Method Of Measuring Viscosity In A Microfluidic System

Abstract

The invention relates to a microfluidic method for measuring viscosity in a micro droplet in a microfluidic system, comprising the steps of i) introducing a fluorescent molecule into a micro droplet otherwise comprising a fluid, ii) in the microfluidic system, exciting the fluorescent molecule in said micro droplet by applying light to the micro droplet, iii) measuring the resulting fluorescence emitted from the micro droplet thereby determining the viscosity of the fluid in the micro droplet. The invention also relates to method of screening for microorganisms or cells that produce viscosity-modulating compounds with desired properties. Finally, the invention also relates to the use of fluorescent molecules for measuring the viscosity of a fluid in a micro droplet in a microfluidic system.

Claims

1. Microfluidic method for measuring the viscosity in a micro droplet in a microfluidic system, comprising the steps of i) providing a micro droplet, wherein the micro droplet comprises a fluid and a fluorescent molecule, ii) in the microfluidic system, exciting the fluorescent molecule in said micro droplet by applying light to the micro droplet, and iii) measuring the resulting fluorescence emitted from the micro droplet thereby determining the viscosity of the fluid in the micro droplet.

2. The method according to claim 1, Wherein the fluorescence emitted by the fluorescent molecule depends on the viscosity of the fluid in the micro droplet.

3. Method according to claim 2, wherein i) the fluorescent molecule undergoes rotational diffusion that is inversely proportional to the viscosity of the fluid in the micro droplet, and/or ii) the fluorescent molecule is a molecular rotor that forms twisted intra molecular charge transfer upon photo excitation and therefore exhibits two competing de-excitation pathways, the relative intensities of which differ depending on the viscosity.

4. The method according to claim 3, wherein measuring the fluorescence emitted from the micro droplet is performed i) by determining the fluorescence anisotropy signal when the fluorescent molecule undergoes rotational diffusion, and/or ii) by measuring the emission intensity when the fluorescent molecule is a molecular rotor.

5. Method according to claim 1, wherein the fluorescent molecule is selected from the group of benzonitrile-based fluorophores benzylidene malononitriles, stilbenes, arimethene dyes, Viscous Blue 1, Viscous Blue 2, Viscous Blue 420, Viscous Green 1, Viscous Green 2, Viscous UV, Viscous Aqua, Viscous Red, Viscous VpH.

6. The method according to claim 1, wherein the fluorescent molecule is introduced into the micro droplet i) during the formation micro droplet, or ii) after the formation of the micro droplet by a method such as nanoinjection of picoinjection.

7. The method according to claim 1, wherein the micro droplet has a volume of between 10 pL and 5000 nL.

8. The method according to claim 1, wherein the micro droplet comprises at least one microorganism.

9. Method according to claim 8, wherein the microorganism influences the viscosity of the fluid in the micro droplet.

10. Method according to claim 9, wherein the microorganism influences the viscosity by secreting a substance into the fluid.

11. A method of screening for microorganisms or cells than produce viscosity-modulating compounds comprises the following steps: a) providing a composition comprising at least one microorganism or cell, b) optionally subjecting said microorganism Or cell to a reaction that leads to a change in the genetic material of at least one microorganism or cell, c) encapsulating the microorganism or cell obtained in step a) or b) into a micro droplet, wherein each micro droplet statistically comprises only one microorganism or cell, d) measuring the viscosity in each of the micro droplets by the method of claim 1 and e) optionally isolating the micro droplets with the desired viscosity, thereby isolating microorganisms or cells that produce viscosity-modulating compounds with the desired properties.

12. The method of claim 10, wherein step b) is performed by a reaction selected from the group of genetic modification, natural transformation, transduction by phage, conjugation and random mutagenesis, or any other means of modifying the genetic code.

13. The method according to claim 11, wherein the micro droplets with the desired viscosity are isolated by fluorescence activated sorting.

14. The method according to claim 11, wherein the microorganisms are bacteria.

15. (canceled)

16. The method according to claim 5, wherein the benzonitrile-based fluorophore is dimethylamino benonitrile (DMABN).

17. The method according to claim 5, wherein the benzylidene malononitrile is 9-(2,2-dicyanovinyl)julidine (DCVJ).

18. The method according to claim 5, wherein the stilbene is (p-DASPMI).

19. The method according to claim 5, wherein the arimethene dye is crystal violet.

Description

FIGURE CAPTIONS

[0065] FIG. 1

[0066] Fluorescent signal from DCVJ (y-axis) from droplets with buffer (low signal on x-axis) and from droplets containing 5 mg/mL of hyaluronic acid.

[0067] FIG. 2

[0068] Droplet-making chip design showing inlet for fluorinated oil, for aqueous solution containing cells (and possibly a fluorescent molecule), and the outlet for droplets.

[0069] FIG. 3

[0070] Drawing of a typical design for a droplet nanoinjection device (2).

[0071] FIG. 4

[0072] Picture of the process of nanoinjection.

