction occurs
Solution Approach 1:
The invention extracts only the necessary information (analytes) from the tissue sample through in situ hybridization and signal amplification, without physically removing or disrupting the tissue structure. This allows analyte recovery while preserving tissue context and morphology for subsequent analysis.
Solution Approach 2:
The patent uses intermediary molecules (complementary DNA probes, amplification reagents) that bind to target analytes within the tissue and carry detectable signals, enabling analyte detection without physical disruption of tissue architecture. The intermediaries facilitate information transfer from the analyte to the detector while leaving the tissue intact.
2Adaptability or versatility
If highly multiplexed tests are performed to detect multiple targets in a single sample, then diagnostic capability is expanded, but sample isolation is required which disrupts tissue context
Solution Approach 1:
The patent employs universal in situ hybridization protocols that can simultaneously detect multiple different analytes (proteins, nucleic acids) using different labeled probes. This multi-functional approach enables highly multiplexed testing while maintaining tissue integrity and context, as all detections occur within the same tissue section without requiring physical isolation.
3Loss of information
If conventional immunohistochemistry is used for protein detection, then tissue context is preserved, but sensitivity and dynamic range are limited
Solution Approach 1:
The patent performs preliminary signal amplification through in situ hybridization and enzymatic amplification steps before final detection. This preliminary action enhances the sensitivity and dynamic range of protein detection while maintaining tissue context, as the amplification occurs in situ within the tissue section rather than requiring analyte extraction.
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
This approach enables sensitive and multiplexed target analysis while preserving the tissue context, allowing for the detection of multiple targets with improved sensitivity and resolution, facilitating next-generation sequencing and array technology applications.
Implementation Method 1
irradiating the selected region of the tissue sample with light of a wavelength and an intensity sufficient to cause the photo-reactive moiety to react, thereby freeing the detection tag for further reaction
Implementation Method 2
the detection tag is an extendable primer, the photo-reactive moiety is a photo-cleavable blocking group, the enzyme is a polymerase, and the change is an extension of the primer sequence
Implementation Method 3
contacting a tissue sample with a probe comprising a photo-reactive moiety, a specific binding moiety to a target, and a detection tag using conditions sufficient to facilitate binding of the specific binding moiety to a target in the sample
Implementation Method 4
adding a template sequence to the tissue sample, wherein the template sequence is configured to specifically hybridize with the detection tag
Data Source
Figure 1(A)~1(E)
Figure 2(A)~2(B)
Figure 3~4
AI summary
The present disclosure concerns novel methods for analyzing biological samples using photo-cleavable moieties that facilitate target detection and/or biomarker isolation. In some embodiments, the method concerns using a probe comprising a photo-cleavable moiety and a selective irradiation technique for activating/cleaving the photo-cleavable moiety thereby providing facile isolation of the probe or probe components. Also disclosed herein is a system and kit for implementing the methods disclosed herein.