A flexible and cost-effective manual droplet operation platform for miniaturized cell assays and single cell analysis.

Herein, we developed a flexible and cost-effective manual droplet operation system (MDOS) for performing miniaturized cell assays as well as single cell analysis. The MDOS consists of a manual x-y-z translation stage for liquid transferring and switching, a high-precision syringe pump for liquid driving and metering, a tapered capillary probe for droplet manipulation, a droplet array chip for droplet loading and reaction, sample/reagent reservoirs for storage, and a microscope for droplet observation, with a total expense of only $4,000. By using the flexible combination of three elementary operations of the x-y-z stage's moving and the pump's aspirating and depositing, the MDOS can manually achieve multiple droplet handling operations in the nanoliter to picoliter range, including droplet generation, assembling, fusion, diluting, and splitting. On this basis, multiple cell-related operations could be performed, such as nanoliter-scale in-droplet cell culture, cell coculture, drug stimulation, cell washing, and cell staining, as well as formation of picoliter single-cell droplets. The feasibility and flexibility of the MDOS was demonstrated in multi-mode miniaturized cell assays, including cell-based drug test, first-pass effect assay, and single-cell enzyme assay. The MDOS with the features of low cost, easy to build and flexible to use, could provide a promising alternative for performing miniaturized assays in routine laboratories, in addition to conventional microfluidic chip-based systems and automated robot systems.

A Microfluidic Droplet Array System for Cell-Based Drug Combination Screening.

In the last few decades, drug combination therapy has been widely applied in oncology and in other complex diseases. Due to its potential advantage of lower drug toxicity and higher therapeutic efficacy, drug combination treatment has been more and more studied in fundamental labs and pharmacy companies. In this chapter, we report cell-based drug combination screening using a microfluidic droplet system based on a sequential operation droplet array (SODA) technique. In this system, an oil-covered two-dimensional droplet array chip was used as the platform for cell culture and analysis. This chip was fixed in an x-y-z translation stage under control of a computer program. A tapered capillary connected with a syringe pump was coupled with the droplet array chip to achieve multiple droplet manipulations including liquid metering, aspirating, depositing, mixing, and transferring. Complex multistep operations for drug combination screening involving long-term cell culture, medium changing, schedule-dependent drug dosage and stimulation, and cell viability testing were achieved in parallel using the present system. The drug consumption for each screening test was substantially decreased to 5 ng-5 µg, corresponding to 10- to 1000-fold reductions compared with traditional drug screening systems with 96- or 384-well plates.

Cell-based drug combination screening with a microfluidic droplet array system.

We performed cell-based drug combination screening using an integrated droplet-based microfluidic system based on the sequential operation droplet array (SODA) technique. In the system, a tapered capillary connected with a syringe pump was used for multistep droplet manipulations. An oil-covered two-dimensional droplet array chip fixed in an x-y-z translation stage was used as the platform for cell culture and analysis. Complex multistep operations for drug combination screening involving long-term cell culture, medium changing, schedule-dependent drug dosage and stimulation, and cell viability testing were achieved in parallel in the semiopen droplet array, using multiple droplet manipulations including liquid metering, aspirating, depositing, mixing, and transferring. Long-term cell culture as long as 11 days was performed in oil-covered 500 nL droplets by changing the culture medium in each droplet every 24 h. The present system was applied in parallel schedule-dependent drug combination screening for A549 nonsmall lung cancer cells with the cell cycle-dependent drug flavopiridol and two anticancer drugs of paclitaxel and 5-fluorouracil. The highest inhibition efficiency was obtained with a schedule combination of 200 nM flavopiridol followed by 100 µM 5-fluorouracil. The drug consumption for each screening test was substantially decreased to 5 ng-5 µg, corresponding to 10-1000-fold reductions compared with traditional drug screening systems with 96-well or 384-well plates. The present work provides a novel and flexible droplet-based microfluidic approach for performing cell-based screening with complex and multistep operation procedures.

Microfluidic cytometer based on dual photodiode detection for cell size and deformability analysis.

Cellular mechanical properties play an important role in disease diagnosis. Distinguishing cells based on their mechanical properties provides a potential method for label-free diagnosis. In this work, a convenient and low-cost microfluidic cytometer was developed to study cell mechanical properties and cell size based on the change of transmission intensity, using a low-cost commercial laser as a light source and two photodiodes as detectors. The cells pass through a narrow microchannel with a width smaller than the cell dimension, integrated in a polydimethylsiloxane chip, below which the laser is focused. The transit time of individual cells is measured by the time difference detected by two photodiodes. This device was used to study the difference in cell mechanical properties between HL60 cells treated with and without Cytochalasin D. Furthermore, it was also applied to distinguish cells with different diameters, HL60 cells and red blood cells, by measuring the transmission intensity.

Microfluidic droplet-array liquid-liquid chromatography based on droplet trapping technique.

We describe the first realization of liquid chromatographic separation in a droplet-based microfluidic system and develop a novel mode for microchip-based chromatography named as droplet-array liquid-liquid chromatography. In this system, two arrays of picoliter-scale droplets immobilized on both sidewalls of a microchannel with droplet trapping technique served as the stationary phase in chromatographic separation, while the other immiscible phase flowing in the microchannel served as the mobile phase. The chromatographic separation was achieved on the basis of multiple extraction and elution of analytes between the droplet array stationary phase and the mobile phase. The proof-of-concept study of the droplet-array LC system was performed in the separation of fluoranthene and benzo[b]fluoranthene. Under the optimum conditions, the two analytes were separated within 26 min with separation efficiencies of 112 µm and 119 µm plate height, respectively. The advantages of the present system include simple structure, low driving pressure, and relatively high sample capacity. It can also provide a useful platform for LC theory study and educational purposes by allowing the researchers and students to directly "see" the continuous extraction and elution process of a chromatographic separation.

Droplet-based microfluidic flow injection system with large-scale concentration gradient by a single nanoliter-scale injection for enzyme inhibition assay.

We described a microfluidic chip-based system capable of generating droplet array with a large scale concentration gradient by coupling flow injection gradient technique with droplet-based microfluidics. Multiple modules including sample injection, sample dispersion, gradient generation, droplet formation, mixing of sample and reagents, and online reaction within the droplets were integrated into the microchip. In the system, nanoliter-scale sample solution was automatically injected into the chip under valveless flow injection analysis mode. The sample zone was first dispersed in the microchannel to form a concentration gradient along the axial direction of the microchannel and then segmented into a linear array of droplets by immiscible oil phase. With the segmentation and protection of the oil phase, the concentration gradient profile of the sample was preserved in the droplet array with high fidelity. With a single injection of 16 nL of sample solution, an array of droplets with concentration gradient spanning 3-4 orders of magnitude could be generated. The present system was applied in the enzyme inhibition assay of ß-galactosidase to preliminarily demonstrate its potential in high throughput drug screening. With a single injection of 16 nL of inhibitor solution, more than 240 in-droplet enzyme inhibition reactions with different inhibitor concentrations could be performed with an analysis time of 2.5 min. Compared with multiwell plate-based screening systems, the inhibitor consumption was reduced 1000-fold.