METHOD FOR CLEANING A MICROFLUIDIC DEVICE USING AN IONIC LIQUID

Abstract

The invention pertains to a method for cleaning a microfluidic device. More specifically the invention pertains to a method for cleaning a microfluidic device, wherein the method comprises removing biological matter from the inside of the device by flushing the microfluidic device with an ionic liquid. The invention further pertains to the use of an ionic liquid to clean mammalian cells and to a kit of parts comprising a microfluidic device and an ionic liquid.

Claims

1. A method for cleaning a microfluidic device comprising removing biological matter from inside of the microfluidic device by flushing the microfluidic device with a cleaning agent, wherein the cleaning agent comprises an ionic liquid.

2. The method according to claim 1, wherein the biological material comprises a component selected from the group consisting of mammalian cells, mammalian cell organelles, mammalian cell residues, extracellular matrix from mammalian cells and combinations thereof.

3. The method according to claim 1, wherein the method for cleaning is an in-situ cleaning method.

4. The method according to claim 1, wherein the ionic liquid comprises at least one cation selected from the group consisting of 1-butyl-1-methyl-pyrrolidinium, 1-ethyl-3-methylimidazolium, and methyltrioctylammonium.

5. The method according to claim 1, wherein the ionic liquid comprises at least one anion selected from the group consisting of chloride, bromide, iodide, acetate, nitrate, sulfate, sulfonate, tosylate, dicyanamide, thiocyanate, borate, and salicylate.

6. The method according to claim 1, wherein a contact time between the microfluidic device and the cleaning agent is at least 5 minutes.

7. The method according to claim 1, wherein the ionic liquid is at a temperature between 15 and 100 C.

8. The method according to claim 1, further comprising a step of reusing the microfluidic device after flushing the microfluidic device with the cleaning agent.

9. The method according to claim 1, wherein the microfluidic device can be cleaned at least 10 times by the method of cleaning without altering the acoustic, hydrodynamic, optical, and/or electrical properties of the device.

10. A method of removing mammalian cells, mammalian cell residues and/or extracellular matrix from a surface, by contacting the surface with a cleaning agent comprising an ionic liquid.

11. The method according to claim 10, wherein the cleaning agent consists of the ionic liquid.

12. The method according to claim 10, wherein the ionic liquid comprises at least one cation selected from 1-butyl-1-methyl-pyrrolidinium, 1-ethyl-3-methylimidazolium, and methyltrioctylammonium.

13. The method according to claim 10, wherein the ionic liquid comprises at least one anion selected from the group consisting of chloride, bromide, iodide, acetate, nitrate, sulfate, sulfonate, tosylate, dicyanamide, thiocyanate, borate, and salicylate.

14. A kit of parts comprising a microfluidic device and a container holding an ionic liquid.

15. The kit of parts according to claim 14, wherein the kit of parts further comprises a container holding an aqueous soap solution.

16. The method according to claim 1, wherein a contact time between the microfluidic device and the cleaning agent is at least 20 minutes.

17. The method according to claim 1, wherein the ionic liquid is at a temperature between 3 and 50 C.

18. The method according to claim 1, wherein the microfluidic device can be cleaned at least 150 times by the method of cleaning without altering the acoustic, hydrodynamic, optical, and/or electrical properties of the device.

19. The method according to claim 1, wherein the biological material comprises a component selected from the group consisting of mammalian cells, mammalian cell organelles, mammalian cell residues, extracellular matrix from mammalian cells and combinations thereof; the ionic liquid comprises at least one cation selected from the group consisting of 1-butyl-1-methyl-pyrrolidinium, 1-ethyl-3-methylimidazolium, and methyltrioctylammonium and the ionic liquid comprises at least one anion selected from the group consisting of chloride, bromide, iodide, acetate, nitrate, sulfate, sulfonate, tosylate, dicyanamide, thiocyanate, borate, and salicylate.

20. The method according to claim 19, wherein the method for cleaning is an in-situ cleaning method.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0082] FIGS. 1A and B representative images of NALM-6 monolayer before and after cleaning with EMIM.sup.+DCA.sup..

[0083] FIGS. 2A and 2B representative images of Skove monolayer before and after cleaning with EMIM.sup.+Ac.sup..

[0084] FIGS. 3A and 3B representative images of Skove monolayer before and after cleaning with EMIM.sup.+Otf.sup..

[0085] FIGS. 4A and 4B representative images of NALM-6 monolayer before and after cleaning with 5% hypochlorite bleach.

[0086] FIG. 5 Graphical representation of results of acoustic resonance measurements.

EXAMPLES

Experiment 1: Cleaning Target Cells in a Microfluidic Device Using Ionic Liquid

[0087] To test the cleaning capability of ionic liquids, an AFS microfluidic device with mimicked use was prepared. The device with mimicked use was prepared by filling a clean device with a cell culture medium comprising target cells. This resulted in the deposition of a cellular monolayer of the target cells.

