Genetically Encoded Biosensors for 3D Human Cell Voltage Monitoring
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Solution Overview
Problem
Current genetically encoded calcium indicators (GECIs) have limitations in resolution, penetration of multi-cell/3D structures, and applicability to various cell types, often requiring two-dimensional microscopic detection and are not representative of human cells, limiting their usefulness in monitoring bioactivities and drug screening.
Innovation Solution
Development of synthetic and genetically encoded biosensors that can detect intracellular changes in human cells, including calcium, voltage, and ATP levels, with specific targeting to cellular compartments, allowing for three-dimensional signal detection in representative cell models, and can be configured for cell-type specificity using promoter sequences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If genetically encoded calcium indicators (GECIs) are used for monitoring intracellular calcium concentration, then calcium signaling can be detected, but the resolution and penetration capability in multi-cell/3D structures is limited
Solution Approach 1:
The patent transitions from 2D monolayer detection to 3D tissue penetration by engineering biosensors with enhanced optical properties. The invention uses fluorescent proteins with improved excitation/emission wavelengths and tissue penetration capabilities, allowing detection through multi-cell layers and 3D structures while maintaining signal resolution.
2Measurement precision
If traditional GECIs are used, then calcium concentration changes can be monitored, but the applicability to various cell types is limited and they require two-dimensional microscopic detection
Solution Approach 1:
The patent creates universal biosensor constructs that can be applied across multiple cell types and detection dimensions. The invention combines modular fluorescent protein domains with calcium-sensing domains in configurations that function in both 2D monolayers and 3D tissue structures, making the system versatile for different cell types and experimental conditions.
3Ease of manufacture
If rodent or immortal cell lines are used for signal transduction studies, then in vitro experiments can be conducted, but the results are not representative of human cells
Solution Approach 1:
The patent employs human-derived induced pluripotent stem cells (iPSCs) differentiated into various human cell types, fundamentally changing the cellular model from rodent/immortal lines to human primary cells. This parameter change in cell origin and differentiation state improves the reliability and representativeness of human cell responses while maintaining in vitro experimental feasibility.
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 accurate monitoring of intracellular changes in human cells, including voltage and ion concentrations, in 2D or 3D models, improving the representation of human cell responses and facilitating drug screening and toxicity evaluations.
Implementation Method 1
a voltage sensing domain sensitive to H+, Na+ and/or K+ concentration
Implementation Method 2
a fluorescent reporter domain. The fluorescent reporter can be adapted to fluoresce at wavelengths in the range from 500 nm to 750 nm
Data Source
AI summary
This invention provides biosensors, cell models, and methods of their use for monitoring heme, oxygen or ATP. Biosensors can include targeting domains, sensing domains and reporting domains. Biosensors can be introduced into cells reprogrammed to represent experimental or pathologic cells of interest. Model cells expressing the biosensors can be contacted with putative bioactive agents to determine possible activities.