Hybrid Protein Nanopore Arrays for Scalable DNA Sequencing
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Solution Overview
Problem
Nanopore technology faces challenges in increasing robustness and parallelization for nucleic acid sequencing, with manual assembly methods being laborious and difficult to scale, and current techniques relying on fragile lipid bilayers that require skilled operators.
Innovation Solution
A system comprising a detection apparatus with tethered protein nanopores surrounded by a membrane, using lipid nanodiscs to stabilize the nanopores and facilitate high-throughput assembly, enabling efficient coupling to solid-state nanopores through chemical engineering and affinity tags for precise nanopore deposition.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If manual Poisson loading method is used to assemble nanopores, then nanopore deposition can be controlled, but the process becomes laborious and difficult to scale
Solution Approach 1:
The invention segments the nanopore assembly process by dividing the array into multiple regions that can be loaded independently and in parallel. Instead of manually loading each aperture sequentially, multiple regions are prepared and loaded simultaneously, dramatically increasing throughput while maintaining control over deposition precision in each region.
Solution Approach 2:
The invention replaces the manual mechanical Poisson loading process with an automated system that uses controlled dispensing mechanisms. This substitution eliminates the need for skilled operators to manually pipette nanopore solutions and enables high-throughput, reproducible nanopore assembly across large arrays.
2Stability of the object's composition
If lipid bilayer membranes are used to surround nanopores, then nanopore stability is improved, but the system becomes fragile and requires skilled operators
Solution Approach 1:
The invention changes the physical parameters of the membrane system by using supported lipid bilayers on rigid substrates instead of suspended bilayers. This parameter change increases mechanical stability and reduces fragility, allowing standard operators to handle the systems without specialized training while maintaining nanopore stability.
Solution Approach 2:
The invention uses thin film supported lipid bilayers that provide the necessary flexibility for nanopore function while being supported by a rigid substrate. This combination maintains the biochemical properties of lipid membranes while eliminating their mechanical fragility, enabling easier operation.
3Length of moving object
If single molecule nanopore sequencing is used, then read length is increased, but parallelization is decreased
Solution Approach 1:
The invention segments the sequencing capacity across thousands of independent nanopores arranged in arrays. Each nanopore processes a single molecule maintaining long read lengths, while the collective array provides massive parallelization. This segmentation allows simultaneous processing of many long reads across the array.
Solution Approach 2:
The invention transitions from single-molecule sequencing in one dimension to array-based parallel sequencing by adding spatial dimensions. Thousands of nanopores are arranged in two-dimensional arrays, enabling parallel processing across the array plane while each individual pore maintains the capability for long-read sequencing.
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 spatial control over nanopore deposition, allows for highly parallel assembly, and achieves nanopore loading efficiencies beyond Poisson statistics, improving the robustness and scalability of nanopore sequencing platforms.
Implementation Method 1
Discrimination of nucleotide bases has been demonstrated by reading the ionic current signal when the DNA molecule is passing through the nanopore
Data Source
Figure 1A~1C
Figure 2A~2B
Figure 3~4
AI summary
Provided is a system for detection of nucleic acid, the system comprising: (i) a detection apparatus, wherein the detection apparatus comprises one or more protein nanopores and (ii) an amplifier configured to amplify electrical signals generated at the nanopore and/or a computer or data acquisition circuit coupled to the detection apparatus to evaluate signals detected from the protein nanopore. In one aspect, the detection apparatus comprises a solid support with an array of solid-state nanopores and a plurality of lipid nanodiscs with protein nanopores, wherein the lipid nanodiscs are disposed on the solid support such that they form seals at the solid-state nanopores. In another aspect, the detection apparatus comprises one or more nanopores that are surrounded by a membrane, wherein each nanopore is tethered to an electrode. Also provided are methods of sequencing nucleic acids using the systems.