BRET Sensor Molecule for Precise Point-of-Care Analyte Quantification
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Solution Overview
Problem
Current ratiometric luminescent sensors face limitations in achieving significant ratio changes and are prone to errors in complex samples like serum due to light absorption, lacking portable and precise point-of-care quantification capabilities for analytes such as drugs and proteins.
Innovation Solution
Development of a bioluminescence resonance energy transfer (BRET) sensor molecule comprising a luciferase enzyme attached to a binding protein, with a synthetic regulatory molecule that changes conformation upon analyte binding, enhancing signal changes and minimizing interference from light-absorbing samples by immobilization on a solid carrier.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional FRET-based ratiometric sensors are used, then sensor readout is independent of sensor concentration, but ratio changes are small and RET efficiency in the closed state cannot be increased
Solution Approach 1:
The patent changes the energy transfer mechanism from FRET to BRET by replacing the fluorescent protein donor with a luciferase enzyme. This parameter change increases the RET efficiency in the closed state, producing larger ratio changes (up to 10-fold or greater) while maintaining the ratiometric measurement capability that eliminates sensor concentration dependence.
Solution Approach 2:
The patent substitutes the fluorescent protein-based FRET mechanism with a bioluminescence-based BRET mechanism. This substitution replaces the need for external light excitation and fluorescent emission with enzyme-catalyzed chemiluminescence, achieving higher RET efficiency and larger signal changes without increasing structural complexity.
2Measurement precision
If ratiometric RET sensors are used for quantification in complex samples like serum, then analyte detection is possible, but light absorption by the sample causes artifacts and unreliable outcomes
Solution Approach 1:
The patent changes the emission wavelength parameter by selecting fluorophores that emit in the red or near-infrared region (e.g., 633 nm, 680 nm, 750 nm). These longer wavelengths experience minimal absorption by biological samples like serum, eliminating the light absorption artifacts that plague sensors using visible wavelength fluorophores while maintaining accurate analyte quantification.
Solution Approach 2:
Instead of trying to make the sample transparent to the detection wavelength, the patent inverts the approach by selecting detection wavelengths that naturally penetrate biological samples. This inversion of the wavelength selection strategy transforms the problematic light absorption issue into a non-issue by operating in the optical window where biological tissues are transparent.
3Measurement precision
If BRET-based sensors are developed for point-of-care quantification, then sensitivity is improved, but portable detection devices with adequate precision are not currently available
Solution Approach 1:
The patent substitutes complex optical excitation systems with a simple bioluminescence readout system. The luciferase enzyme generates light without external excitation, eliminating the need for light sources, excitation filters, and complex optical paths. This substitution enables portable detection devices to achieve high measurement precision using only a camera or simple photodetector to capture the emitted light.
Solution Approach 2:
The sensor system is self-powered through the luciferase-catalyzed chemiluminescence reaction. The sensor molecule itself generates the light signal needed for detection without requiring external light sources or power supplies. This self-service capability dramatically simplifies the portable detection system, enabling precise point-of-care measurements with minimal device complexity.
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
The BRET sensor achieves significantly increased ratio changes and reliable quantification of analytes in complex samples, enabling precise point-of-care monitoring and therapeutic drug monitoring using a portable device like a smartphone.
Implementation Method 1
a bioluminescent donor protein and a fluorescent acceptor... analyzing energy resonance transfer... bioluminescence resonance energy transfer (BRET)
Implementation Method 2
bioluminescence resonance energy transfer (BRET)... energy resonance transfer... RET efficiency
Data Source
AI summary
The invention relates to the field of in vitro detection methods using luminescence. Provided is a sensor molecule for detecting an analyte of interest in a sample using bioluminescence resonance energy transfer (BRET), the sensor molecule comprising a proteinaceous moiety tethered to a synthetic regulatory molecule. Also provided is an analytical device comprising a sensor and methods using the sensor molecule.