Isothermal Nucleic Acid Detection via Displacement Amplification
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current nucleic acid detection methods, such as lateral flow assays, are limited by the need for sufficient sample amounts and lack amplification capabilities, making it difficult to detect small numbers of molecules efficiently and quickly.
Innovation Solution
A test strip device with a displacement area, signal amplification areas, and detection areas connected on a porous material, where the presence of a target analyte triggers a cascade of marker releases without breaking chemical bonds, allowing for near-exponential signal amplification without the need for solution-based reagents.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If lateral flow assays are used for detection, then the assay is simple and rapid, but the sensitivity is insufficient for detecting low concentrations of nucleic acids
Solution Approach 1:
The assay is divided into distinct functional zones on the test strip: a displacement zone containing immobilized nucleic acid probes, and a detection zone containing detection probes. This segmentation allows each zone to perform its specific function independently, enabling signal amplification while maintaining the simplicity of a single-step lateral flow format.
Solution Approach 2:
Nucleic acid probes are pre-immobilized on the test strip in the displacement zone before the assay is performed. This preliminary preparation eliminates the need for complex sample processing or addition of reagents during the assay, allowing direct application of the sample and automatic initiation of the displacement reaction, thus achieving high sensitivity without increasing operational complexity.
2Measurement precision
If thermocycling or isothermal amplification is used to amplify nucleic acid, then the detection sensitivity is improved, but the time required for analysis increases to about 90 minutes
Solution Approach 1:
The displacement reaction is self-initiated when the sample is applied to the test strip. The target nucleic acid automatically displaces the marker from the immobilized probe without requiring external enzymes, energy input, or complex amplification machinery. This self-service mechanism achieves rapid signal amplification in minutes rather than hours, eliminating the time-consuming thermocycling or isothermal amplification steps.
3Measurement precision
If fluorescence detection with labelling is used, then the detection sensitivity is improved, but the cost increases and carcinogenic reagents are required
Solution Approach 1:
The test strip uses disposable immobilized probes with simple markers instead of expensive fluorescent labels. The markers provide sufficient signal for detection without requiring costly instrumentation or carcinogenic reagents like SYBR green. This approach achieves the required detection sensitivity while eliminating harmful substances and reducing costs.
4Measurement precision
If restriction endonucleases are used for sequence-specific cleavage, then the specificity is improved, but the complexity of the assay increases and additional solution-based reagents are required
Solution Approach 1:
The assay extracts and utilizes only the essential specificity-providing element (the immobilized nucleic acid probe sequence) while eliminating the need for complex restriction endonuclease systems. The probe sequence itself provides the sequence-specific recognition and displacement function, removing the requirement for additional solution-based enzymatic reagents and simplifying the overall assay design.
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 the detection of a single molecule with high sensitivity, producing 10^6 to 10^9 detectable markers, allowing for the detection of low concentrations of nucleic acids and other molecules without additional enzymes or reagents, improving sensitivity and specificity.
Implementation Method 1
the displacement occurs by disrupting the binding of a double stranded nucleic acid strand interaction
Implementation Method 2
the first released marker comprises a protease; a second immobilised marker which can be cleaved by the presence of the protease to release a detectable marker
Implementation Method 3
fluid can flow from the displacement area through the signal amplification area(s) and into the detection area(s) upon application of the fluid to the device
Data Source
Figure 1
Figure 2
Figure 3
AI summary
The present invention relates to a method and kit for amplifying and detecting a quantity of nucleic acid. The invention is particularly relevant to isothermal amplification techniques carried out on a flow based assay device. The amplified nucleic acid may be detected on the device using an optical read-out.