METHOD FOR INVESTIGATING MOLECULES SUCH AS NUCLEIC ACIDS

Abstract

A method for manipulating a microdroplet of a reaction medium in an immiscible carrier medium with a target molecule bound to a solid support for the purposes of effecting a chemical transformation is provided. It is characterised by the steps of (a) bringing the microdroplet into contact with the solid support under conditions where the microdroplet and solid support are caused to combine, (b) allowing the reaction medium to react with the target molecule and (c) thereafter exerting a force to induce the reaction medium to become detached from the solid support and reform a microdroplet in the carrier fluid. In one embodiment the solid support is a particle, bead or the like.

Claims

1. A method for manipulating a microdroplet of a reaction medium in an immiscible carrier medium with a target molecule bound to a solid support for the purpose of effecting a chemical transformation comprising the steps of (a) bringing the microdroplet into contact with the solid support under conditions where the microdroplet and solid support are caused to combine; (b) allowing the reaction medium to react with the target molecule and (c) thereafter exerting a force to induce the reaction medium to become detached from the solid support and reform a microdroplet in the carrier medium.

2. The method as claimed in claim 1, wherein the reaction medium is a medium for transforming a nucleic acid analyte and the target molecule comprises a polynucleotide.

3. The method as claimed in claim 2, wherein a nucleotide is released from the analyte and is carried away from the vicinity of the solid support in a microdroplet.

4. The method as claimed in claim 3, wherein multiple microdroplets are brought sequentially into contact with the analyte each capable of carrying way a nucleotide.

5. The method as claimed in claim 1 wherein the chemical transformation is used to determine the sequence of a nucleic acid analyte and the method comprises the steps of: causing a stream of microdroplets of the reaction medium comprising a digesting medium in the immiscible carrier medium to contact the target molecule comprising a nucleic acid analyte bound to the solid support comprising a particle one by one in a pathway thereby causing the analyte to be progressively digested into its constituent nucleotides; removing the microdroplets from around the analyte wherein at least some of the microdroplets contain a single nucleotide; further transporting the microdroplets along the pathway to a detection zone; and identifying the nucleotides within the microdroplets in the detection zone and inferring the sequence of the analyte molecule therefrom.

6. The method as claimed in claim 5, wherein the digesting medium comprises a polymerase and a cofactor which causes the polymerase to release nucleotides one by one from the analyte molecule.

7. The method as claimed in claim 6, wherein the cofactor is a polyphosphate anion or analogue thereof.

8. The method as claimed in claim 7, wherein the polyphosphate is pyrophosphate.

9. The method as claimed in claim 5, wherein the digesting medium comprises an exonuclease and the nucleotide monophosphates thus released are further treated with a kinase to convert them to nucleotide triphosphates.

10. The method as claimed in claim 5, wherein the resulting nucleotides are caused to react in the presence of a polymerase with an oligonucleotide capture system comprised of fluorescence probes which in their unused state are non-fluorescing and which are selective for at least one of the nucleotide types characteristic of the analyte, resulting in the release of fluorophores into a detectable state, and wherein the resulting fluorescence derived from the released fluorophore(s) in each microdroplet is detected using an incident source of first electromagnetic radiation and a photodetector.

11. The method as claimed in claim 10, wherein the release of fluorophores is the result of the action of an endonuclease and/or exonuclease and optionally a ligase, such action being catalysed by the capture of the nucleotide being detected.

12. The method as claimed in claim 10, wherein the oligonucleotide capture system is introduced into the microdroplets downstream of the particle through droplet coalescence or through direct injection.

13. The method as claimed in claim 10, wherein the digesting medium is further treated with a pyrophosphatase prior to reaction with the oligonucleotide capture system.

14. The method as claimed in claim 5, wherein the microdroplets are caused to move over a surface chemically modified to have areas of locally increased level of wettability and wherein such an increase in wettability is used to mediate the wetting of the microdroplet fluid around the target molecule.

15. The method as claimed in claim 5, wherein electrowetting or optically-mediated electrowetting effects are used to locally modify the wettability of a region of the surface around the particle.

16. The method as claimed in claim 5, wherein the microdroplets are transported along the pathway to a detection zone via electrowetting or optically-mediated electrowetting.

