Deblocking Double-Layer Chromatography Cassette for Liquid Biopsy
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Solution Overview
Problem
Existing nucleic acid isolation methods, such as silica-based methods, are not suitable for liquid biopsy samples like urine due to large sample volumes, small nucleic acid sizes, and low nucleic acid concentrations, which result in reduced efficiency and flowability of the isolation process.
Innovation Solution
A deblocking double-layer chromatography and cassette system is developed, featuring a first layer of positively charged or cationic membrane and a second layer of silica membrane. The system includes a deblocking mechanism to address blockage by solid particles, eliminating the need for pre-filtering and enhancing automation and flowability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If silica-based isolation method is used, then nucleic acid binding efficiency is improved, but flowability deteriorates due to blockage by solid particles in large volume samples
Solution Approach 1:
The isolation system is divided into two separate layers: a first layer with high flowability for sample loading and a second silica-based layer for nucleic acid binding. This segmentation allows each layer to perform its specific function optimally without compromising the other, resolving the contradiction between flowability and binding efficiency.
Solution Approach 2:
A coupling solution is introduced as an intermediary between the first and second layers. This coupling solution facilitates the transfer of nucleic acids from the flowable first layer to the binding-optimized second layer, enabling both high flowability during sample loading and high binding efficiency during isolation.
2Quantity of substance
If large sample volumes are processed, then nucleic acid recovery is improved, but processing time increases due to reduced flowability
Solution Approach 1:
By segmenting the isolation system into two layers with different functional optimizations, large sample volumes can be processed quickly through the first layer while nucleic acids are efficiently captured in the second layer, maintaining both high recovery and reduced processing time.
Solution Approach 2:
The system enables periodic processing of large sample volumes through the first layer, with nucleic acids being continuously transferred to the second layer, allowing high-throughput processing without time loss.
3Ease of operation
If pre-filtering is added to remove solid particles, then flowability is improved, but device complexity increases
Solution Approach 1:
The filtering function is merged into the first layer of the isolation system itself, eliminating the need for a separate pre-filtering step. The first layer is designed to handle solid particles while maintaining high flowability, simplifying the overall device architecture.
Solution Approach 2:
The first layer serves multiple functions: it acts as both the sample loading channel and the filtering medium for solid particles. This multi-functionality reduces device complexity by eliminating separate filtering components while maintaining ease of operation.
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 deblocking cassette significantly improves the flowability of the first layer and enhances the recovery rate of nucleic acids, achieving efficient isolation of cf-DNA and cf-RNA from large volume urine samples with small nucleic acid sizes and low concentrations.
Implementation Method 1
a first layer of positively charged or cationic membrane or bead and a second layer of membrane or bead which is coupled with the first layer
Implementation Method 2
The principle of silica-based isolation is based on the high affinity of the negatively charged DNA/RNA backbone towards the positively charged silica surface under concentrated chaotrophic salt conditions
Implementation Method 3
GuTC and GuHCl are commonly used for binding nucleic acids to the silica surface. GuTC at a concentration of 4 M to 6 M works best, while GuHCl is used at a higher concentration of up to 6 M. The binding efficiency is significantly improved in the presence of ethanol or propanol.
Implementation Method 4
reversely flowing a second solution (deblocking solution) through the first layer to which the nucleic acid still remains bound, and deblocking the first layer to increase its flowability
Data Source
AI summary
The instant invention provides a deblocking double-layer chromatography and cassette for isolating nucleic acids from liquid biopsy samples such as urine with large sample volumes, small nucleic acid sizes, and low nucleic acid concentrations. Compared with the prior art of Ding et al. (U.S. Pat. No. 9,163,230) which proved the principle of double-layer chromatography comprising a positively charged DEAE membrane and a silica membrane, we further explored and optimized the membrane compositions and solution compositions for plasma and urine samples. More importantly, we set up a “deblocking” mechanism to overcome a “blocking” problem that the membrane is blocked by solid particles in the plasma and urine samples, thus greatly increasing its flowability. In addition, it omits a pre-filtering step, thus particularly suitable for its automation.