Single-Molecule Optical Detection for Dense Sequencing Substrates
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current analyte detection methods suffer from limitations such as low sensitivity, especially for low copy numbers or concentrations, require large sample volumes, and lack the ability to simultaneously detect and quantify nucleic acids and proteins, leading to errors and inaccuracies in identification and quantification.
Innovation Solution
A method for sequencing polynucleotides at high density on a substrate with single molecule resolution, using reversible terminator nucleotides and optical imaging to detect and deconvolve optical signals, allowing for high sensitivity and accurate identification and quantification of analytes, including proteins and nucleic acids, with error correction.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If sample amplification is used to improve sensitivity, then detection sensitivity is improved, but amplification introduces biases and inaccuracies into quantification
Solution Approach 1:
The patent extracts and eliminates the amplification step from the detection workflow. By using single-molecule detection technology, the system directly detects individual analyte molecules without amplification, thereby maintaining quantification accuracy while achieving high sensitivity through advanced optical detection methods.
Solution Approach 2:
The patent changes the detection parameter from bulk signal measurement to single-molecule resolution detection. By using super-resolution microscopy and advanced imaging techniques, the system achieves sensitivity comparable to amplification methods while preserving the true stoichiometry and concentration information of the original sample.
2Measurement precision
If amplification techniques are used to detect low copy number analytes, then sensitivity is improved, but the process becomes more complex and time-consuming
Solution Approach 1:
The patent removes the complex amplification machinery (polymerases, primers, buffers) from the assay workflow. The simplified system uses direct binding of detection probes to target molecules followed by imaging, dramatically reducing procedural complexity and hands-on time while maintaining sensitivity through single-molecule detection capability.
3Adaptability or versatility
If current detection methods are used, then existing analytes can be detected, but errors such as false positives and misidentification occur
Solution Approach 1:
The patent implements feedback through multiple independent detection channels and verification steps. The system uses combinatorial probing strategies where multiple probes must simultaneously bind to the same target, and employs iterative imaging and analysis to confirm detections, thereby eliminating false positives while maintaining broad analyte coverage.
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
Enables high sensitivity, small sample volume analysis with high multiplexity and large dynamic range, reducing errors in analyte detection and quantification, and allowing simultaneous detection of nucleic acids and proteins.
Implementation Method 1
imaging a field of said surface with an optical system to detect an optical signal from each nucleotide incorporated into said polynucleotides
Data Source
AI summary
Disclosed herein are methods and systems for detection and discrimination of optical signals from a densely packed substrate. These have broad applications for biomolecule detection near or below the diffraction limit of optical systems, including in improving the efficiency and accuracy of polynucleotide sequencing applications.