Lyophilizable Indicator Master Mix for Visual DNA/RNA Detection
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Solution Overview
Problem
Existing lyophilizable master mixes containing Calcein, a fluorescent dye, are not viable due to the use of solvents like DMSO, which prevent lyophilization, resulting in sludge-like substances.
Innovation Solution
A lyophilizable indicator master mix is developed using a hydrophobic nucleic-acid based indicator, such as Calcein, solubilized with an aprotic solvent like DMSO, within a specific ratio to enable lyophilization into a form factor like lyobeads, allowing visual detection without additional instrumentation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If DMSO is used to solubilize Calcein in water-based solutions, then the indicator is effectively solubilized, but the solution cannot be lyophilized and results in sludge-like substance
Solution Approach 1:
The patent changes the chemical composition parameters by replacing DMSO with alternative solvents (acetone, acetonitrile, DMF, DCM, THF, HMF, or crown ethers) that allow lyophilization while maintaining Calcein solubility. This parameter change resolves the contradiction by finding a solvent that satisfies both solubility requirements and lyophilization capability.
Solution Approach 2:
The patent introduces an intermediary substance (alternative solvent acting as a bridge) that enables both Calcein solubility and lyophilization. The alternative solvents serve as intermediaries that facilitate the dissolution of hydrophobic Calcein while allowing the solution to be freeze-dried into a stable lyophilized form without forming sludge.
2Difficulty of detecting and measuring
If Calcein is used as a fluorescent dye indicator, then visual detection is achieved, but the master mix cannot be lyophilized for long-term storage
Solution Approach 1:
The patent changes the solvent parameter to enable lyophilization while preserving Calcein's fluorescent detection capability. By selecting alternative solvents that are compatible with both Calcein solubility and freeze-drying processes, the master mix achieves long-term storage stability without sacrificing the visual detection function.
Solution Approach 2:
The patent utilizes phase transition through lyophilization (freezing followed by sublimation of solvent) to transform the liquid master mix into a stable solid/ amorphous solid state for long-term storage. This phase transition enables the master mix to be stored indefinitely while maintaining the Calcein indicator's functionality when reconstituted.
3Ease of manufacture
If hydrophobic nucleic-acid based indicator is used, then lyophilization is enabled, but the indicator must be solubilized in specific ratio with solvent
Solution Approach 1:
The patent establishes specific parameter ranges for the indicator to solvent ratio (0.01-0.05 g/g) to optimize both lyophilization performance and indicator functionality. By defining this precise parameter range, the patent resolves the contradiction by finding the optimal balance point where sufficient solvent is present for complete indicator solubility but not so much that lyophilization becomes difficult or produces unwanted byproducts.
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
The solution provides a lyophilizable master mix with a long shelf life, enabling efficient, cost-effective, and human-readable detection of DNA/RNA sequences, suitable for widespread screening and Point of Need Diagnostics.
Implementation Method 1
a solvent configured to solubilize the substantially hydrophobic nucleic-acid based indicator
Implementation Method 2
A lyophilized or lyophilizable indicator master mix is provided
Data Source
AI summary
Indicator master mix for performing nucleic acid amplification assays to detect and visually identify the presence or absence of target or template DNA or RNA sequences. The indicator master mix comprises (i) a human readable, substantially hydrophobic nucleic-acid based indicator and (ii) a solvent configured to solubilize the substantially hydrophobic nucleic-acid based indicator, such as DMSO. The relative amount of nucleic-acid based indicator to solvent is within the range of (i) about 0.01 gm nucleic-acid based indictor/gm solvent to (ii) about 0.05 gm nucleic-acid based indicator/gm solvent. The master mix is configured to perform the nucleic acid amplification assay to detect and visually identify the presence or absence of the target or template DNA or RNA sequence, and the substantially hydrophobic nucleic-acid based indicator is visually detectable by a human eye to indicate the presence or absence of the target or template DNA or RNA sequence.