Development of Droplet-Based Microfluidic Techniques for Cancer Biomarkers Analysis

開發微流控液滴技術用於癌症生物標誌物的分析研究

Student thesis: Doctoral Thesis

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Award date27 Sep 2022

Abstract

Cancer biomarkers are biological molecules, cellular components, and cells, including nucleic acids, secreted proteins, antibodies, and tumor cells, that are present in the blood, body fluids, and other tissues. Biomarkers provide valuable information for cancer diagnosis, treatment prediction, and progress monitoring. However, due to the lack of characterization and validation for biomarkers and the robustness of analysis techniques in clinical trials, transferring biomarkers from discovery to routine clinical practice has remained challenging. Therefore, efficient and economic approaches must be developed to promote the analysis of biomarkers, thus permitting the precise assessment and therapeutics of cancer diseases.

Droplet-based microfluidics has gradually become an important branch of microfluidics and contributes to the application of a wide range of lab-on-chip systems in understanding cancer biology. This technique produces and manipulates highly monodisperse droplets in the nanometer to micrometer diameter range, which is suitable for manipulating single genes, cells, or organisms. Droplet-based microfluidics shows a wide range of potential applications in biomarker research, including single-molecule polymerase chain reaction (PCR), single-cell analysis, proteome analysis, and clinical diagnosis of human physiological fluids. In this study, a suite of integrated and automated droplet-based microfluidic platforms was developed for the analysis of potential cancer biomarkers, including nucleic acids and circulating tumor cells (CTCs).

1. An multi-functional droplet-based microfluidic platform was developed and applied in in-situ digital DNA amplification for the absolute quantification of nucleic acid and rare mutation detection. Compared with existing digital polymerase chain reaction (dPCR) methods and platforms, the current platform integrates the traditional workflow of digital DNA amplification consisting of droplet generation, transition, and signal detection into a one-step microfluidic device. The platform was used to identify epidermal growth factor receptors (EGFR) by detecting dPCR and human papillomavirus (HPV) via digital loop-mediated isothermal amplification (dLAMP), thus demonstrating the capability and feasibility of the integrated device. Given its advantages of high throughput, easy operation, and low cost, this platform shows potential in detecting mutations and performing single-cell PCR amplification for the guidance of early diagnostics and molecularly targeted therapies.

2. A label-free method was developed for the deterministic encapsulation of single cells in microgels based on cell metabolism activity and phase transition of microgels without the assistance of an external force field. Different cells, including tumor cell lines, white blood cells, and cell mixtures from tissue samples, were distributed in sodium alginate droplets using a microfluidic device. Accumulating cellular metabolism inside droplets decreases the droplets' pH value and triggers microgels' polymerization. Cell debris and dead cells were easily removed during the demulsification process. Finally, a pure population of living cell-containing microgels was obtained. This platform provides a universal, simplified approach for single-cell analysis and is promising in tissue engineering and regenerative medicine applications.

Furthermore, this platform was applied to enrich and isolate tumor cells for subsequent molecular and cellular analysis. Owing to the difference in metabolism pathway between tumor cells and WBCs, droplets containing living tumor cells have a relatively low extracellular pH value and a more acidic environment. As a result, target tumor cells encapsulated inside the microgels were successfully isolated. Compared with traditional methods based on size or immunoaffinity, this approach provides a label- and equipment-free strategy to detect living tumor cells for early clinical detection and disease prognosis.

3. An aptamer functionalized DNA hydrogel glue was generated via rolling circle amplification (RCA) for the specific capturing and release of living CTCs. CTCs are captured inside the porous 3D nanoflower DNA hydrogel network via binding with aptamer. The encapsulated CTCs are then released from the DNA hydrogel glue with minimal damage and subcultured into tumor spheres for molecular characterization. Furthermore, CTCs were successfully isolated from blood samples from patients with different types of cancers, indicating that the DNA hydrogel glue is an attractive tool for the enrichment of CTCs for tumor diagnostics and prognosis in clinical settings.

In summary, the series of droplet-based microfluidic platforms provide competitive advantages of efficiently detecting genetic mutations for molecular diagnosis and simply manipulating single cells for cellular analysis in cancer biology. Further work should be conducted to investigate single-cell molecular analysis inside the droplets and microgels. These platforms have significant contributions to the development of cancer biomarkers. They ultimately could be applied in predictive diagnostics and cancer prognosis in clinical trials, thus effectively increasing the opportunities for treatment and cancer mortality reduction.