Sequential Multiplexed Biological Sample Analysis
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Solution Overview
Problem
Current methods for analyzing biological samples can only detect a limited number of targets simultaneously, requiring multiple samples and limiting the ability to determine the presence, absence, concentration, and spatial distribution of multiple biological targets, especially when sample quantity is limited.
Innovation Solution
A method involving a tissue section where fluorophore-labeled antibody probes and control probes are bound to targets, followed by selective inactivation of the fluorophore labels using an oxidizing agent, allowing for sequential detection of multiple targets in the same sample without stripping the probes, enabling the detection of up to four or more targets using fluorescent-based detection systems.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If multiple targets are detected simultaneously using fluorescent-based detection systems, then the number of detectable targets increases, but the limitation is imposed by the detection system's capacity to resolve multiple fluorescent signals
Solution Approach 1:
The detection process is segmented into sequential steps where fluorophore labels are inactivated between detections. This allows the same detection system to measure multiple targets one at a time without requiring simultaneous signal resolution, thereby overcoming the detection system's inherent limitation in resolving multiple fluorescent signals at once.
Solution Approach 2:
The method employs periodic inactivation of fluorophore labels using oxidizing agents between sequential detections. This periodic action enables repeated use of the same detection channel for different targets, effectively increasing the number of detectable targets without requiring proportional increases in detection system complexity.
2Measurement precision
If additional biological samples are used to analyze multiple targets, then the ability to determine relative characteristics improves, but the sample quantity available for analysis is reduced
Solution Approach 1:
The method performs preliminary inactivation of fluorophore labels after each detection step, preparing the sample for the next detection without requiring additional physical samples. This preliminary chemical modification enables sequential analysis of multiple targets in the same original sample, preserving sample quantity while maintaining measurement precision for relative characteristics.
Solution Approach 2:
The fluorophore labels are selectively inactivated and discarded after serving their detection purpose, while the sample and probes are recovered for reuse. This allows the same biological sample to be analyzed multiple times for different targets, maximizing the utility of limited sample quantity while obtaining comprehensive relative characteristics.
3Adaptability or versatility
If fluorophore labels are stripped from probes to enable sequential detection, then multiple targets can be detected in the same sample, but the probes are damaged and cannot be reused
Solution Approach 1:
The inactivation process is applied locally and selectively to the fluorophore labels while leaving the probes intact. By targeting only the fluorophore portion for oxidation and inactivation, the method enables sequential detection without damaging the probe structure, thereby maintaining probe reliability and enabling its reuse for subsequent detections.
Solution Approach 2:
Oxidizing agents serve as intermediaries that selectively modify the fluorophore labels to inactivate them, while the probes act as carriers that remain unaffected. This intermediary mechanism allows sequential detection capability to be achieved without directly damaging the probes, preserving their integrity for repeated use.
4Loss of information
If multiple targets are analyzed in a single sample, then spatial distribution information is obtained, but existing methods require multiple separate samples
Solution Approach 1:
The method merges multiple detection operations into a single sample by sequentially inactivating fluorophore labels and performing repeated detections. This combining approach enables analysis of multiple targets and preservation of spatial distribution information in one original sample, eliminating the need for multiple separate samples that would be required by conventional simultaneous detection methods.
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
Enables the analysis of multiple targets in a single biological sample with minimal impact on sample integrity, allowing for spatial information and efficient use of limited sample quantities, facilitating multiplexed analysis and overcoming the limitations of existing detection methods.
Implementation Method 1
applying to the sample in step (d) a solution comprising an oxidizing agent that selectively inactivates the fluorophore label(s) of the at least one fluorophore-labeled antibody probe and not the control probe(s)
Data Source
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AI summary
Methods and kits for probing multiple targets in a biological sample are provided. The methods include the steps of (a) providing a biological sample containing multiple targets, (b) binding at least one fluorescent probe to one or more targets present in the sample; (c) binding at least one control probe to one or more targets present in the sample; (d) detecting at least one signal from the at least one fluorescent probe bound in step (b) and detecting at least one control signal from the at least one control probe bound in step (c); (e) applying to the sample in step (d) a solution comprising an oxidizing agent that selectively inactivates the fluorescent probe and not the control probe; (f) binding at least one fluorescent probe to one or more targets present in the sample of step (e); and (g) detecting at least one signal from the at least one fluorescent probe bound in step (f). An associated kit is also provided.