Oligonucleotide-Mediated Multiplexed Analyte Detection
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Solution Overview
Problem
Conventional methods for detecting and quantifying multiple target analytes in biological samples are limited by the ability to distinguish and amplify signals from different dye probes, leading to challenges in multiplexed analysis and sample integrity, especially when performing multiple labeling and detection cycles.
Innovation Solution
The method involves using oligonucleotides conjugated to binding species and reactive species to selectively label target analytes, allowing for serial cycles of labeling and detection without disrupting antibody-biomarker binding, using mild dehybridization conditions to remove agents and maintain sample integrity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If multiple dye-conjugated antibodies are used to detect multiple target analytes simultaneously, then the ability to identify multiple analytes is improved, but the signal distinction and amplification capability deteriorates
Solution Approach 1:
The detection process is segmented into multiple sequential cycles. In each cycle, only one or a few specific dye-conjugated antibodies are applied to detect specific target analytes, rather than applying all antibodies simultaneously. This segmentation allows for clear signal distinction in each cycle while maintaining the ability to detect multiple different analytes across multiple cycles, thereby resolving the contradiction between multiplexed analysis capability and signal distinction
Solution Approach 2:
The patent implements periodic action through repeated cycles of antibody application, detection, and removal. Each cycle consists of applying specific antibodies, detecting signals, removing antibodies, and then repeating with different antibodies. This periodic cycling enables multiple analytes to be detected over time with clear signal distinction in each period, solving the contradiction between detecting multiple analytes and maintaining signal clarity
2Adaptability or versatility
If multiple labeling and detection cycles are performed to detect more target analytes, then the multiplexed analysis capability is improved, but the sample integrity deteriorates
Solution Approach 1:
The patent applies preliminary action by using oligonucleotide-conjugated antibodies that hybridize to complementary oligonucleotides on the target analytes before detection. This preliminary hybridization creates a stable complex that allows for repeated binding and removal cycles without disrupting the antibody-target binding, thus enabling multiple labeling cycles while maintaining sample integrity
Solution Approach 2:
The patent introduces oligonucleotides as intermediary molecules between the antibodies and target analytes. These oligonucleotide intermediaries enable reversible binding through hybridization, allowing antibodies to be removed after detection without affecting the target analytes. This intermediary mechanism permits multiple detection cycles while preserving sample integrity, resolving the contradiction between multiplexed analysis and sample reliability
3Ease of operation
If conventional antibody removal methods are used after labeling, then the second agents can be removed, but the first agents remain bound and sample integrity is compromised
Solution Approach 1:
The patent extracts the removal mechanism from general antibody-antigen binding and applies it specifically to oligonucleotide-oligonucleotide hybridization. By using oligonucleotide intermediaries, the patent enables selective removal of antibody-oligonucleotide complexes through DNA/RNA denaturation conditions that do not affect the protein-based antibody-target binding. This selective extraction of removable components allows easy agent removal while preserving sample integrity
Solution Approach 2:
The patent utilizes parameter changes in the chemical state of oligonucleotides to enable removal. By changing conditions such as temperature or chemical environment to induce oligonucleotide denaturation, the patent allows reversible separation of oligonucleotide-conjugated antibodies from their targets. This parameter-based control enables easy removal of second agents while maintaining the stability of first agents and sample integrity
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 the selective and accurate identification and quantification of multiple target analytes with minimal cross-species labeling, allowing for extended cycles of analysis and improved signal amplification, thereby overcoming the limitations of conventional methods.
Implementation Method 1
The second oligonucleotide hybridizes to the first oligonucleotide of one of the first agents, localizing the second agent in the sample at positions corresponding to the target analyte associated with that first agent
Implementation Method 2
A reaction between the reactive species and a labeling agent that is introduced deposits the labeling agent in proximity to that target analyte
Implementation Method 3
The second agent can then be removed by dehybridization under relatively mild conditions, ensuring that each of the first agents remains bound to the sample
Data Source
AI summary
The disclosure features methods that include: contacting a biological sample having a first target analyte with a first agent, where the first agent includes a first binding species that specifically binds to the first target analyte, and a first oligonucleotide conjugated to the binding species; contacting the biological sample with a second agent, where the second agent includes a first reactive species and a second oligonucleotide conjugated to the first reactive species, to hybridize at least a portion of the second oligonucleotide to at least a portion of the first oligonucleotide; and contacting the biological sample with a first labeling species, where the first labeling species reacts with the first reactive species to deposit the first labeling species or a derivative thereof in the biological sample.