Multiplexed Fluorescence Detection Using Temporal Segmentation
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Solution Overview
Problem
Current multiplexed fluorescence detection systems face challenges with cross-talk between channels, limited sensitivity, and dynamic range, particularly in time-resolved fluorescence (TRF) detection, due to overlapping emission signals from lanthanide-based probes.
Innovation Solution
The method employs fluorescent labels with distinct fluorescence emission lifetimes and excitation/emission wavelengths, utilizing spectral and temporal differences to minimize cross-talk, allowing for the simultaneous detection of multiple analytes with reduced background interference and improved sensitivity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If lanthanide-based probes are used for time-resolved fluorescence detection, then sensitivity is improved, but cross-talk between detection channels increases
Solution Approach 1:
The invention segments the detection process by introducing a temporal delay between excitation and measurement. The system excites the lanthanide-based probes and waits for a predetermined period (typically microseconds to milliseconds) before measuring fluorescence emission. This temporal segmentation allows short-lived background fluorescence to decay completely before detection, while the long-lived lanthanide signal is still present, thereby achieving high sensitivity without cross-talk interference
Solution Approach 2:
The invention employs periodic pulsed excitation followed by delayed periodic measurement. The system uses periodic light pulses to excite the probes and implements periodic measurement cycles with predetermined delays. This periodic action pattern enables the system to repeatedly capture the long-lived fluorescence signal after background decay, maintaining high sensitivity while avoiding cross-talk between different detection channels
2Adaptability or versatility
If multiple fluorophores are detected simultaneously, then multiplexing capability is improved, but cross-talk between channels increases
Solution Approach 1:
The invention adds a temporal dimension to the detection process by implementing time-resolved measurement. Instead of relying solely on spectral separation in the wavelength domain, the system introduces time as an additional dimension for signal discrimination. By measuring fluorescence at different time points after excitation and applying mathematical transformations, the system can resolve multiple fluorophores with overlapping spectra, achieving high multiplexing capability with minimal cross-talk
3Object-affected harmful factors
If time-resolved fluorescence detection is used, then background rejection is improved, but detection complexity increases
Solution Approach 1:
The invention changes the temporal parameter of fluorescence detection by introducing a predetermined delay between excitation and measurement. Instead of measuring fluorescence immediately, the system waits for a specific time period allowing background fluorescence to decay. This parameter change in the time domain enables effective background rejection while maintaining relatively simple detection hardware and procedures
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 achieves low cross-talk levels below 1%, enabling accurate multiplexed detection of up to four independent fluorophores with enhanced sensitivity and dynamic range, improving quantitation and stability in protein analysis.
Implementation Method 1
exciting the first fluorescent label with a first excitation light having the first excitation wavelength, wherein the first fluorescent label emits a first detection signal having the first emission wavelength
Implementation Method 2
the first fluorescent label has a first fluorescence emission lifetime which is at least 3 times longer than background fluorescence emission lifetimes
Data Source
AI summary
The present invention is directed to multiplexed fluorescence detection, including time-resolved fluorescence (TRF) detection. A combination of spectral and temporal differences in fluorescence emission and spectral differences in excitation is used to enhance the ability to separate signals in an assay from multiple fluorescent labels. Different classes of labels may be utilized, including upconversion phosphors as well as lanthanide chelates and transition metal chelates. The methods may be implemented in optical plate readers, including cartridge-based multi-mode readers.