Enzyme-Assisted Biosensor for Rapid Small Molecule Detection
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Solution Overview
Problem
Current methods for detecting small molecules in vitro are complex, time-consuming, and require multiple preparation steps, often involving nucleic acid amplification, which increases cost and complexity, and are not feasible for one-pot assays due to separation of sensing and amplification steps.
Innovation Solution
The development of an in vitro biosensor using enzyme-assisted nucleic acid reactions that integrate allosteric proteins or aptamers with Toehold-mediated Strand Displacement reactions, enabling electrochemical, fluorescent, or colorimetric outputs in a one-pot assay without the need for nucleic acid amplification, and can be adapted for various preservation techniques and modalities like microfluidic devices.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional biosensor methods using allosteric transcription factors are used, then detection of small molecules can be achieved, but the process requires multiple preparation steps including incubation, equilibration, washes, and centrifugation which increases complexity and time
Solution Approach 1:
The invention merges the sensing step (allosteric protein-ligand interaction) and the signal transduction step (nucleic acid amplification) into a single integrated system. The allosteric protein is directly coupled to the nucleic acid template, eliminating the need for separate preparation steps such as incubation, equilibration, washes, and centrifugation that were required in traditional methods.
Solution Approach 2:
The invention creates a universal platform where the same basic architecture (allosteric protein-nucleic acid complex) can detect various small molecules by simply changing the specific allosteric protein or aptamer used, without requiring different assay protocols or preparation steps for each analyte.
2Measurement precision
If nucleic acid amplification techniques are used for signal amplification, then detection sensitivity is improved, but the cost and complexity of the test increase
Solution Approach 1:
The system uses self-service amplification through toehold-mediated strand displacement reactions that automatically amplify the signal without requiring external enzymes or complex amplification protocols. The nucleic acid templates self-assemble and self-amplify through the inherent thermodynamic driving force of the toehold-mediated displacement mechanism.
3Measurement precision
If multiple preparation steps are required for biosensor assays, then detection accuracy can be maintained, but the total time from reaction preparation to results increases beyond 100 minutes
Solution Approach 1:
The allosteric protein is pre-complexed with the nucleic acid template in a stable configuration that is ready for immediate detection. This preliminary assembly eliminates the need for time-consuming incubation and equilibration steps that were traditionally required to allow the components to associate and reach equilibrium.
4Measurement precision
If allosteric transcription factors are modified and/or immobilized for biosensor platforms, then the sensing capability is improved, but flexibility is reduced due to variable processing and efficiency depending on the aTF
Solution Approach 1:
The invention creates a universal platform where the same basic architecture (allosteric protein-nucleic acid complex) can detect various small molecules by simply changing the specific allosteric protein or aptamer used, without requiring different assay protocols or preparation steps for each analyte.
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 simplifies the detection process, reducing turnaround time and increasing sensitivity, while allowing for flexible and modular biosensor design, capable of detecting small molecules quickly and accurately in biological and environmental samples.
Implementation Method 1
contacting an endonuclease, DNA template, sensor molecule, and an invading probe with the sample... digesting the DNA template with the endonuclease
Implementation Method 2
hybridizing the invading probe to the DNA template... the invading probe initiates a toehold-mediated strand displacement reaction
Implementation Method 3
Toehold-mediated Strand Displacement reactions, enabling electrochemical, fluorescent, or colorimetric outputs
Implementation Method 4
the sensor molecule excludes the endonuclease from a restriction site specific to said endonuclease in the DNA template
Implementation Method 5
binding or hybridizing the sensor molecule to the DNA template, wherein the sensor molecule excludes the endonuclease from a restriction site... in the absence of the analyte or displacing the sensor molecule from the DNA template when the analyte binds to the sensor molecule
Implementation Method 6
detecting a signal emitted from the invading probe, wherein detection of said signal is indicative of the presence of the analyte
Data Source
AI summary
The subject invention pertains to composition and methods of using said composition as an in vitro biosensor of small molecules in biological and/or environmental samples using enzyme-assisted nucleic acid reactions. The methods and compositions can be used to sense and/or transduce the signal of a sensing event mediated by allosteric proteins, endonucleases and nucleic acid reactions. This invention allows the rapid development and setup of one-pot assays to provide results in minutes. The methods and compositions may be used to generate an electrochemical, fluorescent, colorimetric, and/or luminescent output and the methods can be performed in different modalities, including a solution-based or paper-based assay.