[0088] The microfluidic device with mimicked use was subsequently cleaned. According to the protocol, the culture medium was first removed from the device using negative pressure supplied by a syringe. As a precaution, the monolayer was visually checked using an optical microscope. If a homogeneous layer was observed, the microfluidic device with mimicked use was considered to be a good representation for a used device. The device was subsequently filled with an ionic liquid, heated to the specified temperature, and left for the specified time. Next the device was flushed with an aqueous surfactant solution and subsequently with deionized water. After flushing, the device was visually inspected using an optical microscope to determine the cleanliness of the inner surface.

[0089] In Table 1, the experimental setup (i.e. the substrate+coating, the target cells, the ionic liquid, the temperature of the experiment, and the holding time) of the experimental program is summarized. In the last column of this table, the result of the cleaning is presented. Representative images of surfaces with mimicked use before and after cleaning are given in FIGS. 1-3.

TABLE-US-00001 TABLE 1 Summary of experimental program and result Surface Ionic Liquid Substrate Coating Target Cells Cation Anion T ( C.) t (min) Result Glass PLL NALM-6 EMIM.sup.+ DCA.sup. 50 30 Clean Glass PLL NALM-6 EMIM.sup.+ DCA.sup. 50 300 Clean Glass PLL NALM-6 EMIM.sup.+ DCA.sup. 15 1 Clean Glass PLL NALM-6 EMIM.sup.+ DCA.sup. 50 30 Clean Glass PLL NALM-6 EMIM.sup.+ DCA.sup. 50 210 Clean Glass PLL NALM-6 EMIM.sup.+ DCA.sup. 50 60 Clean Glass PLL Raji EMIM.sup.+ DCA.sup. 50 30 Clean Glass PLL NALM-6 EMIM.sup.+ DCA.sup. 37 30 Clean Glass PLL HeLa EMIM.sup.+ DCA.sup. 50 30 Clean Glass PLL Skove EMIM.sup.+ DCA.sup. 50 30 Clean Glass PLL NALM-6 EMIM.sup.+ DCA.sup. 50 30 Clean Glass PLL Skove EMIM.sup.+ DCA.sup. 50 30 Clean Glass PLL Skove EMIM.sup.+ DCA.sup. 50 60 Clean Glass PLL Skove EMIM.sup.+ Ac.sup. 50 30 Clean Glass PLL Skove EMIM.sup.+ Ac.sup. 50 60 Clean Glass PLL Skove EMIM.sup.+ Otf.sup. 50 30 Clean Glass PLL Skove EMIM.sup.+ Otf.sup. 50 60 Clean

Experiment 2: Cleaning Cells in a Microfluidic Device Using Water or Bleach (Representative)

[0090] The above experiment was repeated using water as cleaning medium and using bleach as cleaning medium. It was found that water does lyse at least part of the cells over time (>20 min) but does not solubilize all the released contents. Cleaning with only water hence leaves debris on the glass.

[0091] An experiment with bleach was performed using a 5% sodium hypochlorite solution in water at 37 C. for 30 min and a NALM-6 monolayer. It was found that bleach does lyse at least part of the cells over time (>10 min) but does not solubilize all the released contents. Cleaning with only water hence leaves debris on the glass. The visual results of this experiment are shown in FIG. 4.

Experiment 3: Effect of Cleaning on Acoustic Resonance

[0092] The effect of cleaning on the acoustic properties of a microfluidics device was compared when cleaning was performed using a method with harsh chemicals or using ionic liquid (EMIM.sup.+ DCA.sup.).

[0093] To determine the effect on acoustic properties when cleaning with ionic liquid, a series of cleaning runs with ionic liquid was performed. The resonance frequency of the device was determined at the beginning (i.e. at n=0 cleaning runs), after 16 cleaning runs and after 110 cleaning runs. To perform a cleaning run, the device was filled with EMIM.sup.+DCA.sup., heated to 40 C., and left for 60 minutes. Next, the device was flushed with an aqueous surfactant solution and subsequently with deionized water.

[0094] To determine the effect on acoustic properties when cleaning with harsh chemicals, a series of cleaning runs with harsh chemicals was performed. The resonance frequency of the device was determined at the beginning (i.e. at n=0 cleaning runs) and throughout the experiment. To perform a cleaning run, the device was filled with bleach and left for 20 minutes at room temperature. Subsequently the device was flushed with water, further rinsed with 12M HCl (required to remove cellular debris), and again flushed with water. Then the device was filled with NaOH 1M and left for 1 h at room temperature. After subsequently flushing with deionized water, the cleaning run was completed.

[0095] The resonance frequency of the device was determined by applying a frequency sweep around the expected response frequency and determining the system response. This was performed using the known method such as published in PCT/NL2021/050575 and by Kamsma in section 4.3.1 of his PhD thesis (Kamsma, D 2018, Acoustic Force Spectroscopy (AFS): From single molecules to single cells, PhD, Vrije Universiteit Amsterdam). Section 4.3.1 is hereby incorporated by reference. According to good practice, the same method and settings for measuring the resonance frequency was used throughout the experiment allowing for a good comparison.

[0096] The results of the experiments are summarized in FIG. 5, where the change in resonance frequency (kHz) is shown as a function of the number of cleaning steps (n). As can be seen, the resonance frequency hardly changes when ionic liquid is used for cleaning. However, harsh chemicals do significantly change the resonance frequency.