DROPLET GENERATOR AND GENERATION METHOD

Abstract

A method of generating droplets from a liquid sample is described and is characterised by the steps of a) locating the sample at a first electrowetting location and applying an electrowetting force to cause a region of the sample to become elongated in a direction in which the electrowetting force is applied; b) temporarily altering the electrowetting force on the sample and accumulating electrostatic surface charge on the sample to counter surface tension forces between the bulk of the sample and the elongated sample region; c) restoring the electrowetting force; d) further elongating the charged elongated sample region using the electrowetting force and e) severing a droplet from the charged elongated sample region. A corresponding droplet generator is also described.

Claims

1-19. (canceled)

20. A method of generating droplets from a liquid sample, comprising the steps of: a) locating the sample at a first electrowetting location and applying an electrowetting force to cause a region of the sample to become elongated in a direction in which the electrowetting force is applied; b) temporarily altering the electrowetting force on the sample and accumulating electrostatic surface charge on the sample to counter surface tension forces between the bulk of the sample and the elongated sample region by means of charge-charge repulsion; c) thereafter restoring the electrowetting force; d) further elongating the charged elongated sample region using the electrowetting force and e) severing a droplet from the further elongated sample region.

21. The method of claim 20, further comprising the step: f) transporting the severed droplet away from the first electrowetting location along a pathway comprised of the first electrowetting location and at least one second location where further electrowetting forces are applied.

22. The method of claim 20, wherein the electrowetting force is applied by means of a drive voltage and the electrostatic surface charge is applied by means of a charging voltage.

23. The method of claim 22, wherein the charging voltage is a constant DC voltage.

24. The method of claim 22, wherein the charging voltage is a monotonically increasing voltage.

25. The method of claim 20, wherein the electrowetting forces arise from an AC drive voltage being directed to the electrowetting location(s) by means of either a specific electrode located at each location, or by means of a virtual electrode at each location created by means of a semiconductor layer activated at corresponding locations by impingement of electromagnetic radiation.

26. The method of claim 20, wherein steps (a) to (d) are repeated multiple times until sufficient elongation of the elongated sample region has occurred for step (e) to take place.

27. The method of claim 20, wherein the droplets are less than 1 millimetre in diameter.

28. The method of claim 21, wherein steps (a) to (f) are repeated multiple times to generate a stream of droplets.

29. The method of claim 20, wherein the sample is portioned into multiple droplets of equal volume.

30. The method of claim 20, wherein multiple electrowetting forces are applied to a single liquid sample to cause it to be split into multiple droplets simultaneously.

31. The method of claim 20, wherein the droplet or at least some of the droplets contain one or more biological molecules, cells or functionalised microparticles.

32. The method of claim 20, wherein switching between application of the electrowetting force and the electrostatic charging is achieved using a microprocessor applying algorithms.

33. The method of claim 20, wherein the sample is progressively charged to a degree which is less than the Rayleigh limit for the sample medium.

34. The method of claim 20, wherein the sample contains cells and is initially charged beyond the Rayleigh limit to lyse or otherwise disrupt the membranes of the cells.

35. A droplet generator comprising: a first electrowetting location adapted to receive a liquid sample; at least one second electrowetting location arranged so that the first and second electrowetting locations define a pathway along which droplets severed from the sample can be transported using directional electrowetting forces; an AC drive circuit arranged at the first electrowetting location and comprised of either an electrode and an associated AC electrical circuit or a semiconductor zone activated by the impingement of electromagnetic light thereon; and a DC charging circuit arranged at the first electrowetting location and adapted to electrostatically charge the surface of the sample.

36. The droplet generator of claim 35, further comprising a control circuit for switching between the drive and the charging circuits.

37. The droplet generator of claim 35, further comprising an analyser for analysing the contents of each droplet produced.

38. The droplet generator of claim 37, wherein the analyser is arranged to detect fluorescence emitted by the droplets when interrogated with electromagnetic radiation.

Description

[0025] The invention is now illustrated with reference to the following.

[0026] An optically-activated electrowetting substrate of the type described in our earlier patent application (PCT/EP2018/066573) was employed. It comprised top and bottom glass plates each 500 μm thick and coated with transparent layers of conductive Indium Tin Oxide (ITO) having a thickness of 130 nm. Each layer was connected to an A/C drive circuit and a DC charging circuit. One of the plates was coated with an intermediate layer of amorphous silicon which was 800 nm thick. Both plates were then coated with 160 nm thick layers of high purity alumina which in turn were coated with an 80 nm thick layer of Teflon AF to render the final surfaces in contact with the sample hydrophobic. The two plates were then spaced 20 μm apart using spacers and a liquid sample inserted.

[0027] At a given location where the sample was to be located, the silicon-containing plate was illuminated by an LED from beneath with visible light (wavelength 660 or 830 nm) at a level of 0.01 Wcm.sup.2 in order to activate the desired electrowetting force via the drive circuit. The sample employed was comprised of an aqueous culture of immortalised cells in a growth medium.

[0028] Daughter droplets were then progressively drawn off of the sample by periodically interrupting application of the electrowetting force and replacing it with a ramped-up charging force supplied by the DC circuit in a series of alternating pulses at a pre-determined repetition rate. In the process, the sample was progressively elongated in the direction of the applied electrowetting force until such time as the daughter droplet was broken off. After break-off, the daughter droplet was moved away from the vicinity of the sample using pathways of other electrowetting structure arranged around the site holding the sample as explained in our earlier patent application.

[0029] This methodology was used to investigate the characteristics of the daughter droplet severance process in terms of minimum droplet size and break-off distance. The results are summarised in the following table which shows that using the method of the invention a substantial reduction in the minimum daughter droplet volume can be obtained whilst at the same time minimisation the elongation distance required to reach the point of break-off.

TABLE-US-00001 TABLE Minimum Shortest Pulsatile charging Number of volume break-off conditions dropletisation detached distance No charging 7 12,000 pL 1.1 mm Ramp 0 to 100 V linear 4 5,000 pL 800 um increase over 50 ms; 1 Hz repetition rate Ramp 0 to 30 V linear 12 1,200 pL 400 um increase in 30 ms, 1 Hz repetition rate

[0030] FIG. 1 is a pair of photomicrographs of a droplet being detached from the input liquid plug whilst under pulsatile charging.