Single Molecule Placement via Capture Primer Hybridization
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Solution Overview
Problem
Current methods for placing single molecules on substrates are inefficient due to reliance on high-resolution lithography and size exclusion, resulting in low loading efficiency and incompatibility with large-scale production, particularly in loading nanopores into lipid bilayers.
Innovation Solution
A substrate with immobilized capture primers and target polynucleotides, where the target molecule is linked to one end of the polynucleotide, allowing for kinetic exclusion amplification to fill the substrate, increasing loading throughput and compatibility with large-scale production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If molecular size exclusion effect through limiting anchoring space is used, then single molecule placement is achieved, but loading efficiency is low
Solution Approach 1:
The patent introduces a polynucleotide tether as an intermediary between the target molecule and the substrate. This tether allows the target molecule to be delivered to the substrate surface and then released through strand displacement, enabling efficient loading while maintaining single molecule placement. The intermediary resolves the contradiction by decoupling the delivery mechanism from the final positioning mechanism.
2Measurement precision
If high resolution lithography is used for single molecular placement, then placement precision is improved, but device complexity and manufacturing difficulty increase
Solution Approach 1:
The patent replaces the mechanical lithography system with a biochemical system based on polynucleotide hybridization and strand displacement. Instead of using complex lithographic equipment to position molecules, the system uses complementary base pairing between capture primers and polynucleotide tethers to achieve precise placement, significantly reducing device complexity while maintaining placement precision.
3Measurement precision
If monitoring ionic current change during loading is used, then single nanopore loading is achieved, but compatibility with large scale production is lost
Solution Approach 1:
The patent employs a self-service mechanism where capture primers immobilized on the substrate automatically capture polynucleotide tethers through hybridization, and subsequent strand displacement automatically releases the target molecules. This self-assembling, self-releasing system eliminates the need for continuous ionic current monitoring, enabling parallel processing and large-scale production while maintaining precise nanopore loading.
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
The method enables efficient placement of single molecules on substrates with higher loading efficiency and scalability, overcoming the limitations of existing techniques.
Implementation Method 1
hybridizing a plurality of first and second capture primers immobilized to a feature on a substrate with at least one target polynucleotide
Implementation Method 2
the target polynucleotide includes a target region flanked by first and second capture primer binding regions complementary to the first and second capture primers
Implementation Method 3
amplifying the at least one target polynucleotide at an average amplification rate that exceeds an average transport rate of a target polynucleotide to a feature to produce a plurality of clonal amplicons
Data Source
Figure 1
Figure 2
Figure 3A~3D
AI summary
Provided herein is a substrate comprising a plurality of first and second capture primers immobilized to a feature on the substrate; at least one target polynucleotide, one end attached to one of said capture primers and the other end linked to a target molecule, wherein said target polynucleotide comprises a target region flanked by first and second capture primer binding regions complementary to said first and second capture primers, said second capture primer binding region comprising a base pair mismatch to said second capture primer, and a plurality of clonal amplicons complementary to said target polynucleotide immobilized to said feature. Provided is a method for placing single target molecules on the substrate.