Microfluidic Droplet Sequence Production for High-Throughput Reaction Screening

By AZoNano.com Staff Writers

Topics Covered

Introduction
Experimental
Modes of Operation
Mode 1 Setup
Results
Conclusion
About Dolomite Microfluidics

Introduction

There is an increasing demand for screening large numbers of biological reactions, increased screening rate, and reduced reagent consumption. The bulk of the cost is due to screening libraries and reagents. The throughput and speed requirements can be addressed by conventional liquid handling systems - however, these have limitations on working volume, especially below 1µl.

Improvements to throughput and speed of reaction screening are essential for applications requiring screening of high value or scarce reagents. Present day microplate technologies operate on 1-100µl volumes, but it will be necessary to scale assays down to volumes under 1µl, to reduce the costs of reagents.

Droplet-based assays are well suited for high throughput screening owing to their ability to operate in miniscule volumes. Mitos Dropix technology introduces liquid sampling and processing over a very wide 10nl – 50µl volume range using droplet technology.

This article shows the droplet sequence production for library generation using simple fluids to help readers understand the concept, and to be able to assess its applicability to specific lab protocols.

Mitos Dropix was developed by Dolomite under exclusive sub-licence with Drop-Tech Ltd. having won Dolomite’s 2012 Productizing Science® competition.

Experimental Procedure

A droplet sequence of six different aqueous droplets spaced by FC-40 carrier fluid was produced in 50 iterations in a test case.

In a two hour period, a library of approximately 2000 aqueous droplets was created with 6 variable compositions. Bright field imaging was used for visualizing and characterizing droplet sizes.

Droplets with volumes as low as 30nl and as high as 1000nl were produced. The maximum volume of sample loaded in the sample strip is 50µl. An accuracy of ±10% was achieved over the entire dynamic range.

The main aspects incorporated in the Mitos Dropix technology are exploiting fluid surface tension and buoyancy. A user calibration feature ensures accuracy with fluids over a wider range of properties.

Modes of Operation

Four basic modes of operation are suggested.

  • Mode 1: Droplet sequence production or droplet library generation. Applications that need a large reference droplet library are able to utilize this mode. A single Mitos Dropix can store 24 different fluids, and therefore the library consists of 24 different compositions.

  • Mode 2: Mixing or concentration gradients from two or more wells using a merging chip. Using a droplet merging chip, ratiometric mixing from up to 24 wells, results in effectively infinite droplet combinatorial reactions.

  • Mode 3: Either in-line with mode 2, or independently, Mode 3 extends Mitos Dropix from nanoliter technology to picoliter technology. This is most effectively achieved with the use of a droplet production microfluidic device.

  • Mode 4: Synchronized multiple Mitos Dropix. Applications requiring more than 24 starting solutions will benefit from using multiple Mitos Dropix.

Mode 1 Setup

In this article, droplet-on-demand sequencing and library generation is shown with the Mitos Dropix setup in Mode 1.

Six aqueous samples were loaded into the Mitos Dropix and a sequence of nanoliter volume droplets was generated with preset volume and composition.

To differentiate between the different water samples, food dye was added to each sample before it was loaded into the Dropix Sample Strip. The volume of each water sample was 50µl.

The Dropix Fluid Reservoir - PMMA was filled with FC40, which is a inert and biocompatible fluorocarbon oil. The Mitos Dropix was connected to a Mitos Duo-XS Pump which aspirated the droplet and carrier stream into the Droplet Storage Coil.

A series of droplets were generated separated by the carrier oil. The XS pump because of its dual syringe function, has the added benefit of constantly refilling the carrier fluid bath during operation, thereby maintaining a steady fluid level. Figure 1 shows the four modes of operation.

Figure 1. Modes of Operation

The Dropix Sample Hook assembly has two vertical positions:

  • Down position – in this position the carrier fluid, oil, is aspirated.
  • Up position – in this position the aqueous sample is aspirated, creating the droplet.

Figure 2. Schematic showing Dropix Sample Hook assembly motion relative to the Sample Strip. Left: In ‘down’ position aspirating carrier fluid. Right: In ‘up’ position asiprating droplet fluid.

The Dropix sample strip can be disposed and designed around the format of a 192 well plate. The Dropix Sample Strip Holder enables interfacing with other fluid handling automation.

