Fluorescent Protein Complex for Dual Chemiluminescence and Fluorescence Detection
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Solution Overview
Problem
Current bioluminescent enzymes are unstable to heat and lack both chemiluminescence and fluorescence capabilities, limiting their application in simultaneous detection and measurement.
Innovation Solution
Development of a novel fluorescent protein (bFP) with chemiluminescence activity, composed of a calcium-binding photoprotein, coelenteramid, and calcium ions, which also exhibits fluorescence, allowing for both chemiluminescence and fluorescence detection, and its derivative (gFP) that can be converted back to a calcium-binding photoprotein for instantaneous light emission.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If conventional bioluminescent enzymes are used, then chemiluminescence detection is possible, but they are unstable to heat and lack fluorescence capability
Solution Approach 1:
The patent merges chemiluminescence activity and fluorescence capability into a single photoprotein complex. The photoprotein contains both a luminescent substrate (coelenterazine) and a fluorescent chromophore (coelenteramide), enabling the same molecule to perform both chemiluminescence catalysis and fluorescence emission, thereby resolving the contradiction between detection versatility and thermal stability
Solution Approach 2:
The invention creates a composite photoprotein structure consisting of apoprotein, coelenterazine, and coelenteramide in specific stoichiometric ratios. This composite material combines the heat-resistant properties of the protein framework with the dual optical capabilities of the embedded chromophores, achieving both thermal stability and multi-mode detection
2Adaptability or versatility
If conventional bioluminescent enzymes are used, then chemiluminescence activity is achieved, but simultaneous fluorescence detection is not possible
Solution Approach 1:
The photoprotein complex serves multiple functions simultaneously: it acts as a chemiluminescence catalyst (luciferase activity), a fluorescent marker, and a calcium indicator. The coelenteramide chromophore within the complex emits fluorescence when excited by light, while also being the product of the chemiluminescence reaction, enabling dual detection modes from a single molecular entity
3Illumination intensity
If calcium-binding photoprotein reacts with calcium ions, then instantaneous light emission occurs, but the reaction is too fast for controlled observation
Solution Approach 1:
The patent uses a substoichiometric amount of calcium ions relative to the photoprotein concentration. This partial action approach ensures that not all photoprotein molecules react simultaneously, creating a sustained, controlled light emission and fluorescence signal that persists longer than the instantaneous burst, allowing for extended observation and measurement
Solution Approach 2:
The reaction system exhibits periodic or sustained signal emission rather than a single instantaneous burst. The controlled calcium binding produces a sustained chemiluminescence and fluorescence signal that can be observed over an extended period, enabling temporal resolution and kinetic analysis
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 novel fluorescent proteins provide a stable, heat-resistant means for simultaneous chemiluminescence and fluorescence detection, enabling long-term observation and detection of target substances with enhanced sensitivity and thermal stability.
Implementation Method 1
a novel complex that not only has function (enzymatic function) to make a luminescence substrate emit light but also emits fluorescence in response to excitation of light
Implementation Method 2
Luminescence is the release of energy in the form of light (photons), produced when excited oxyluciferin molecules generated immediately after the oxidation reaction of luciferin return to the ground state
Implementation Method 3
calcium ion-binding photoproteins react specifically with calcium or strontium ions etc. and emit light instantaneously
Data Source
AI summary
A fluorescent protein (bFP) having chemiluminescence activity is a complex composed of the apoprotein of a calcium-binding photoprotein, coelenteramid or an analog thereof, and calcium ions or divalent or trivalent ions that can be substituted for the calcium ions. In the complex, the ratio of the number of molecules of the apoprotein to that of the coelenteramid is 1:1 and the ratio of the number of molecules of the apoprotein to that of the divalent or trivalent ions is 1:1 to 1:4. The fluorescent protein is used as a marker because it catalyzes luminescence of coelenterazine and has fluorescence capability. Removal of calcium ions etc. from this fluorescent protein (bFP) having luminescence activity provides a novel fluorescent protein (gFP). Mixing this gFP with the coelenterazine provides a calcium-binding photoprotein, which emit light instantaneously, enabling use as a marker.