Photoactivated Borate Bleaching for Multiplex Biological Detection
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Solution Overview
Problem
Current methods for analyzing biological samples are limited in their ability to detect multiple targets simultaneously due to fluorescence-based detection systems, requiring additional samples and struggling with the stability and efficiency of borate compounds used for signal bleaching, especially with xanthene dyes.
Innovation Solution
A novel method employing a signal cycling process with a borate compound acting as an electron transfer agent for photo-induced chemical bleaching, allowing the reuse of fluorescent dyes to detect multiple markers in a biological sample by irradiating with visible light, effectively bleaching xanthene dyes without the need for special storage or handling.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If anionic borates with cationic counter ion are used for photobleaching, then bleaching efficiency is improved, but stability to oxidation deteriorates and special storage/handling is required
Solution Approach 1:
The patent changes the chemical parameters of the borate compound by introducing bulky hydrophobic groups (such as t-butyl groups) and adjusting the charge distribution. This modifies the borate's chemical stability while maintaining its photobleaching capability, allowing it to resist oxidation without requiring special storage conditions.
Solution Approach 2:
The patent creates a composite structure within the borate molecule by combining electron-withdrawing groups with bulky hydrophobic groups. This composite molecular structure achieves both high bleaching efficiency and oxidation stability, resolving the contradiction between productivity and reliability.
2Productivity
If multiple targets are detected simultaneously using fluorescence-based detection, then analysis throughput is improved, but the number of detectable targets is limited by the detection system
Solution Approach 1:
The patent employs periodic photobleaching cycles where fluorophores are sequentially activated and then bleached. This periodic action allows the same detection channel to be reused multiple times for different targets, enabling detection of many more targets than the number of simultaneously detectable fluorophores.
Solution Approach 2:
The patent temporarily discards the fluorophore signal by photobleaching it after detection, then recovers the detection channel for reuse with a different fluorophore. This cycle of discarding and recovering allows multiplexed detection beyond the limits of simultaneous fluorophore detection.
3Quantity of substance
If additional biological samples are used for further analysis, then sufficient sample material is available, but sample availability is limited and sample integrity must be maintained
Solution Approach 1:
The patent performs preliminary detection of multiple targets in a single sample using sequential photobleaching cycles before any further analysis is needed. This preliminary multiplexed detection maximizes the information obtained from the limited sample, eliminating the need for additional samples.
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 high-throughput multiplex sample analysis, allowing for the sequential detection of multiple targets with improved stability and efficiency, reducing the need for additional samples and maintaining the integrity of the biological sample.
Implementation Method 1
a photo-induced chemical bleaching (PICB) step allows the same signal generators, e.g., fluorophores, to be reused in the subsequent cycle to detect additional markers
Implementation Method 2
The PICB step can include applying a borate compound of Formula I, which acts as an electron transfer agent, and initiating a photoreaction
Implementation Method 3
initiating a photoreaction, e.g., by irradiating the sample with visible light
Data Source
Figure 1(a)~1(e)
Figure 2(a)~2(b)
Figure 3(a)~3(b)
AI summary
Methods comprising the use of photoactivated chemical bleaching for detecting multiple targets in a biological sample are provided. The methods include the steps of providing a biological sample containing multiple targets, binding at least one probe to one or more target present in the sample, and observing a signal from the probe. The method further includes the steps of contacting the sample comprising the bound probe with a cationic or zwitterionic borate compound and irradiating the sample, thereby initiating a photoreaction that substantially inactivates the probe by photoactivated chemical bleaching. The method further includes the steps of binding at least one probe to one or more target present in the sample, and observing a signal from the probe. The process of binding, observing and bleaching may be iteratively repeated.