Optical Nanopore Sequencing Polarization Control
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Solution Overview
Problem
Nanopore sequencing faces challenges in reliable nanostructure fabrication, control of DNA translocation rates, unambiguous nucleotide discrimination, and low signal-to-noise ratios in optical detection due to difficulties in aligning fluorescent labels for optimized signal generation and detection.
Innovation Solution
The method involves directing an excitation beam with a predetermined polarization state through a nanopore to orient fluorescent labels, making them unresponsive during translocation, and detecting changes in fluorescence as nucleotides exit the nanopore, allowing for unambiguous nucleotide identification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If fluorescent labels are used for nucleotide detection in nanopore sequencing, then nucleotide identification capability is improved, but signal-to-noise ratio deteriorates due to inability to align absorption dipoles with excitation light
Solution Approach 1:
The nanopore pre-orients fluorescent labels before detection by spatially constraining them during translocation. This preliminary orientation action ensures that absorption dipoles are positioned favorably relative to the excitation beam polarization, maximizing signal generation before the nucleotide reaches the detection zone and reducing the need for post-positioning adjustments.
Solution Approach 2:
The invention changes the spatial orientation parameter of fluorescent labels by utilizing the nanopore's physical constraints. As nucleotides translocate through the nanopore, the confined space forces absorption dipoles into specific orientations that are unresponsive to certain polarization directions of the excitation beam, thereby modulating the optical response and improving signal discrimination.
2Object-affected harmful factors
If nanopore spatial orientation of fluorescent labels is implemented, then signal-to-noise ratio is improved, but device complexity increases due to requirements for polarization control and precise nanopore positioning
Solution Approach 1:
The nanopore structure itself performs the orientation function that would otherwise require complex external alignment mechanisms. The physical confinement and electrostatic environment within the nanopore automatically orient the fluorescent labels based on their intrinsic properties, eliminating the need for active feedback control systems or complex mechanical alignment apparatus.
Solution Approach 2:
The invention replaces complex mechanical alignment systems with optical field interactions. Instead of using mechanical devices to physically rotate or position fluorescent labels, the system uses the polarization state of light and the nanopore's electrostatic field to achieve orientation, substituting mechanical complexity with optical field control.
3Illumination intensity
If fluorescent labels are aligned with excitation light polarization, then fluorescence signal intensity is improved, but nucleotide discrimination accuracy deteriorates due to loss of orientation information
Solution Approach 1:
The system employs periodic modulation of the excitation light polarization state to probe different orientation components of the fluorescent labels. By cycling through different polarization angles and measuring the corresponding fluorescence intensity variations, the system simultaneously maximizes overall signal intensity while extracting orientation-dependent information for nucleotide discrimination.
Solution Approach 2:
The invention utilizes the dynamic translocation process where fluorescent labels continuously change their position and orientation as they move through the nanopore. This dynamic behavior provides time-varying fluorescence signals that encode both intensity and orientation information, allowing simultaneous optimization of signal strength and discrimination accuracy through temporal analysis.
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 approach enhances the signal-to-noise ratio in nanopore sequencing by spatially constraining and orienting fluorescent labels, enabling effective detection and identification of nucleotides, thereby improving the accuracy and efficiency of polynucleotide analysis.
Implementation Method 1
nucleotides of the polynucleotide are labeled with fluorescent labels having absorption dipoles and wherein the nanopore spatially orients the fluorescent labels so that during translocation the absorption dipoles are substantially unresponsive to the excitation beam
Implementation Method 2
detecting changes in fluorescent signals generated by the fluorescent labels as nucleotides with fluorescent labels exit the nanopore and absorption dipoles thereof become responsive to excitation by the excitation beam
Data Source
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AI summary
In some aspects the invention is directed to methods of analyzing a polynucleotide which include steps of directing to a nanopore an excitation beam having a predetermined polarization state; translocating a polynucleotide through the nanopore, wherein nucleotides of the polynucleotide are labeled with fluorescent labels having absorption dipoles and wherein the nanopore spatially orients the fluorescent labels so that during translocation the adsorption dipoles are substantially unresponsive to the excitation beam; detecting changes in fluorescent signals generated by the fluorescent labels as nucleotides with fluorescent labels exit the nanopore and absorption dipoles thereof become responsive to excitation by the excitation beam with the predetermined polarization state; and identifying nucleotides exiting the nanopore from the changes in fluorescent signals.