Aptamer-Based Fluorescence Polarization Detection for Extracellular Vesicles
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Solution Overview
Problem
Current methods for detecting and quantifying extracellular vesicles (EVs) are limited by their complexity, sensitivity, and specificity, particularly in clinical settings, as they often require advanced instrumentation and struggle with distinguishing EVs from other biomolecules, leading to inaccurate quantification and high detection limits.
Innovation Solution
An aptamer-based fluorescence polarization detection method that immobilizes EVs using antibodies and subsequently uses fluorescently labeled aptamers to quantify them, allowing for sensitive and specific detection without the need for extensive sample preparation, leveraging the high affinity and small size of aptamers to target EV markers.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional fluorescence intensity-based methods are used to detect EVs, then the detection process is simple, but the sensitivity and specificity are insufficient leading to high detection limits
Solution Approach 1:
The patent changes the detection parameter from fluorescence intensity to fluorescence polarization. This parameter transformation enables the detection method to achieve higher sensitivity and specificity for EVs while maintaining operational simplicity. The fluorescence polarization parameter provides a new measurement dimension that effectively distinguishes EVs from other biomolecules.
Solution Approach 2:
The patent introduces fluorescently labeled aptamers as intermediaries to bridge the detection target (EVs) and the detection signal (fluorescence polarization). These aptamers specifically bind to EV surface markers, enabling selective detection through polarization measurements while the fluorescent label provides the necessary optical signal.
2Measurement precision
If antibodies are used to detect EV markers, then high specificity is achieved, but the large size (about 150 kDa) makes them unsuitable for fluorescence polarization detection
Solution Approach 1:
The patent uses aptamers as synthetic copies that replicate the binding function of antibodies. These nucleic acid-based probes can be fluorescently labeled and are much smaller in size, making them suitable for fluorescence polarization detection while maintaining the high specificity needed for EV marker detection.
Solution Approach 2:
The patent replaces the protein-based antibody binding mechanism with a nucleic acid-based aptamer binding mechanism. This substitution enables the use of smaller, fluorescently labelable probes that are compatible with fluorescence polarization detection while preserving the ability to specifically recognize EV markers.
3Weight of moving object
If lipophilic dyes are used as probe substances, then the molecular weight is small (～1 kDa), but they exhibit non-specific binding with non-EV particles and low labeling efficiency
Solution Approach 1:
The patent creates a composite probe structure by combining fluorescent labels with aptamers. This composite design integrates the small size and fluorescence properties of dye molecules with the high specificity of aptamer-EV interactions, achieving both appropriate molecular weight for FP detection and high detection accuracy through specific binding.
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 method enables accurate and sensitive detection of oncogenic EVs in cell culture media and human plasma, differentiating them from other biomolecules, with a lower limit of detection and broader dynamic range compared to traditional fluorescence intensity-based methods, making it suitable for clinical applications.
Implementation Method 1
an aptamer-based fluorescence polarization detection method for extracellular vesicles
Implementation Method 2
uses fluorescently labeled aptamers to quantify them
Implementation Method 3
immobilizes EVs using antibodies
Data Source
AI summary
The present invention relates to technical field of C12N15/115, and particularly relates to an aptamer-based fluorescence polarization detection method for extracellular vesicles (EVs) and its application. The method comprises the following steps: S1. immobilizing EVs by interacting with antibodies against surface-biomarker proteins of EVs or surface cancer markers thereof; rapidly washing them to remove free EVs, proteins, membrane fragments, and lipids; S2. respectively adding aptamers matched with EV markers or cancer cell markers therein and cultivating them the aptamers are fluorescently labeled; S3. performing fluorescence polarization detection on the products from Step S2 to achieve qualitative and quantitative analysis of EVs secreted by cancer cells. This invention can specifically detect extracellular vesicles secreted by cancer cells in blood, and detection process is not interfered with by free tumor marker proteins, tumor cell membrane fragments, or tumor cell extracellular vesicle membrane fragments in blood. The detection results are accurate and effective.