Sequential Biological Target Detection via Fluorescence Bleaching
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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.
Innovation Solution
The method involves using probes with binders coupled to enzymes and fluorescent signal generators, where the bound probes are reacted with enzyme substrates and an oxidizing agent, allowing for iterative detection and analysis of multiple targets in a single sample by modifying and reusing the signal generators.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If multiple targets are detected simultaneously using fluorescence-based detection systems, then the number of detectable targets increases, but the complexity of the detection system and sample preparation requirements increase
Solution Approach 1:
The detection process is segmented into sequential steps where one target is detected, the signal is destroyed, and then the next target is detected. This segmentation allows multiple targets to be detected using a single detection channel without requiring complex multi-channel systems, thus reducing device complexity while maintaining the ability to detect multiple targets.
Solution Approach 2:
The method employs periodic action by cycling through detection-destroy-reaction sequences for each target. The fluorescent signal generator is periodically activated and then destroyed, allowing the detection system to focus on one target at a time in a rhythmic sequence, enabling multiple target detection without simultaneous signal interference.
2Measurement precision
If multiple biological samples are used to analyze different targets, then the ability to determine relative characteristics improves, but the sample availability and analysis efficiency deteriorate
Solution Approach 1:
A single biological sample is made universal for analyzing multiple different targets through sequential detection. The same sample serves multiple functions by allowing repeated detection cycles with different probes, eliminating the need to use multiple separate samples and thereby improving analysis efficiency while maintaining measurement precision.
Solution Approach 2:
The fluorescent signal generator is intentionally destroyed after each detection cycle to prevent signal interference in subsequent detections. This controlled discarding of the signal generator allows the sample to be reused for the next target detection, improving productivity while maintaining the ability to analyze relative characteristics through sequential measurements.
3Productivity
If fluorescent signal generators are reused for multiple detections, then sample utilization improves, but signal interference between detections increases
Solution Approach 1:
The potential harmful effect of signal persistence is converted into a benefit by intentionally destroying the fluorescent signal generator after each detection. This controlled destruction eliminates signal interference between detections, allowing the same sample to be utilized for multiple targets without contamination from previous signals, thus improving both productivity and measurement accuracy.
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 detection of multiple targets in a single biological sample with minimal sample disruption, providing spatial information and allowing for multiple analyses without stripping probes, thus overcoming the limitations of existing techniques.
Implementation Method 1
applying to the sample a solution containing an oxidizing agent that substantially inactivates both the fluorescent signal generator and the enzyme
Implementation Method 2
reacting the bound probe with an enzyme substrate coupled to a fluorescent signal generator
Implementation Method 3
enzyme substrate coupled to a fluorescent signal generator
Implementation Method 4
binding at least one probe having a binder coupled to an enzyme to one or more target present in the sample
Data Source
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AI summary
Methods for probing multiple targets in a biological sample are provided. The methods include the steps of providing a sample containing multiple targets, binding at least one probe having a binder coupled to an enzyme to one or more target present in the sample, and reacting the bound probe with an enzyme substrate coupled to a fluorescent signal generator. The methods include the steps of observing a signal from the fluorescent signal generator and applying to the sample a solution containing an oxidizing agent that substantially inactivates both the fluorescent signal generator and the enzyme. The methods further include the steps of binding at least one probe having a binder coupled to an enzyme to one or more target present in the sample of step, reacting the bound probe with an enzyme substrate coupled to a fluorescent signal generator; and observing a signal from the fluorescent signal generator. The methods disclosed herein also provide for multiple iterations of binding, observing, and oxidizing for deriving information about multiple targets in a single sample. An associated kit is also provided.