Planar Waveguide Evanescent Field Sequencing
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Solution Overview
Problem
Current nucleic acid sequencing methods by synthesis face limitations in sensitivity and accuracy, particularly when sequencing multiple nucleic acid molecules in parallel, leading to shorter read lengths and higher costs due to the need for high-powered lasers and limited fluorophore options.
Innovation Solution
The use of a planar waveguide technology enhances detection sensitivity by generating an evanescent field for fluorescently labeled nucleotides, allowing for accurate identification of incorporated nucleotides over multiple cycles, thereby increasing read lengths and reducing the power requirements for lasers, which expands the range of usable fluorophores.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If high-powered lasers are used to excite fluorophores in traditional sequencing methods, then detection sensitivity is improved, but cost of optical equipment increases and laser power requirements increase
Solution Approach 1:
The patent introduces an evanescent field as an intermediary between the laser light source and the fluorophores. This evanescent field is generated by a planar waveguide and confines the excitation light to a thin region near the waveguide surface, creating a highly localized and intense field that efficiently excites fluorophores without requiring high overall laser power. The evanescent field acts as a mediator that concentrates optical energy where it is needed most.
Solution Approach 2:
The patent applies local quality by creating a non-uniform distribution of excitation intensity through the evanescent field. The field intensity is highest at the waveguide surface where fluorophores are located and decays exponentially with distance. This localized intensity distribution allows efficient excitation of fluorophores while minimizing overall energy consumption and enabling the use of lower-powered lasers.
2Productivity
If traditional imaging methods are used for sequencing multiple nucleic acid molecules in parallel, then throughput is improved, but detection sensitivity and read length are reduced
Solution Approach 1:
The patent merges multiple sequencing reactions into a single field of view on the planar waveguide surface. Multiple nucleic acid molecules are immobilized in close proximity on the waveguide, allowing simultaneous excitation and detection of fluorophores from many molecules using a single evanescent field and a single detector (such as a CCD camera). This merging approach maintains high throughput while improving detection sensitivity through the enhanced local field intensity.
3Adaptability or versatility
If traditional fluorescence detection is used, then fluorophore selection is limited by laser wavelength requirements, but the patent expands fluorophore options
Solution Approach 1:
The planar waveguide evanescent field system provides a universal excitation platform that can efficiently excite a wide variety of fluorophores with different excitation wavelengths. The evanescent field's confined geometry and high intensity make it compatible with multiple laser wavelengths, allowing the same basic optical setup to support diverse fluorophore choices without requiring complex wavelength-specific optical paths for each fluorophore type.
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 significantly improves the sensitivity of nucleotide detection, enabling longer read lengths and reducing the cost of optical equipment, allowing for more accurate sequencing and broader fluorophore usage, particularly beneficial in de novo genome sequencing and SNP analysis.
Implementation Method 1
A liquid sample is brought into contact with the waveguiding layer as a superstrate and excitation light coupled into the waveguiding layer produces an evanescent field at the interface between the waveguiding layer and the superstrate.
Implementation Method 2
Luminescence produced by substances having luminescent properties either in the liquid sample or immobilised on the waveguiding layer is then measured.
Data Source
Figure 1a~1b
Figure 2~3
Figure 4~5
AI summary
The present invention is concerned with improvements to methods of imaging nucleotides incorporated into polynucleotides and in particular with improved methods of determining the sequence of template nucleic acid molecules using multiple cycle nucleic acid "sequencing-by-synthesis" reactions.