EV miRNA Detection via RBCM Vesicle Fusion
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Solution Overview
Problem
Current methods face challenges in detecting low-abundance microRNAs (miRNAs) carried by extracellular vesicles (EVs) due to their small size, high sequence similarity, ease of degradation, and low abundance, which results in low sensitivity and specificity.
Innovation Solution
The method involves subjecting red blood cell membrane-derived vesicles (RVs) to membrane fusion with EVs, delivering a specific hairpin probe designed for the target miRNA into the EVs to complete DNA self-assembly, thereby achieving in situ fluorescence detection of the miRNA carried by the EVs.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional detection methods are used for miRNA in EVs, then the detection process is simple, but the detection sensitivity is low due to low miRNA abundance and easy degradation
Solution Approach 1:
The patent employs a nested structure where hairpin probes are delivered inside EVs, which then interact with target miRNAs in a confined intravesicular space. This nested configuration concentrates detection reagents within the EV lumen, enhancing local probe-target collision frequency and detection sensitivity without requiring complex external delivery systems
Solution Approach 2:
The patent introduces an intermediary delivery system using RVs (red blood cell-derived vesicles) that fuse with target EVs to deliver hairpin probes. This intermediary approach enables probe delivery without directly disrupting EV integrity, maintaining the confined space architecture while achieving reagent introduction through a mediating vesicular system
2Productivity
If EV membrane structure is disrupted to deliver probes, then probe delivery efficiency increases, but EV structural integrity is compromised
Solution Approach 1:
The patent merges the delivery function with the target structure itself by using RV-EV fusion. Instead of disrupting EVs, the delivery vesicles (RVs) merge with target EVs, allowing probe delivery while preserving the EV membrane structure. This merging approach enables dual functionality: delivery vehicle and target containment remain intact
Solution Approach 2:
The patent performs preliminary probe loading into delivery vesicles (RVs) before they encounter target EVs. This pre-preparation of delivery vehicles with embedded hairpin probes allows for efficient probe transfer upon fusion, achieving high delivery efficiency without requiring post-fusion manipulation that could compromise EV integrity
3Measurement precision
If probe concentration is increased to improve detection sensitivity, then detection limit improves, but background noise increases
Solution Approach 1:
The patent implements local quality by concentrating probes within the confined EV intravesicular space rather than distributing them throughout the entire sample volume. This localized concentration achieves high effective probe-target interaction probability and low detection limits while maintaining low overall probe concentration in the bulk solution, thereby minimizing background noise
Solution Approach 2:
The nested configuration of probes within EVs creates a confined reaction space where probe-target interactions occur exclusively inside vesicles. This spatial confinement prevents free probe molecules from contributing to background noise in the bulk solution, allowing high local probe concentration for sensitive detection without proportional increase in global background signal
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
This approach significantly increases the detection sensitivity of miRNA-21 in EVs by maintaining the integrity of the EV membrane structure during probe delivery, allowing for efficient DNA self-assembly and fluorescence detection, with a desirable linear relationship between fluorescence intensity and miRNA-21 concentration from 50 pM to 40 nM.
Implementation Method 1
delivering a specific hairpin probe designed for a target miRNA into the EVs to complete DNA self-assembly
Implementation Method 2
sequences of the three specific hairpin probes each include a self-complementary sequence and a complementary palindromic sequence
Implementation Method 3
two of the specific hairpin probes each are modified with a fluorophore and a quencher
Implementation Method 4
achieving in situ fluorescence detection of the miRNA carried by the EVs
Data Source
AI summary
The present disclosure belongs to the technical field of detection, and specifically relates to a method, a system, and a kit for in situ detection of a micro ribonucleic acid (miRNA) carried by extracellular vesicles (EVs). In the method for in situ detection of a miRNA carried by EVs, fluorescence detection probes are delivered without disrupting a membrane vesicle structure of the EVs using an innovative strategy of conducting membrane fusion between red blood cell membrane (RBCM)-derived vesicles (RVs) and the EVs. Therefore, an obvious increase in a local concentration of the fluorescence probes in a confined space leads to a sharp increase in a probability of collisions between the probes, thereby increasing a detection sensitivity of a target. There is a desirable linear relationship between a detected fluorescence intensity and a miRNA-21 concentration at 50 pM to 40 nM.