Single cell analysis has emerged as a paradigm shift in cell biology to understand the heterogeneity of individual cells in a clone for pathological interrogation. Microfluidic droplet technology is a compelling platform to perform single cell analysis by encapsulating single cells inside picoliter-nanoliter (pL-nL) volume droplets. However, one of the primary challenges for droplet based single cell assays is single cell encapsulation in droplets, currently achieved either randomly, dictated by Poisson statistics, or by hydrodynamic techniques. In this paper, we present an interfacial hydrodynamic technique which initially traps the cells in micro-vortices, and later releases them one-to-one into the droplets, controlled by the width of the outer streamline that separates the vortex from the flow through the streaming passage adjacent to the aqueous-oil interface (d). One-to-one encapsulation is achieved at a d equal to the radius of the cell, whereas complete trapping of the cells is realized at a d smaller than the radius of the cell. The unique feature of this technique is that it can perform 1. high efficiency single cell encapsulations and 2. size-selective capturing of cells, at low cell loading densities. Here we demonstrate these two capabilities with a 50% single cell encapsulation efficiency and size selective separation of platelets, RBCs and WBCs from a 10× diluted blood sample (WBC capture efficiency at 70%). The results suggest a passive, hydrodynamic micro-vortex based technique capable of performing high-efficiency single cell encapsulation for cell based assays.
The rapid progress of droplet microfluidics and its wide range of applications have created a high demand for the mass fabrication of low-cost, high throughput droplet generator chips aiding both biomedical research and commercial usage. Existing polymer or glass based droplet generators have failed to successfully meet this demand which generates the need for the development of an alternate prototyping technique. This work reports the design, fabrication and characterization of a mass manufacturable thermoplastic based microfluidic droplet generator on cyclic olefin copolymer (COC). COC chips with feature size as low as 20 µm have been efficiently fabricated using injection molding technology leading to a high production of inexpensive droplet generators. The novelty of this work lies in reoptimising surface treatment and solvent bonding methods to produce closed COC microchannels with sufficiently hydrophobic (contact angle of 120°) surfaces. These COC based droplet generators were shown to generate stable monodisperse droplets at a rate of 1300 droplets/second in the dripping regime. These new mass manufacturable, disposable and cheap COC droplet generators can be custom designed to cater to the rapidly increasing biomedical and clinical applications of droplet microfluidics.
Characterization of single cell metabolism is imperative for understanding subcellular functional and biochemical changes associated with healthy tissue development and the progression of numerous diseases. However, single-cell analysis often requires the use of fluorescent tags and cell lysis followed by genomic profiling to identify the cellular heterogeneity. Identifying individual cells in a non-invasive and label-free manner is crucial for the detection of energy metabolism which will discriminate cell types and most importantly critical for maintaining cell viability for further analysis. Here, we have developed a robust assay using the droplet microfluidic technology together with the phasor approach to Fluorescence Lifetime Imaging Microscopy (FLIM) to study cell heterogeneity within and among the leukemia cell lines (K-562 and Jurkat). We have extended these techniques to characterize metabolic differences between proliferating and quiescent cells—a critical step toward label-free single cancer cell dormancy research. The result suggests a droplet-based non-invasive and label-free method to distinguish individual cells based on their metabolic states, which could be used as an upstream phenotypic platform to correlate with genomic statistics.
In this work, we describe the mechanism of particle trapping and release at the flow-focusing microfluidic droplet generation junction, utilizing the hydrodynamic microvortices generated in the dispersed phase. This technique is based solely on our unique flow-focusing geometry and the flow control of the two immiscible phases and, thus, does not require any on-chip active components. The effectiveness of this technique to be used for particle trapping and the subsequent size selective release into the droplets depends on the fundamental understanding of the nature of the vortex streamlines. Here, we utilized theoretical, computational, and experimental fluid dynamics to study in detail these microvortices and parameters affecting their formation, trajectory, and magnitude.
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