Absolute Nucleic Acid Quantification Without Microfluidic Partitioning
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Solution Overview
Problem
Current nucleic acid quantification methods, such as digital PCR (dPCR) and rolling-circle amplification (RCA), face limitations including high cost, limited scalability, narrow dynamic range, and operational complexity due to reliance on microfluidic systems and Poisson statistics, hindering widespread adoption.
Innovation Solution
A microfluidics-free workflow integrating padlock-probe ligation, enzymatic cleanup, isothermal rolling-circle amplification, centrifugal deposition, and digital imaging-based enumeration, using standard laboratory equipment to achieve high-throughput, cost-effective absolute quantification of nucleic acids.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If digital PCR (dPCR) is used for absolute quantification of nucleic acids, then measurement precision is improved, but device complexity and cost increase due to reliance on microfluidic systems and specialized consumables
Solution Approach 1:
The invention extracts and eliminates the microfluidic partitioning step from the dPCR workflow. Instead of using microfluidic devices to create droplets or chambers, the method uses conventional PCR amplification followed by digital imaging to count individual amplification products, thereby removing complex microfluidic hardware while maintaining absolute quantification capability
Solution Approach 2:
The invention uses optical copying/imaging to capture and count amplification products. A fluorescence microscope or imaging system creates digital copies of the physical amplification products in the well, allowing enumeration without physical partitioning. This replaces the need for microfluidic physical separation with optical detection
2Measurement precision
If digital PCR (dPCR) is used for absolute quantification, then measurement precision is improved, but productivity decreases due to limited throughput of 16-96 samples per run
Solution Approach 1:
The invention makes the quantification method universal by adapting it to work with conventional PCR plates and imaging systems that are already widely used in laboratories. The same workflow can handle anywhere from single samples to full 384-well plates, providing flexibility and high throughput without requiring specialized microfluidic equipment
Solution Approach 2:
The invention transitions from physical partitioning in three-dimensional microfluidic droplets to two-dimensional planar distribution of amplification products in a well that can be imaged. This dimensional change allows parallel processing of many more samples simultaneously using standard plate formats
3Measurement precision
If rolling-circle amplification (RCA) is used for signal amplification, then sensitivity is improved, but device complexity increases due to reliance on microfluidic components and specialized consumables
Solution Approach 1:
The invention extracts RCA from its traditional microfluidic implementation and relocates it to conventional PCR tubes or wells. The isothermal amplification reaction proceeds in a simple liquid environment without requiring microfluidic channels, pumps, or specialized consumables, thereby eliminating the complexity barrier while preserving RCA's sensitivity advantages
Solution Approach 2:
The invention uses fluorescence-labeled detection probes as intermediaries to bridge the RCA amplification reaction and the imaging detection system. These probes convert the biochemical amplification into optical signals that can be captured by standard fluorescence microscopes, eliminating the need for specialized microfluidic detection components
4Measurement precision
If microfluidic partitioning is used in dPCR, then absolute quantification accuracy is improved, but cost per sample increases due to specialized consumables
Solution Approach 1:
The invention replaces expensive, specialized microfluidic consumables with inexpensive, disposable conventional PCR plates and tubes that are already widely available in laboratory settings. The low cost of these standard consumables dramatically reduces per-sample expenses while the imaging-based detection maintains quantification accuracy
Solution Approach 2:
The invention uses optical imaging to create digital copies of the amplification products for enumeration. This optical copying approach eliminates the need for expensive physical microfluidic consumables, as the detection is performed using light and cameras rather than specialized physical partitions
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 method enables accurate, scalable, and cost-effective absolute quantification of nucleic acids across a broad dynamic range without microfluidic partitioning, reducing assay complexity and cost per sample, and improving throughput and precision.
Implementation Method 1
A padlock probe is hybridized to a target nucleic acid
Implementation Method 2
circularized by a thermostable DNA ligase
Implementation Method 3
amplified through rolling-circle amplification using a phi29-family DNA polymerase to generate single-stranded DNA concatemers
Implementation Method 4
substantially all amplification products are centrifugally deposited onto the planar surface of a multi-well plate
Implementation Method 5
RCA amplicons are fluorescently labeled and visualized
Data Source
AI summary
The invention provides methods, systems, and reagent kits for absolute quantification of nucleic acids using a high-throughput, microfluidics-free workflow that integrates padlock-probe ligation, enzymatic cleanup, rolling-circle amplification, centrifugal deposition, and digital fluorescence imaging. Amplified products are deposited onto a planar multi-well plate and digitally enumerated by automated image analysis to determine absolute copy number without statistical partitioning or reference standards. The platform supports both DNA- and RNA-based workflows and multiplex detection using spectrally distinct or barcoded probes, enabling simultaneous quantification of multiple nucleic-acid targets with high precision, broad dynamic range, and low cost.