Isothermal Biomolecule Detection via Polymerase Activity
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Solution Overview
Problem
Current methods for detecting biomolecules, such as nucleic acids, are either slow and prone to artifacts or require complex and time-consuming laboratory procedures, limiting their sensitivity and speed in diagnosing pathogenic organisms.
Innovation Solution
A method involving a reaction mixture with a nucleic acid probe, a nucleic acid template, and a polymerase that extends the probe, detecting the polymerase activity through ATP or pyrophosphate generation, allowing for rapid and sensitive detection of target nucleic acid sequences without the need for thermal cycling.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If qPCR/qRT-PCR is used for detection, then sensitivity is improved, but time required and device complexity increase
Solution Approach 1:
The invention extracts and eliminates the thermal cycling step from the PCR process, using isothermal amplification instead. This removes the time-consuming heating and cooling cycles while maintaining amplification capability, directly resolving the contradiction between sensitivity and time requirement
Solution Approach 2:
The invention changes the temperature parameter from cyclic variation to constant isothermal conditions. By maintaining a single temperature (e.g., 65°C) throughout the reaction, the method achieves rapid amplification without the time loss associated with thermal cycling, while preserving detection sensitivity
2Measurement precision
If qPCR/qRT-PCR is used for detection, then sensitivity is improved, but device complexity increases
Solution Approach 1:
The invention extracts and removes the thermal cycler from the required equipment list. By using isothermal amplification, the method eliminates the need for complex temperature cycling equipment, reducing device complexity while maintaining sensitivity through alternative detection chemistry
Solution Approach 2:
The invention replaces the mechanical thermal cycling system with a simpler isothermal chemical reaction system. The constant temperature reaction requires only basic heating equipment rather than complex programmable thermal cyclers, significantly reducing device complexity
3Loss of time
If isothermal amplification is used, then time required is reduced, but sensitivity decreases
Solution Approach 1:
The invention introduces an intermediary signaling system using fluorescently labeled nucleotides and exonuclease digestion. This intermediary mechanism amplifies the signal from isothermal amplification, compensating for the reduced sensitivity and enabling detection of low-abundance targets despite the simplified isothermal process
Solution Approach 2:
The invention uses fluorescent color changes as a detection mechanism. Fluorescently labeled nucleotides incorporated during isothermal amplification emit light signals that can be detected, providing sensitive quantification without requiring complex thermal cycling. The fluorescent signal intensity correlates with amplification product amount, maintaining measurement precision
4Measurement precision
If exponential amplification is used, then detection sensitivity is improved, but artifacts increase
Solution Approach 1:
The invention uses periodic addition of reagents (nucleotides, exonuclease) in a controlled sequence during isothermal amplification. This periodic action allows the reaction to proceed in controlled stages, reducing non-specific amplification and artifacts while maintaining sensitivity through the cumulative effect of repeated cycles of nucleotide incorporation and digestion
Solution Approach 2:
The invention incorporates feedback through real-time fluorescent detection during isothermal amplification. The fluorescent signal provides continuous information about amplification progress, allowing the reaction to be monitored and controlled to prevent artifacts. The detection system feeds back on the reaction state, enabling adjustment to maintain reliability
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 rapid and sensitive detection of biomolecules, reducing the time required for diagnostics and minimizing artifacts, while being less dependent on complex laboratory equipment.
Implementation Method 1
a polymerase capable of extending the 3′ end of the nucleic acid probe
Implementation Method 2
incorporation of the nucleotide by the polymerase results in release of a molecule of ATP
Implementation Method 3
measuring the amount of pyrophosphate generated by the polymerase
Implementation Method 4
the ATP is measured by luminescence
Implementation Method 5
P is annealed to Pc in the absence of target nucleic acid
Implementation Method 6
a first sequence complementary to a template nucleic acid sequence
Data Source
AI summary
Provided are methods, compositions and devices for high sensitivity detection of biomolecules such as nucleic acids in biological samples. The methods rely on target detection, nucleic acid amplification, and sensitive detection to provide a signal which can be conveniently measured in a lab assay or device, including with portable and point-of-care instrumentation.