Nanopore PEG Tag Detection for Multiplex Protein Quantification
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Solution Overview
Problem
Existing protein detection methods struggle with multiplex analysis, requiring high concentrations, variable antibody affinities, and fluorescence saturation, limiting sensitivity and dynamic range, especially for low copy number proteins.
Innovation Solution
A method using cleavable PEG tags attached to proteins via nanopores, where proteins are captured, tagged analytes are bound to antibodies near nanopores, and cleaved tags are detected electronically, generating unique current blockade signals for quantification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If fluorescent labeling is used for protein detection, then detection sensitivity is improved, but fluorescence saturation and overlap limit the dynamic range and number of distinguishable proteins
Solution Approach 1:
The patent replaces the optical detection system (fluorescence) with an electrical detection system (nanopore current measurement). By measuring ionic current blockades as tagged proteins pass through the nanopore, the system achieves single-molecule sensitivity without the saturation and overlap problems inherent in fluorescent detection, enabling a much wider dynamic range from single to thousands of copies per cell
Solution Approach 2:
The patent changes the detection parameter from optical intensity (fluorescence) to electrical conductance (ionic current). This parameter change allows for digital quantification of protein molecules based on the number and duration of current blockades, providing a linear relationship between signal and molecule count across a wide dynamic range without saturation effects
2Productivity
If multiple proteins are detected simultaneously using protein arrays, then throughput is improved, but variable antibody affinities make quantification difficult
Solution Approach 1:
The patent extracts the detection tag (polyethylene glycol tag) from the protein-antibody interaction complex and detects it separately through the nanopore. This separation allows the antibody binding step to occur with variable affinities while the subsequent tag detection provides a standardized, affinity-independent signal for quantification, resolving the quantification accuracy problem in multiplex detection
Solution Approach 2:
The patent introduces a cleavable polyethylene glycol tag as an intermediary between the protein and the detection system. The tag serves as a universal detection element that can be attached to different proteins via specific antibodies, allowing multiplex detection while the nanopore measures a consistent electrical signal for each tag regardless of which antibody captured the protein, enabling accurate quantification across multiple targets
3Reliability
If high concentrations of proteins are required for detection, then signal reliability is improved, but detection of low copy number proteins becomes impossible
Solution Approach 1:
The patent replaces bulk optical detection with single-molecule electrical detection through nanopores. The nanopore system can detect individual protein molecules passing through, providing single-molecule sensitivity that enables detection of low copy number proteins while maintaining signal reliability through digital counting of molecular events
Solution Approach 2:
The patent employs a self-amplifying detection mechanism where each protein molecule carries multiple detectable tags (polyethylene glycol tags), and each tag generates a measurable current blockade signal. This allows the system to amplify the signal from low-abundance proteins inherently, as even a single protein molecule with multiple tags produces a detectable signal, eliminating the need for high concentration thresholds
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 digital protein quantification at the single molecule level with a wide dynamic range, overcoming fluorescence limitations and providing scalable, high-throughput protein detection.
Implementation Method 1
detecting tag moieties bound to said at least one nanopore, wherein said tag moieties generate unique electrical signals
Data Source
Figure 1(A)~1(B)
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AI summary
This invention provides methods for detecting the presence of a plurality of predetermined compounds in a sample using a plurality of tag moieties and at least one nanopore. This invention also provides methods for determining the quantity of each of a plurality of predetermined compounds in a sample using a plurality of tag moieties and at least one nanopore. This invention further provides methods for detecting interaction of at least two predetermined compounds using at least one tag moiety and at least one nanopore.