Multiplexed Fluorescence Detection Using Infrared Dyes
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Solution Overview
Problem
Current multiplexed fluorescence detection systems face challenges with cross-talk between lanthanide-based probes, limiting their ability to detect multiple signals from the same spatial location due to overlapping emission peaks, resulting in only one truly sensitive channel while the other is limited by background signals.
Innovation Solution
The use of fluorescent labels with distinct fluorescence emission lifetimes and excitation/emission wavelengths, such as combining long-lifetime lanthanide chelates with upconverting phosphors and infrared dyes, allows for time-domain and wavelength-domain resolved multiplexed time-resolved fluorescence detection, minimizing cross-talk to below 1% by exploiting temporal and spectral differences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If lanthanide-based probes are used for multiplexed fluorescence detection, then sensitivity is improved, but cross-talk between probes increases due to overlapping emission peaks
Solution Approach 1:
The patent transitions from spectral-domain separation to temporal-domain separation by utilizing time-resolved fluorescence detection. Different fluorophores are differentiated by their fluorescence emission lifetimes rather than by emission wavelength, enabling multiplexed detection without spectral cross-talk
Solution Approach 2:
The invention changes the detection parameter from emission wavelength to fluorescence emission lifetime. By measuring the temporal decay characteristics of fluorescence signals, the system can distinguish between multiple fluorophores with overlapping spectra, achieving high sensitivity while eliminating cross-talk
2Adaptability or versatility
If multiple fluorophores with different emission wavelengths are used for multiplexing, then the ability to detect multiple analytes is improved, but spectral overlap increases causing cross-talk
Solution Approach 1:
The patent moves the discrimination dimension from spectral (wavelength-based) to temporal (time-based). By exploiting differences in fluorescence lifetimes, multiple analytes can be detected simultaneously without the spectral overlap problems that plague wavelength-based multiplexing approaches
Solution Approach 2:
The invention introduces time as an intermediary parameter for signal separation. Rather than directly separating signals by wavelength, the system uses temporal decay characteristics as a mediating property to distinguish between fluorophores, enabling clean signal separation even when emission spectra overlap
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 improved quantitation and reduced background interference, allowing for the simultaneous detection of multiple analytes with high sensitivity and dynamic range, overcoming the limitations of existing systems by achieving low cross-talk and enhanced signal separation.
Implementation Method 1
fluorescent labels with distinct fluorescence emission lifetimes and excitation/emission wavelengths, such as combining long-lifetime lanthanide chelates with upconverting phosphors and infrared dyes, allows for time-domain and wavelength-domain resolved multiplexed time-resolved fluorescence detection
Implementation Method 2
combining long-lifetime lanthanide chelates with upconverting phosphors and infrared dyes
Data Source
AI summary
Methods and systems for time-domain and wavelength domain resolved multiplexed time-resolved fluorescence of a sample having at least one of a first fluorescent label bound to a first analyte and a second fluorescent label bound to a second analyte. The sample also includes a third fluorescent label bound to a third analyte and a fourth fluorescent label bound to a fourth analyte. The first and second fluorescent labels emit wavelengths having a first and second lifetime. The third fluorescent label comprises an upconverting phosphor (UCP) emitting at a wavelength having a third lifetime, and the fourth fluorescent label comprises an infrared dye emitting at wavelength having a fourth lifetime. The methods and systems measure as a function of time intensities of at least one of more of the first, second, third, and fourth fluorescent labels.