A single-cell assay and a biosample enrichment method for analyzing single cell secretion
- Author(s): Cai, Wei
- Advisor(s): Lo, Yu-Hwa
- et al.
One of the key challenges of biology is to understand how individual cells process information and respond to perturbations. However, most of the existing single cell analysis methods can only provide a glimpse of cell properties at specific time points and are unable to provide cell secretion and protein analysis at the single cell resolution. This thesis offers the description of a single-cell assay as well as a CO2-induced enrichment method for the analysis of single cells secretions.
The single-cell assay introduced in this thesis enables the accommodation of different cellular types, allows for easy and efficient single cell loading and culturing, and is suitable for studying the efforts of in-vitro environmental factors in combination with drug screening. One salient feature of the assay is the non-invasive collection and survey of single cell secretions at different time points, producing unprecedented insight of single cell behaviors based on the biomarker signals from individual cells under given perturbations. In addition, the open-well design of the assay allows for simple collection of cells with standard tools such as pico-pipette for downstream processes in relating the single-cell secretions with gene analysis. Above all, the acquired information is quantitative. For example, measured by the number of exosomes each single cell secretes for a given time period, exosomal miRNA carried by exosomes secreted by single cell. Therefore, this single-cell assay provides a convenient, low-cost, and robust tool for quantitative, time lapsed studies of single cell properties.
Another challenge for single cell secretion analysis is the limit-of-detection (LOD) and sensitivity. Thus, sample enrichment is an important step in the work flow of biosensing for disease detection and numerous biological or clinical processes. Most current techniques require devices that are tailored to specific chemical or physical characteristics of the target objects to enrich or capture them from the sample. The complexity within these devices all serve to, increase cost and may even limit the enrichment factor. Here, a technique of using a CO2 laser to drive targets towards the laser spot via mass transport without requiring any device fabrication processes or special reagents was introduced. To prove the concept, single-stranded DNA (ssDNA) has been enriched by more than 100,000-fold in less than 4 minutes. The temperature and evaporation rate profile at the enriched area are measured alongside theoretical analyses and modeling to monitor and understand the physical process. The formation of aggregates comprised of streptavidin Q-dots and biotin labeled exosomes with this method was demonstrated to show the capability of biosample detection, purification, and quantification. The method is not only simple and highly efficient, but also applicable to all types of biomolecules and bioparticles. Thereby promising a simple, cost effective and efficient solution for biological sample preparation for sensing, analytics, and diagnostics.