EXAMPLES

Example 1: Measuring the Viscosity Inside a Droplet by Measuring the Fluorescence

[0073] We have undertaken proof of concept experiments for the molecular rotor DCVJ (9-(2,2-Dicyanovinyl)julolidine). We have incorporated this fluorophore in droplets containing buffer and in droplets containing 5 mg/mL hyaluronic acid (solution of 5 mg/mL of hyaluronic acid is highly viscous). To be able to distinguish different droplets, the droplets with buffer were additionally marked with low concentration of sulforhodamine, while the droplets with 5 mg/mL of hyaluronic acid were marked with high concentration of sulforhodamine.

[0074] The results are shown in FIG. 1.

[0075] The results show that the DCVJ fluorescence is clearly different in droplets containing 5 mg/mL of hyaluronic acid compared to the droplets containing buffer. This shows that the viscosity of the fluid inside the droplet can be calculated by measuring the fluorescence emission of an appropriate fluorophore.

Example 2: Preparation of a Droplet Generating Device (1)

[0076] Examples 2 to 5 provide one experimental setup that can be useful to perform microfluidic viscosity and screening experiments according to the invention.

[0077] Soft-lithography in poly(dimenthylsiloxane) (PDMS) was used to prepare the droplet generating device (1). A SU-8 photoresist mould was used to prepare the PDMS. To prepare the SU-8 mold, a layer of SU-8 was spin coated on a silicon wafer. The wafer was covered by a designed mask and exposed to UV for a certain period of time. After full development and baking the wafer, the SU-8 mould was ready for PDMS. The SU-8 thickness for droplet making chip in this example was 200 m. The droplet volume generated by the chip depends on the SU-8 thickness. To generate nanoliter droplets, the thickness can vary from 80 m to 500 m.

[0078] The thickness of the SU-8 mould for different types of PDMS chip varies. The SU-8 thickness for droplet nanoinjection chip, droplet sorting chip can for example respectively be 180 m and 350 m.

[0079] The droplet volume generated by the chip depends on the SU-8 thickness. To generate nanoliter droplets, the thickness can vary from 80 m to 500 m.

[0080] After preparation of the SU-8 mould, PDMS was cast on the mould and bound to a glass side. The inside part of microfluidic channel was treated by a commercial surface coating agent (Trichloro-(1H,1H,2H,2H-perfluorooctyl)-silane, Sigma-Aldrich) to make the channel surface hydrophobic.

Example 3: Generation of Fungi Spore-Containing Droplets

[0081] To generate droplets on a chip, the PDMS chip was connected via tubing to an oil phase reservoir, an aqueous phase reservoir and an outlet tubing. A possible chip design is shown in FIG. 2. In this case, the oil phase consists of perfluorocarbon oil (HFE7500, 3M) with 5% (w/w) of a surfactant, made by coupling oligomeric perfluorinated polyethers (PFPE) with polyethyleneglycol (PEG) (Biocompatible surfactants for water-in-fluorocarbon emulsions, Lab Chip, 2008, 8, 1632-1639). However, any phase that is immiscible with the droplets, which in this case are made of an aqueous phase, could have been used (any oil or gas phase). The aqueous phase consists of fungi spore suspension as an example, but is not limited to fungi spores. In other examples, mammalian cells, bacterial cells, yeast cells etc. could be used. The flow rate was controlled by syringe pumps (PHD2000, Havard Apparatus). The flow rate of oil phase was 4 mL/h, and the flow rate of aqueous phase was 3 mL/h. The droplet volume generated here was 20 nL (diameter=0.336 mm). The droplets encapsulate the fungi spores during the droplet generation process. The droplets were collected in a vial and incubated at 30 C. over 48 hours for germination and growth of the fungi.

Example 4: Nanoinjection of a Fluorescent Molecule into the Droplets

[0082] After incubation, the droplets were reinjected into a chip that is capable of nanoinjection. Nanoinjection is employed in order to add the fluorophore directly prior to the start of the assay into droplets. A typical design of nanoinjection device (2) is shown in FIG. 3 and a typical nanoinjection process is pictured in FIG. 4. The flow rate of spacing oil was 0.6 mL/h, the flow rate of droplet reinjection was 0.5 mL/h, and the flow rate of aqueous phase for nanoinjection was 0.1 mL/h. A high voltage with 20,000 V and 20,000 Hz was added to help nanoinjection. Other technologies, such as acoustic wave technology can also be used to add reagents to droplets. The spacing oil phase consists of perfluorocarbon oil. The droplets contain the grown fungi after 48 hours of incubation.

[0083] A poly(tetrafluoroethylene) (PTFE) tubing (inner diameter=0.3 mm, outer diameter=0.56 mm) was connected to the outlet (3) of the nanoinjection device (2). The tubing is a delay line (4) in which the droplets that comprise the fungi and the fluorescent molecule are all incubated by moving through a delay line.

Example 5: Temperature Control of Droplet Incubation

[0084] After nanoinjection, the droplets flowed in the PTFE tubing (the delay line (4)). The droplets were continuously moving in the tubing. The length of the tubing in this example was 6 meters, but can also be significantly shorter or longer (e.g. up to 100 m) depending on the incubation time needed. The tubing was incubated at 30 C. in the present example. The temperature setting however can be adapted to the needs of each specific assay. Temperature control was obtained by submerging the tubing containing the droplets for assay incubation into a bed of heated metal beads or in a water bath. Other arrangements like a tubing coil surrounding a peltier element could be another option.