In Situ Hybridization Detection Using Nucleic Acid Segmentation
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current detection systems for targets in samples, such as in situ hybridization, face limitations due to the complexity and specificity of antibody-based amplification methods, which are laborious, limited by availability of secondary antibodies, and not adaptable for multiple targets or varying signal intensities.
Innovation Solution
The use of nucleic acid hybridization-based compositions and methods that separate probes and detectable labels into distinct units, allowing for flexible interaction through specific hybridization, with adaptor units enhancing signal amplification and compatibility with various targets and labels, enabling detection of multiple targets with differential amplification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Power
If antibody-based amplification methods are used to enhance signal from targets, then signal amplification is achieved, but the method becomes laborious and complex
Solution Approach 1:
The invention divides the detection system into separate functional units: a probe unit that binds to the target and a detectable label unit that provides the signal. These units are separated rather than pre-conjugated, allowing independent optimization and simplifying the overall system design while maintaining signal amplification capabilities through multiple probe bindings.
Solution Approach 2:
The invention creates universal binding units with standardized interfaces (such as biotin-streptavidin or antibody-antigen interactions) that can work with multiple different probes and detectable labels. This multi-functionality eliminates the need for custom conjugation for each probe-label pair, reducing complexity while preserving signal enhancement.
2Power
If secondary antibodies are used for signal amplification, then the number of probes recognizing each target increases, but availability and cross-reactivity limitations arise
Solution Approach 1:
The invention introduces universal intermediary binding units (such as streptavidin or Fc receptors) that mediate between diverse probes and detectable labels. These intermediaries provide standardized binding interfaces, eliminating cross-reactivity issues between different species while maintaining the ability to amplify signals through multiple binding events.
Solution Approach 2:
The system employs universal binding units with broad specificity (e.g., streptavidin binding to biotin, or Fc receptors binding to various antibody isotypes) that can interface with multiple different probe types. This universality ensures availability across different target types while preventing unwanted cross-reactivity through highly specific standardized interactions.
3Power
If antibodies are conjugated to enhance signal, then signal amplification is achieved, but the size of antibodies reduces penetration of reagents
Solution Approach 1:
The invention segments the detection system into smaller, separate units: compact probe molecules (such as nucleic acid probes or small antibodies) and distinct detectable label units. This segmentation eliminates the need for large pre-conjugated antibodies, improving penetration into tissue samples while maintaining signal amplification through multiple separate binding events.
Solution Approach 2:
The invention shifts from a single large conjugated antibody to multiple smaller units that can bind in sequence or in parallel. This dimensional change from one large molecule to multiple smaller interacting molecules improves penetration depth and distribution throughout the sample while achieving equivalent or enhanced signal amplification.
4Power
If multiple layers of molecular interactions are added to increase probes per target, then signal amplification increases, but the system becomes limited to staining of one, two or three different targets
Solution Approach 1:
The invention employs universal binding interfaces (such as biotin-streptavidin or Fc-receptor interactions) that allow the same detectable label unit to work with multiple different probe types. This universality enables simultaneous detection of multiple different targets, each with its own probe, while maintaining signal amplification through the standardized multi-layer interaction framework.
Solution Approach 2:
The system segments detection of different targets into independent probe units that all interface through a common universal binding mechanism. This segmentation allows each target to be detected with its own specific probe while using the same amplification and detection infrastructure, enabling simultaneous multi-target detection without cross-interference.
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 provides a flexible and efficient method for detecting multiple targets in samples, enhancing signal amplification, and normalizing signal intensity, compatible with various detection systems and formats, including in situ hybridization, immunohistochemistry, and flow cytometry.
Implementation Method 1
at least one nucleic acid analog segment of the recognition unit specifically hybridizes to at least one nucleic acid analog segment of the detection unit
Data Source
AI summary
The invention provides compositions and methods for the detection of targets in a sample; in particular, an in situ hybridization (ISH) sample. Probes and detectable labels may be provided in multiple layers in order to increase the flexibility of a detection system, and to allow for amplification to enhance the signal from a target. The layers may be created by incorporating probes and detectable labels into larger molecular units that interact through nucleic acids base-pairing, including peptide-nucleic acid (PNA) base-pairing. Optional non-natural bases allow for degenerate base pairing schemes. The compositions and methods are also compatible with immunohistochemistry (IHC), immunocytochemistry (ICC), flow cytometry, enzyme immuno-assays (EIA), enzyme linked immuno-assays (ELISA), blotting methods (e.g. Western, Southern, and Northern), labeling inside electrophoresis systems or on surfaces or arrays, and precipitation, among other general detection assay formats. The invention is also compatible with many different types of targets, probes, and detectable labels.