Figure 3. Left: Dropix Sample Strip Holder with multiple Dropix Sample Strips in a 192 well format. These are filled using a micropipette. Right: Sample Strip transferred from the Dropix Sample Strip Holder onto the Dropix.

A single Mitos Dropix can carry up to 24 different reagents. As the Dropix Sample Hook assembly rises in a well, the tube orifice transfers from the carrier fluid into the droplet fluid while the syringe continually aspirates.

Based on an assumption of an inviscid fluid, the target droplet volume translates into timing for the rise and fall of the Dropix Sample Hook assembly.

Results

As droplets flow through the tubing and into the flexible FEP tubing, they can then be imaged. Images of the droplets were recorded using a DSLR camera with macro-lens and brightfield imaging. The droplets appear as elongated slugs in the semi-transparent tubing. The slug length was measured to estimate droplet volumes. Figure 3 shows seven droplets – green (200nl), blue (150nl), red (100nl), yellow (100nl), clear (100nl) and brown (200nl).

The droplets were collected in the Droplet Storage Coil. This coil holds an FEP tube with an O.D. of 0.80mm, I.D. of 0.25mm and a length of 1.0m. As the FEP tubing is semi-transparent, the Droplet Storage Coil can be used as an observation cell for optical diagnostics such as brightfield or fluorescence microscopy. The Droplet Storage Coil can also be placed in an incubator. Safe temperature limits are available in the user manual.

To demonstrate consistency over iterations, the data is presented in terms of the variation relative to the target droplet volume. This is indicative of the dispersity of the droplet size. The subset of droplet sizes in the above test are 100, 150 and 200nl, performance results of which are presented in the Table 1.

Table 1. Variations in droplet volume with Mitos Dropix system

Target Droplet Volume (with offset included) Number of droplets in test Measured droplet volume (average of 15 droplets) Variation of droplet volume around target for 15 droplets Variation of droplet volume around target for 15 droplets
100 nL (150 nL) 15 103nL Max = 110
Min = 97
100nL -3/+10
-3%/+10%
150 nL (250 nL) 15 138nL Max = 145
Min = 134
150nL -16/-5
-10%/-3%
200 nL (300 nL) 15 178nL Max = 188
Min = 170
200nL -30/22
-15%/-7%

Conclusion

Assembly, testing and droplet sequencing of the Mitos Dropix system was performed, and droplet sequencing was demonstrated successfully. Six 50µl aqueous samples were loaded into the Dropix Sample Strip and converted into a sequence of nanoliter droplets in a fluorocarbon oil carrier stream.

The range of droplet volumes generated was 50nl – 1000nl. The variation in droplet volume for a set of 10 samples was typically within ±10% around the target value. This result includes a fixed volume offset of 50nL which is independent of flow rate and droplet volume. It does however depend on the viscosity and surface tension of fluids.

The Dropix Sample Strip can hold 24 aqueous samples, each with a maximum volume of 50 µl. Each 50µl aqueous sample can be split into between 50 - 1000 droplets based on a droplet volume range of 50nl – 1000nl. It is possible to refill the sample strip during operation without significant disturbance to droplet production.

About Dolomite Microfluidics

Dolomite is a world leader in Productizing Science™ and an innovator in creating microfluidic devices and solutions. We sell the coolest microfluidic products around the world, often working with partner companies to extend the range of technology available to our customers. Productizing Science™ means creating marketable and commercially successful products from scientific discovery, and Dolomite excels in commercialising microfluidic products which exceed expectations.

We offer modular, standard microfluidic systems benefiting a wide range of applications, always adhering to the principles of having multiple functionalities, scalability, user-friendly design and a cost-effective, flexible solution for our customers.

Moreover, we offer Productizing Science™ as a service, which is a product development & manufacturing partnership creating microfluidic solutions for problems which span an extremely wide range of applications. Customers come to Dolomite with their technical challenges, and Dolomite helps solve these problems using its extensive background technology.

Dolomite also designs & manufactures a wide range of world leading standard components such as OEM products, microfluidic connectors & interfaces, chips, pumps, valves, detectors, sensors & accessories. Finally, we offer design consultancy to create customized chips or connectors and/or a prototyping service for the supply of glass, metal or polymer devices, and custom microfluidic connectors.

This information has been sourced, reviewed and adapted from materials provided by Dolomite Microfluidics.

For more information on this source, please visit Dolomite Microfluidics.

Date Added: Apr 18, 2014 | Updated: Apr 24, 2014
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