Engineered Red Blood Cell Biosensors Using Bimolecular Complementation
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Solution Overview
Problem
Existing technologies lack efficient methods for detecting extracellular ligands using engineered red blood cells (eRBCs) that can form functional protein complexes to provide detectable signals upon ligand binding.
Innovation Solution
Engineered red blood cells (eRBCs) are equipped with modular extracellular sensors comprising ligand-binding domains and transmembrane domains, allowing them to form ternary complexes with functional protein fragments that reconstitute enzymatic or fluorescent activities upon ligand binding.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Difficulty of detecting and measuring
If engineered red blood cells are designed to detect extracellular ligands, then detection capability is improved, but cell complexity increases
Solution Approach 1:
The sensor is divided into two separate exogenous sensors, each expressing a different fragment of the functional protein (first fragment and second fragment). These fragmented sensors are integrated into the red blood cell membrane independently, allowing the cell to detect ligands through the reconstitution of functional protein activity when both sensors bind the same ligand, thereby improving detection capability while managing cellular complexity through modular design
Solution Approach 2:
The extracellular ligand acts as an intermediary that brings the two separate sensor fragments into proximity. When the ligand binds to both extracellular domains simultaneously, it mediates the interaction between the first and second fragments of the functional protein, enabling signal generation without requiring direct integration of complex sensing machinery within the cell
2Measurement precision
If two exogenous sensors are integrated into red blood cells, then detection sensitivity is improved, but manufacturing complexity increases
Solution Approach 1:
The detection system is segmented into two independently expressible sensor components, each encoding a fragment of the functional protein fused to an extracellular ligand-binding domain. This segmentation allows for separate production and characterization of each sensor component before integration into red blood cells, improving detection sensitivity through the requirement of dual binding events while simplifying manufacturing by enabling modular production approaches
Solution Approach 2:
The sensor design utilizes universal components including extracellular ligand-binding domains that can recognize various targets and intracellular protein fragments that reconstitute functional activity. This multi-functionality allows the same basic sensor architecture to detect different ligands by simply changing the extracellular binding domain, thereby improving detection sensitivity across multiple targets while maintaining consistent manufacturing protocols
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 eRBCs effectively detect extracellular ligands by forming functional protein complexes, enabling sensitive and specific molecular signaling through bimolecular complementation, thereby enhancing the detection capabilities of red blood cells.
Implementation Method 1
the extracellular ligand binding domain of the first exogenous sensor and the ligand binding domain of the second exogenous sensor bind to a ligand to form a ternary complex
Implementation Method 2
the first fragment of the functional protein and the second fragment of the functional protein interact in the ternary complex to reconstitute functional activity of the functional protein
Implementation Method 3
fluorescent proteins that emit fluorescence when the ligand binding domain of the first exogenous sensor and the ligand binding domain of the second exogenous sensor bind to the same ligand
Implementation Method 4
enzymatic proteins that exhibit enzymatic activity when ligand binding domain of the first exogenous sensor and the ligand binding domain of the second exogenous sensor bind to the same ligand. Substrates for the enzymatic proteins may include substrates that are luminescent after they are metabolized by the enzymatic protein
Data Source
AI summary
Disclosed are systems and methods for detecting extracellular ligands. The disclosed systems and method for detecting extracellular ligands typically comprise or utilize engineered red blood cells (eRBCs) that comprises modular extracellular sensors. The eRBCs may comprise: (i) a first exogenous extracellular sensor; the first extracellular sensor comprising: a) a ligand binding domain, b) a transmembrane domain, and c) a first fragment of a functional protein, and (ii) a second exogenous extracellular sensor; the second extracellular sensor comprising: a) a ligand binding domain, b) a transmembrane domain, and c) a second fragment of the functional protein. In the eRBCs, the ligand binding domain of the first exogenous sensor and the ligand binding domain of the second exogenous sensor bind to the same ligand to form a tertiary complex, and the first fragment of the functional protein and the second fragment of the functional protein interact in the tertiary complex to reconstitute functional activity of the functional protein. Suitable functional proteins for the disclosed eRBCs may include fluorescent proteins that emit fluorescence when the ligand binding domain of the first exogenous sensor and the ligand binding domain of the second exogenous sensor bind to the same ligand, and enzymatic proteins that exhibit enzymatic activity when ligand binding domain of the first exogenous sensor and the ligand binding domain of the second exogenous sensor bind to the same ligand.