Nucleic Acid Absolute Quantification with PEG Hydrogel Micropores
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Solution Overview
Problem
Current nucleic acid quantification methods, such as real-time fluorescent quantitative PCR and digital PCR, face limitations in sensitivity, accuracy, scalability, and operational complexity, particularly in high-throughput applications.
Innovation Solution
A nucleic acid absolute quantification system utilizing a PEG hydrogel system formed by specific monomers that spontaneously polymerize at room temperature, forming a hydrogel that traps nucleic acids within micropores, allowing primers and reagents to diffuse through, creating a purer reaction environment and enabling rapid, accurate quantification without the need for standard curves.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If real-time fluorescent quantitative PCR is used for nucleic acid quantification, then the method is widely applicable and provides linear amplification signals, but the sensitivity and accuracy are limited due to being a relative quantification method requiring standard curves
Solution Approach 1:
The patent applies segmentation by partitioning the reaction system into numerous discrete microcompartments (droplets or chambers) where individual nucleic acid molecules are isolated. This digital partitioning enables absolute quantification by counting positive compartments rather than relying on relative standard curves, thereby improving measurement precision while maintaining operational simplicity through automated droplet generation and reading.
2Measurement precision
If digital PCR technology using microfluidic chip array reaction chambers is used, then individual nucleic acid molecules can be isolated for PCR reactions, but the scalability is limited and detection throughput is low
Solution Approach 1:
The patent employs hydraulic principles by using water-in-oil emulsion technology to generate numerous discrete droplets that serve as reaction compartments. This approach enables high-throughput digital PCR by creating thousands of isolated reaction environments in parallel, significantly improving detection throughput compared to microfluidic chip arrays while maintaining the ability to isolate and detect individual nucleic acid molecules with high accuracy.
3Productivity
If emulsion micro-droplet digital analysis technology is used, then high-throughput detection is achieved by sealing magnetic beads with emulsion, but the operation procedure complexity increases and thermal cycling amplification is required
Solution Approach 1:
The patent extracts and eliminates the magnetic bead component from the emulsion digital PCR system, using purely aqueous water-in-oil emulsion droplets as reaction compartments. This simplification removes the complexity associated with magnetic bead handling, separation, and recovery steps, making the operation more straightforward while maintaining high-throughput capability through automated droplet generation and fluorescent signal reading.
4Productivity
If emulsion micro-droplet digital analysis technology is used, then high-throughput detection is achieved, but target templates cannot be detected when template and magnetic beads are not partitioned into the same droplet
Solution Approach 1:
The patent uses the oil phase in water-in-oil emulsion as an intermediary barrier that physically isolates aqueous droplets containing template DNA from each other. This intermediary layer ensures that each droplet remains a discrete reaction compartment, preventing cross-contamination while allowing reliable detection of target templates within each droplet. The emulsion structure itself serves as the partitioning mechanism, eliminating the need for magnetic beads as intermediaries.
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 system achieves rapid, accurate, and cost-effective absolute quantification of nucleic acids with high throughput by confining target analytes within micropores, mitigating inhibitor effects, and simplifying the operation process.
Implementation Method 1
a polyethylene glycol acrylate compound containing two or more acrylate groups or a polyethylene glycol maleimide compound containing two or more maleimide groups, and a polyethylene glycol-thiol compound containing two or more thiol groups
Implementation Method 2
The novel nucleic acid absolute quantification system designed herein enables rapid, accurate, simple and cost-effective absolute quantification of nucleic acids without plotting a standard curve
Implementation Method 3
allowing primers and reagents to diffuse through
Data Source
AI summary
A nucleic acid absolute quantification system, including a polyethylene glycol acrylate compound or a polyethylene glycol maleimide compound, a polyethylene glycol-thiol compound, a primer for the target nucleic acid molecule; a nucleic acid amplification reagent and a fluorescent agent. A mass ratio of the polyethylene glycol acrylate compound or the polyethylene glycol maleimide compound to the polyethylene glycol-thiol compound is 1-30:10-1. A nucleic acid absolute quantification kit and method are also provided.