Hydrogel Post Array Primer Immobilization for Microfluidic PCR
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Solution Overview
Problem
Current microfluidic devices face challenges in performing seamless PCR and melting curve analysis within defined microfluidic volumes without immobilizing primers, and there is a need for efficient post-PCR analysis and the ability to establish different primer combinations within a hydrogel post array.
Innovation Solution
A method involving a hydrogel post array with a cell-free, enzymatic nucleic-acid amplification system, where at least two non-immobilized nucleic acids promote synthesis of amplified nucleic acid products, using fluorescent markers like LC Green or SYBR Green for detection, and employing a polyacrylamide hydrogel with photo-polymerization to create a system for real-time PCR and MCA in microfluidic volumes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If primers are immobilized within the gel, then PCR reliability is improved, but device complexity and manufacturing difficulty increase
Solution Approach 1:
The patent extracts the primer immobilization step from the gel formation process. Primers are added to the gel after polymerization, allowing the gel matrix to be formed first without incorporated primers, then primers are introduced separately. This separates the manufacturing steps and reduces complexity while maintaining PCR reliability.
Solution Approach 2:
The gel matrix is formed in advance through photo-polymerization before primer addition. This preliminary gel formation creates the structural foundation without the complexity of incorporating primers during gel synthesis, and primers are subsequently added as a separate step to the prepared gel.
2Adaptability or versatility
If multiple primer combinations are used in a post array, then adaptability is improved, but manufacturing precision and homogeneity control become more difficult
Solution Approach 1:
The post array is segmented into multiple positions, each capable of receiving different primer combinations. This segmentation allows each post to be independently configured with specific primer pairs for different detection targets, achieving versatility while maintaining precision through individual post control.
Solution Approach 2:
Different primer combinations are introduced into different locations within the post array. Each post or region of the gel can contain specific primer pairs tailored to detect particular nucleic acid sequences, creating local quality variations that enable multiplex detection while maintaining manufacturing precision through localized primer addition.
3Productivity
If real-time PCR and MCA are performed in microfluidic volumes, then productivity is improved, but measurement precision and detection accuracy become more challenging
Solution Approach 1:
Fluorescent intercalating dyes are used to detect PCR products in real-time. The dyes bind to double-stranded DNA and produce fluorescent signals that can be detected and measured during the amplification process, enabling real-time monitoring and measurement of product formation in microfluidic volumes with high precision.
Solution Approach 2:
The patent replaces traditional mechanical detection methods with optical detection using fluorescent dyes. This substitution allows for real-time, non-invasive measurement of PCR products through fluorescence emission, improving measurement precision while maintaining the miniaturization benefits of microfluidic volumes.
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 real-time PCR and MCA with high accuracy and efficiency in small volumes, allowing for simultaneous testing of multiple nucleic acids and primer combinations, reducing cross-contamination and improving diagnostic capabilities in clinical and molecular characterization.
Implementation Method 1
Performing PCR in a colloidal hydrogel matrix (hereafter termed 'gel') may confer a multitude of advantages. For example, the DNA, polymerase enzyme and other PCR reagents a) have reduced access to the device materials' surfaces where they may be adsorbed, absorbed, poisoned or otherwise rendered inactive
Implementation Method 2
The hydrogel posts contain a fluorescent marker, wherein said fluorescent marker has different fluorescence properties when interacting with double-stranded nucleic acids than with single-stranded nucleic acids and in a still further aspect said fluorescent marker is LC Green or SYBR Green
Implementation Method 3
the hydrogel post is comprised of cross-linked polyacrylamide of 2.2%-3.1% weight per unit volume, and photo-polymerized in the absence of APS
Data Source
AI summary
The present invention provides for a novel system and method for amplification and detection of nucleic acids within a microfluidic device wherein multiple nucleotides capable of priming PCR are present within the system and substantially sequestered within separate hydrogel posts therein.