Membrane-Coupled Nanopore Sequencing at Ultra-Low Analyte Levels
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Solution Overview
Problem
Existing nucleic acid sequencing technologies are slow and expensive due to reliance on amplification techniques and require high quantities of fluorescent chemicals, and nanopore-based sequencing faces challenges with efficient capture of analytes at low concentrations, particularly in samples like cancer cell DNA.
Innovation Solution
Coupling the analyte to a membrane where the detector is present, allowing for enhanced interaction and reducing the required analyte concentration by several orders of magnitude, thereby increasing the sequencing duty cycle and reducing temporary or permanent nanopore blocking.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If nanopore-based sequencing is used to detect analytes at low concentrations, then sequencing capability is maintained, but capture rates are insufficient and duty cycle is reduced
Solution Approach 1:
The patent transitions from three-dimensional bulk solution detection to two-dimensional membrane surface detection. By coupling analytes to a membrane surface, the system confines analyte movement to a two-dimensional plane, increasing the probability of interaction with nanopores embedded in the same membrane. This dimensional reduction solves the capture rate problem at low concentrations by ensuring analytes remain in close proximity to detection sites.
Solution Approach 2:
The membrane acts as an intermediary platform that couples analytes to the detection system. Instead of relying on random collisions in solution, the membrane provides a structured interface where analytes are presented to nanopores in a controlled manner. This intermediary structure enhances the efficiency of analyte-nanopore interactions without requiring high analyte concentrations.
2Quantity of substance
If amplification techniques are used to increase analyte quantity, then sufficient signal is obtained, but sequencing speed decreases and cost increases
Solution Approach 1:
The patent extracts the amplification step from the sequencing workflow by implementing direct detection of native nucleic acids. By using membrane-coupled nanopore detection, the system obtains sufficient signal without requiring PCR or other amplification techniques, thereby maintaining sequencing speed while reducing the quantity of nucleic acid needed.
Solution Approach 2:
The patent replaces the mechanical/chemical amplification process with an enhanced electrical detection system. The membrane-coupled nanopore configuration provides signal amplification through electrical measurement rather than through biological or chemical amplification, eliminating the time and complexity associated with traditional amplification methods.
3Measurement precision
If high quantities of fluorescent chemicals are used for signal detection, then detection sensitivity is improved, but cost and complexity increase
Solution Approach 1:
The patent replaces optical detection using fluorescent chemicals with electrical detection using nanopores. This substitution eliminates the need for fluorescent labeling and associated chemicals, reducing cost and complexity while maintaining or improving detection sensitivity through direct electrical measurement of nucleic acid translocation.
Solution Approach 2:
The patent uses inexpensive nanopore proteins (such as alpha-hemolysin) that can be easily produced and replaced, eliminating the need for expensive fluorescent dyes and complex optical detection systems. The nanopores serve as disposable or regenerable detection elements that provide sensitive detection without the ongoing cost of specialized chemicals.
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 significantly enhances nanopore sequencing efficiency by increasing capture rates by approximately four orders of magnitude, allowing sequencing at ultra-low analyte concentrations and improving sequencing systems' duty cycle.
Implementation Method 1
When a potential is applied across a nanopore, there is a drop in the current flow when an analyte, such as a nucleotide, resides transiently in the barrel for a certain period of time. Nanopore detection of the analyte gives a current blockade of known signature and duration.
Implementation Method 2
In one embodiment, the analyte is coupled to the membrane via a hydrophobic anchor present in the membrane.
Data Source
AI summary
The invention relates to a new method of determining the presence, absence or characteristics of an analyte. The analyte is coupled to a membrane. The invention also relates to nucleic acid sequencing.