Droplet Microfluidics for Nucleic Acid Quantification and Single Cell Analysis

Abstract

Droplet microfluidics provides controlled generation of monodispersed droplets of the order of a few picoliters in multiphase microfluidic systems. These droplets are employed as micro-reactors to conduct chemical/biochemical reactions and assays in a controlled and high-throughput manner that find applications in point-of-care and lab-on-chip platforms. While the microfluidic devices are compact, the existing solutions to control fluid flow operations have a significant footprint that effects their portability, logistic viability and economics. As an alternate to the existing instrumentation-intensive flow-rate driven control for droplet generation, we studied and standardised suction driven fluid-flow control. In multiphase and multi-channel devices with suction-based flow control, microchannel geometry and suction pressure at the outlet determine the flow rates in individual channels. It is thus critical to understand the role of geometry along with suction pressure in the dynamics of droplet generation. We propose a governing parameter, called as modified capillary number, that captures droplet generation behaviour and outlines the design requirements for a suction driven droplet generation. As droplet microfluidics allows capture and analysis of individual cells with unprecedented control and throughput, single cell studies with microdroplets are gaining popularity. However, such analysis requires microfluidic devices with multiple unit operations that become a challenge with suction driven fluid-flow due to limited pressure head and lack of independent control over dispersed and continuous phase flow rates. To demonstrate single cell analysis, we defined and developed individual unit operations integrated in a multi-operation suction microfluidic device designed to quantify the low copy number RNA from single cells. The device, droplet digital Single cell Nucleic Acid Quantifier (dd-ScNAQ), successively performs encapsulation of single cells in droplets, cell lysis and cellular lysate/RNA distribution in smaller secondary droplets followed by on-chip temperature controlled isothermal nucleic acid amplification for quantification using fluorescence microscopy in a continuous flow geometry. Finally, to bypass the optical methods for detection of cells in droplets, which limits ‘in-field’ microfluidic applications; we developed a low-cost microfluidic impedance cytometry (MIC) approach for single cell quantification. We devised a rapid micro-fabrication protocol for flow devices with integrated coplanar in-contact field’s metal (icFM) electrodes to conduct MIC. With a single-step photolithography protocol, our icFM electrodes provide a cost-effective alternate to the conventional clean-room intensive microelectrode fabrication processes. The performance of icFM electrodes, averaged over a frequency range of 0.5 to 4 MHz, is found to be comparable to the widely used platinum electrodes in the detection of single human erythrocytes in a feedback-controlled suction driven MIC setup. However, the two electrodes show frequency dependent variability during impedance measurements that is attributed to the contrast between double layer capacitance and effective charge densities at their respective surfaces. We also demonstrate quantification of single cells entrapped in water-in-oil droplets using our icFM MIC devices as a non-optical and label-free method for droplet based single cell analysis. Together, this study extends the reach of existing microfluidic technologies to address the needs of the rapidly growing lab-on-chip industry by presenting novel methods to conduct microfluidic operations for single cell studies.