Droplet Fluorochrome Diffusion Control in Real-Time PCR
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Solution Overview
Problem
The existing real-time PCR method in a droplet-based closed system faces challenges with fluorochrome diffusion from the reaction droplet into the oil, leading to reduced fluorescence detection due to hydrophobic interactions, which inhibits accurate detection of amplified nucleic acid products.
Innovation Solution
Incorporating a fluorochrome into the oil phase of the droplet encapsulating medium to maintain a stable concentration within the droplet, ensuring effective detection of amplified products by compensating for diffusion and preventing inhibition of the PCR reaction.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If a fluorochrome is added to the reaction liquid for PCR to enable real-time detection, then fluorescence detection capability is improved, but the fluorochrome diffuses into the oil phase due to hydrophobic interaction, reducing detection accuracy
Solution Approach 1:
A surfactant is introduced as an intermediary substance between the fluorochrome and the oil phase. The surfactant reduces hydrophobic interaction and prevents fluorochrome diffusion into the oil, maintaining stable fluorochrome concentration in the aqueous droplet while enabling real-time fluorescence detection
Solution Approach 2:
The chemical composition parameters of the oil phase are modified by adding a surfactant. This changes the interfacial properties and hydrophobicity of the oil, preventing fluorochrome diffusion while maintaining the closed droplet system for real-time PCR detection
2Illumination intensity
If the fluorochrome concentration in the reaction liquid is increased to compensate for diffusion loss, then fluorescence signal strength is improved, but the PCR reaction is inhibited due to excessive fluorochrome concentration
Solution Approach 1:
The surfactant acts as a protective intermediary that prevents fluorochrome loss to the oil phase. This allows the fluorochrome concentration to be maintained at optimal levels for both PCR reaction efficiency and fluorescence signal detection, without needing excessive concentrations
3Temperature
If a thermal cycler with metal block temperature control is used, then temperature stability is improved, but the reaction time becomes excessively long (1 hour or more)
Solution Approach 1:
The traditional mechanical thermal cycler system is replaced with a droplet-based microfluidic system. Temperature is controlled by moving the droplet between heat sources and cooling zones, enabling rapid temperature cycling (several minutes) while maintaining adequate temperature stability for PCR amplification
Solution Approach 2:
The droplet is subjected to periodic movement between heating and cooling zones, creating rapid periodic temperature changes that drive the PCR amplification cycles. This periodic thermal action achieves both speed and stability requirements
4Speed
If the droplet is repeatedly moved in the oil for PCR amplification, then temperature control speed is improved, but fluorochrome diffusion into the oil increases due to manipulation
Solution Approach 1:
The surfactant serves as a protective intermediary that remains effective during droplet manipulation. It prevents fluorochrome diffusion into the oil phase even when the droplet is repeatedly moved for rapid temperature cycling, maintaining both speed and fluorochrome stability
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 enables accurate and efficient real-time nucleic acid amplification and fluorescence detection in a closed system, facilitating rapid and precise genetic testing for clinical diagnostics.
Implementation Method 1
a droplet composed of a reaction liquid for PCR containing magnetic particles in the non-aqueous liquid and repeatedly moving the droplet to or from a heat source or vicinity thereof with the use of a magnet provided under the container
Implementation Method 2
a molecule of SYBR (Registered Trade Mark) GREEN I escapes through an interface between the droplet and the oil into the oil due to hydrophobic interaction between the hydrophobic parts of the molecule itself and the oil
Implementation Method 3
hydrophobic interaction between the hydrophobic parts of the molecule itself and the oil
Implementation Method 4
using a fluorochrome such as SYBR (Registered Trade Mark) GREEN I or the like that specifically binds to double-stranded DNA
Data Source
AI summary
The present invention provides a real time nucleic acid amplification reaction method comprising performing a nucleic acid amplification reaction in a droplet present in a container. The droplet is composed of a nucleic acid amplification reaction liquid including a nucleic acid to be amplified and magnetic particles. The container holds a droplet encapsulating medium immiscible with the nucleic acid amplification reaction liquid forming the droplet, and has a transport surface having a temperature gradient. Fluorochrome is initially contained in the droplet encapsulating medium, and optionally in the droplet, at start of the nucleic acid amplification reaction. The droplet is transported together with the magnetic particles by generating and applying a magnetic field so that the droplet is placed on the transport surface at a temperature point at which the nucleic acid synthesis reaction is started and maintained, thereby controlling a temperature of the reaction liquid.