Cavity-Separated Multi-Nanopore Protein Sequencing
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Solution Overview
Problem
Current protein sequencing methods, such as Edman degradation and mass spectrometry, are limited by low throughput, dynamic range, and sensitivity, making it difficult to analyze proteins at low concentrations and in complex environments, and nanopore-based approaches face challenges in distinguishing amino acids by ion current alone.
Innovation Solution
A cavity-separated multi-nanopore (CSMP) device that records both ionic currents and the time peptides take to traverse through multiple nanopores, providing additional information on fragment size and facilitating protein sequencing by measuring specific current and dwell times.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional protein sequencing methods (Edman degradation, mass spectrometry) are used, then protein identification can be achieved, but throughput and dynamic range are limited
Solution Approach 1:
The invention divides the protein sequencing process into multiple independent nanopore channels, each capable of simultaneously analyzing peptide fragments. This segmentation enables parallel processing of multiple samples, dramatically increasing throughput while maintaining single-molecule sensitivity for detecting low-abundance proteins
Solution Approach 2:
The invention transitions from single-channel to multi-channel nanopore systems, adding the dimension of parallel processing. Multiple nanopores operate simultaneously to analyze different peptide fragments or multiple samples, exponentially increasing throughput without sacrificing detection sensitivity
2Measurement precision
If fluorescent fingerprinting is used for single-molecule protein identification, then detection sensitivity improves, but error rates remain high (20-30%)
Solution Approach 1:
The invention changes the detection parameter from fluorescent signal intensity to ionic current blockade characteristics. By measuring the electrical current disruption caused by peptides translocating through nanopores, the system achieves both single-molecule sensitivity and reduced error rates through more reliable electrical signal discrimination
Solution Approach 2:
The invention replaces the optical detection system (fluorescence microscopy) with an electrical detection system (ionic current measurement). This substitution eliminates the limitations of fluorescent detection while maintaining single-molecule sensitivity, achieving both high precision and low error rates
3Quantity of substance
If nanopore-based approaches are used for protein sequencing, then single-molecule detection is achieved, but amino acid distinction by ion current alone is challenging
Solution Approach 1:
The invention segments the protein into smaller peptide fragments that are sequentially analyzed through nanopores. This segmentation creates distinct translocation events for different amino acid sequences, enhancing the ability to discriminate between amino acids through their unique ionic current signatures
Solution Approach 2:
The invention introduces peptide fragmentation as an intermediary step between protein sample preparation and nanopore analysis. By cleaving proteins into characteristic peptide fragments, the system creates more distinguishable translocation events that improve amino acid discrimination while maintaining single-molecule detection capability
4Adaptability or versatility
If mass spectrometry is used for protein identification, then database search capability is available, but dynamic range is limited to 10^4 or 10^5
Solution Approach 1:
The invention adds the dimension of temporal resolution to mass spectrometry-like analysis by measuring translocation time of peptide fragments through nanopores. This additional temporal parameter extends the dynamic range beyond conventional MS capabilities, enabling detection across 11 orders of magnitude while maintaining protein identification capability
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
Enhances protein sequencing by improving detection capabilities and reducing errors, enabling single-cell analysis and identifying proteins with higher precision and sensitivity compared to existing methods.
Implementation Method 1
The nanopores can separate to fluidic compartments... electrodes in each compartment can facilitate an application of a voltage bias across the CSMP, and therefore drive charge carriers and other charged molecules through CSMP. The resulting ionic current can be recorded over time
Implementation Method 2
drive charge carriers and other charged molecules through CSMP... While translocating through the constrictions of the CSMP device, a protein/polypeptide/peptide/amino acid can cause a specific pattern of ionic current modulation
Data Source
AI summary
An exemplary system and method can be provided, e.g., for detecting a molecular size and charge. The exemplary system can comprise a cavity, nanopores separated by the cavity, and electrolyte reservoirs. Each of the reservoirs can be provided on a side of a respective nanopore, and within the cavity. A plurality of such systems can be integrated in a surface of a complementary metal-oxide-semiconductor (CMOS) integrated circuit, which can comprise transimpedance amplifiers configured to measure a conductance through the nanopores. Further an exemplary device can be provided for protein sequencing, and can comprise a first compartment with a first electrode, a second compartment with a second electrode, and a channel between the first and second compartments. Each of the compartments can be fluidly coupled to the channel using a nanopore. A detector can also be provided which is configured to record at least one parameter in the channel by applying a voltage bias across the first and second electrodes so that charged molecules pass through the nanopore fluidly coupled to the first and second compartments.