Transgenic Mammal FRET Sensor for ATP Quantification
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for measuring ATP levels in cells are prone to large measurement errors, destroy subcellular compartments, and fail to quantify time-dependent changes or distribution of ATP in single living cells.
Innovation Solution
A transgenic non-human mammal expressing a fusion protein comprising the ε subunit of ATP synthase and two distinct fluorescent proteins that function as donor and acceptor in FRET, allowing for precise measurement of ATP levels and distribution without damaging subcellular compartments.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If luciferase assay is used to measure cellular ATP levels, then ATP levels can be measured, but large measurement errors occur and subcellular compartments are destroyed
Solution Approach 1:
The patent replaces the mechanical homogenization process with a fluorescent imaging approach. Instead of physically breaking down cells to measure ATP, the system uses fluorescent proteins that bind to ATP molecules in living cells, allowing non-invasive optical detection of ATP levels and distribution without destroying cellular structures.
Solution Approach 2:
The patent introduces fluorescent proteins as intermediary molecules that mediate between ATP and the detection system. These fluorescent proteins bind to ATP and emit light signals, serving as a bridge that allows indirect but non-destructive measurement of ATP levels, eliminating the need for direct mechanical disruption of cells.
2Measurement precision
If mass spectroscopy is used to measure ATP levels, then ATP levels can be measured, but large numbers of cells are required and subcellular compartments are destroyed
Solution Approach 1:
The patent replaces mass spectroscopy with optical detection using fluorescent proteins. This substitution enables measurement at the single-cell level through fluorescence imaging, eliminating the need to process large numbers of cells and preserving subcellular compartment integrity that would be destroyed by mass spectroscopy preparation protocols.
Solution Approach 2:
The patent creates a fluorescent copy or signal representation of ATP molecules through fluorescent protein binding. Instead of directly analyzing ATP mass or requiring large cell populations, the system generates optical signals that copy the ATP distribution pattern, enabling precise measurement in individual cells without destroying their structure.
3Measurement precision
If homogenization is performed to measure ATP levels, then ATP levels can be measured, but subcellular compartments including mitochondria are destroyed
Solution Approach 1:
The patent substitutes mechanical homogenization with optical detection methods. By using fluorescent proteins that can be visualized through microscopy, the system measures ATP levels and subcellular distribution without applying mechanical force that would destroy mitochondria and other organelles, thereby maintaining cellular composition integrity.
Solution Approach 2:
The patent employs fluorescent proteins as intermediary indicators that bind to ATP molecules without disrupting cellular structures. These intermediaries allow detection of ATP in its native cellular environment, eliminating the need for homogenization that would destroy subcellular compartments while maintaining measurement precision.
4Measurement precision
If conventional ATP measurement methods are used, then ATP levels can be measured, but time-dependent changes and distribution in single cells cannot be quantified
Solution Approach 1:
The patent replaces endpoint measurement methods with real-time optical imaging. The fluorescent protein system allows continuous or repeated imaging of the same cell over time, enabling quantification of time-dependent ATP changes and dynamic distribution patterns that conventional methods cannot capture because they require cell lysis or multiple separate measurements.
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 quantitative measurement of ATP levels and distribution in single cells with high precision and low measurement error, unaffected by oxygen concentration, and allows for detailed investigation of ATP distribution in developing embryos and various disease models.
Implementation Method 1
The method utilizes a fusion protein that includes a cyan fluorescent protein (CFP) and a yellow fluorescent protein (YFP) which are fused to the CBS domain of inosine monophosphate dehydrogenase 2 (IMPDH2). IMPDH2 can react with adenosine nucleic acids such as ATP, adenosine diphosphate (ADP) and adenosine monophosphate (AMP).
Data Source
AI summary
Provided are a transgenic non-human mammal expressing a fusion protein, wherein the fusion protein comprises an ε subunit of an ATP synthase and two distinct fluorescent proteins as a donor and an acceptor for FRET, one of the fluorescent proteins being placed at an amino terminal moiety of the ε subunit and the other being placed at a carboxyl terminal moiety of the ε subunit, and a method of screening for an agent for preventing or treating diseases in a mammal in need thereof, comprising using an above transgenic non-human mammal.