Signaling Conjugates for Multiplexed In Situ Detection
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Solution Overview
Problem
Current in situ hybridization (ISH) and immunohistochemistry (IHC) techniques face challenges in preserving tissue context and specificity, leading to false positives/negatives due to contamination and limited multiplexing capabilities, with traditional chromogenic substrates experiencing background interference and color limitations.
Innovation Solution
Development of signaling conjugates with latent reactive moieties and chromogenic moieties that absorb light selectively, enabling enhanced detection sensitivity and multiplexing capabilities, compatible with automated slide staining instruments, and allowing for the use of bright-field microscopy for target detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If traditional chromogenic substrates are used for ISH and IHC detection, then the detection method is simple and cost-effective, but background interference occurs and color options are limited
Solution Approach 1:
The patent modifies the chromogenic substrate by conjugating it to a carrier protein, fundamentally changing its physical and chemical parameters. This conjugation increases molecular size, alters solubility characteristics, and prevents aggregation, thereby eliminating background interference while preserving the simplicity of chromogenic detection.
Solution Approach 2:
The invention creates a composite chromogenic substrate by combining small molecule chromogens with carrier proteins. This composite structure merges the high chromogenicity of small molecules with the solubility and non-aggregating properties of proteins, resolving the contradiction between simple detection and background-free signals.
2Ease of manufacture
If traditional chromogenic substrates are used, then the detection method is cost-effective, but multiplexing capability is limited
Solution Approach 1:
By changing the molecular parameters of chromogenic substrates through protein conjugation, the patent enables distinct spectral properties while maintaining cost-effectiveness. Multiple conjugated substrates with different chromogenic moieties can be used simultaneously in the same tissue section, enabling multiplexing without requiring expensive fluorescent instruments.
Solution Approach 2:
The carrier protein-conjugated chromogenic substrates serve multiple functions: they provide cost-effective chromogenic detection, enable multiplexing through spectral differentiation, and maintain compatibility with standard IHC/ISH protocols. This multi-functionality resolves the contradiction between cost-effectiveness and versatility.
3Measurement precision
If mRNA extraction is performed for PCR or microarray analysis, then gene expression can be quantified, but tissue specificity and cellular context are lost
Solution Approach 1:
The patent replaces the mechanical process of mRNA extraction and amplification with a direct chromogenic detection method. By using enzyme-conjugated chromogenic substrates that deposit color directly at the target location in situ, the method eliminates the need for tissue dissociation and mRNA extraction, thereby preserving tissue context while enabling precise gene expression quantification.
4Loss of information
If conventional IHC/ISSH protocols are used, then tissue context is preserved, but false positive or negative results occur due to contaminating non-tumor cells
Solution Approach 1:
The patent changes the detection parameters by using highly specific enzyme-conjugated chromogenic substrates with optimized binding characteristics. This increases the signal-to-background ratio and reduces cross-reactivity with non-tumor cells, thereby improving reliability while maintaining tissue context.
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 signaling conjugates provide improved detection sensitivity and multiplexing capabilities, reducing background interference and enabling the detection of multiple targets with distinct colors, thereby enhancing the accuracy and reproducibility of ISH and IHC assays.
Implementation Method 1
chromogenic moieties that absorb light selectively, enabling enhanced detection sensitivity and multiplexing capabilities
Implementation Method 2
The enzyme catalyzes conversion of the latent reactive moiety into a reactive moiety which covalently binds to the biological sample
Data Source
AI summary
Disclosed herein are embodiments of a signaling conjugate, embodiments of a method of using the signaling conjugates, and embodiments of a kit comprising the signaling conjugate. The disclosed signaling conjugate comprises a latent reactive moiety and a chromogenic moiety that may further comprise a linker suitable for coupling the latent reactive moiety to the chromogenic moiety. The signaling conjugate may be used to detect one or more targets in a biological sample and are capable of being covalently deposited directly on or proximally to the target. Particular disclosed embodiments of the method of using the signaling conjugate comprise multiplexing methods.