Porous Membrane Platform for Single Cell Analysis
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Solution Overview
Problem
Current methods for isolating and analyzing circulating tumor cells (CTCs) face challenges such as low specificity, high risk of cell loss and contamination, and limited throughput due to complex workflows and multiple handling steps, which complicate the reliable detection and characterization of these cells.
Innovation Solution
A method involving a porous membrane with adjustable pore size and surface properties for selective cell trapping, combined with a well plate containing reagents for analysis, allows for integrated cell enrichment and analysis, minimizing handling steps and ensuring high specificity and throughput by trapping CTCs while allowing smaller cells to pass through, and enabling microscopic verification and analysis of retained cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If marker independent separation methods relying on size/deformability are used, then throughput is improved, but selectivity deteriorates due to significant size overlaps between CTCs and WBCs
Solution Approach 1:
The patent applies local quality by functionalizing specific regions of the porous membrane with EpCAM antibodies. The membrane has heterogeneous properties: some areas are coated with antibodies for specific CTC binding, while other areas remain uncoated to allow passive size-based filtration. This local differentiation enables both high throughput (via uncoated regions) and high selectivity (via antibody-coated regions capturing EpCAM-positive CTCs).
Solution Approach 2:
The invention uses a composite structure combining a porous filtration membrane with immobilized EpCAM antibodies. The composite material integrates the size-exclusion properties of the porous matrix with the specific binding properties of the antibody coating, achieving both high throughput filtration and high selectivity for CTCs expressing EpCAM markers.
2Adaptability or versatility
If complex workflows with multiple handling steps are used for CTC isolation and analysis, then analysis capability is improved, but cell loss and contamination risk increase
Solution Approach 1:
The patent merges CTC isolation and single-cell analysis into a single integrated platform. The porous membrane with EpCAM antibodies directly captures CTCs in an enriched state, and the captured cells remain on the membrane for immediate downstream analysis including microscopy, flow cytometry, or molecular assays. This consolidation eliminates multiple transfer steps, reducing cell loss and contamination while maintaining comprehensive analysis capability.
Solution Approach 2:
The invention performs preliminary CTC enrichment and capture on the porous membrane before analysis. By pre-concentrating and immobilizing CTCs on the membrane in an enriched state, the system prepares samples for downstream analysis without requiring subsequent cell manipulation steps, thereby preventing cell loss and contamination that would occur during transfer operations.
3Measurement precision
If EpCAM based isolation method is used, then specificity for epithelial CTCs is improved, but coverage of all CTC types deteriorates due to EMT causing down-regulation of epithelial markers
Solution Approach 1:
The patent employs parameter changes by utilizing EpCAM antibody coating on the porous membrane to specifically target and capture epithelial CTCs expressing EpCAM markers. The antibody coating density and pore size parameters are optimized to enhance specific binding while maintaining filtration efficiency, achieving high specificity for the intended cell population.
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 reliable, efficient, and high-throughput analysis of CTCs with reduced risk of contamination and cell loss, allowing for direct single-cell analysis without complex manipulation, thereby improving the accuracy and efficiency of cancer diagnosis and treatment strategies.
Implementation Method 1
filtering a liquid cell suspension comprising cells of interest through the porous membrane
Data Source
Figure 1A~1D
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AI summary
The invention relates to a method for analyzing one or more single cells of interest, comprising a) provision of (i) a porous membrane comprising at least one pore, wherein the pore is adjusted to trap a single cell of interest when the cell is applied to the membrane in a liquid medium (ii) a well plate comprising at least one well, wherein the well diameter is larger than the pore diameter and the at least one well contains reagents for analyzing at least one component of the cells of interest, wherein the reagents can be provided in liquid or dry form, (iii) means for assembling the porous membrane and the well plate in an orientation to position the at least one pore opposite of the at least one well, wherein the means for assembling are part of the porous membrane and/or the well plate, (iv) a cover for sealing the at least one well with the at least one pore localized opposite of the at least one well after assembling the porous membrane and the well plate; and b) filtering a liquid cell suspension comprising cells of interest through the porous membrane; c) assembling the porous membrane and the well plate in an orientation to position the at least one pore opposite of the at least one well; d) sealing the at least one well and the at least one pore localized opposite of the well with the cover, and e) analysis of at least one cellular component of a retained single cell in the at least one sealed well.