17. The method as claimed in claim 5, wherein at least part of the pathway is an optically-mediated electrowetting microfluidic space comprising: a first composite wall comprised of a first substrate; a first transparent conductor layer on the first substrate having a thickness in the range 70 to 250 nm; a photoactive layer activated by electromagnetic radiation in the wavelength range 400-1000 nm on the first transparent conductor layer having a thickness in the range 300-1000 nm; and a first dielectric layer on the photoactive layer having a thickness in the range 120 to 160 nm; and a second composite wall comprised of a second substrate; a second transparent conductor layer on the second substrate having a thickness in the range 70 to 250 nm and optionally a second dielectric layer on the second transparent conductor layer having a thickness in the range 25 to 50 nm; wherein exposed surfaces of the first and second dielectric layers are disposed less than 50 μm apart.

18. The method as claimed in claim 17, wherein the microfluidic space is activated to transport the microdroplets by means of (a) an AC source providing a potential difference across the first and second composite walls connecting the first and second conductor layers and (b) at least one source of second electromagnetic radiation having an energy higher than the bandgap of a photoexcitable layer adapted to impinge on the photoactive layer to induce corresponding ephemeral first electrowetting locations on the surface of the first dielectric layer.

19. The method as claimed in claim 18, wherein the first and second sources of electromagnetic radiation are the same.

20. The method as claimed in claim 5, further comprising an initial step of transporting the particle to a location where digestion takes place either in a microdroplet using electrowetting or optically-mediated electrowetting or magnetically.

21. The method as claimed in claim 5, wherein the particle is held magnetically at the location where digestion takes place while the stream of microdroplets is contacted with it.

22. The method as claimed in claim 10, wherein fluorescence is detected by the steps of introducing the microdroplets into an inlet of at least one capillary tube and causing the first electromagnetic radiation to impinge on the microdroplets as they emerge from outlet(s).

23. A method for pyrophosphorolysing a nucleic acid analyte comprising the step of contacting in turn each microdroplet in a stream of aqueous microdroplets suspended in a carrier medium and containing a pyrophosphorolysing enzyme with a particle to which the analyte is attached whereby the enzyme is caused to release a nucleoside triphosphate molecule from the analyte into at least some of the microdroplets before they are removed from around the particle.

Description

[0068] A method of the present invention suitable for sequencing a DNA analyte according to the preferred third aspect of the invention is now illustrated by the following Figures and associated description.

[0069] FIG. 1 shows a plan of a microfluidic chip comprised of a microdroplet preparation zone 1 and a microdroplet manipulation zone 3 integrated into a single chip made of transparent plastic. 1 comprises regions containing fluid 7, 8 and 9 attached to inlets 4, 5 and 6 which respectively introduce into the chip a pyrophosphorolysing stream, an inorganic pyrophosphatase stream and a stream of the various detection chemicals and enzymes required to identify nucleoside triphosphates in accordance with one of our earlier patent applications. These streams are then delivered respectively to orifices 7a, 8a and 9a from which various microdroplets 7b, 8b and 9b are produced (for example using a droplet-dispensing head or by cutting from a larger intermediate droplet). 10 is a reservoir containing paramagnetic-polymer composite microbeads 11 to some or all of which are attached a single molecule of the polynucleotide analyte to be sequenced. Each of 11 is transported in microdroplets along electrowetting pathway 12 to a location 7c where it is held by magnetism and contacted with a stream of 7b. At this location, the analyte attached to 11 is progressively pyrophosphorolysed at a rate such that after each 7b is disengaged from 11 it is either empty or contains only one single nucleoside triphosphate molecule. After disengagement, 7b along with microdroplets 8b and 9b, are caused to move to 3 where they are manipulated along various optically-mediated electrowetting paths (defined here by square electrowetting locations 25) at a temperature of 30-40° C. in accordance with the scheme illustrated in FIG. 2. In the process, 7b and 8b are first caused to coalesce at first coalescing points 12 to generate intermediate microdroplets 13 which are thereafter caused to coalesce with 9b at second coalescing points 14 to generate a plurality of streams of final microdroplets 15 which move forward through an incubation region 16 maintained at a temperature in the range 60-75° C. in order to allow the contents to incubate and the necessary chemical and enzymatic reactions to take place. Thereafter, 15 are transported to final locations in detection zone 2 where they are interrogated with light from a LED source and any fluorescence emitted by each microdroplet detected using a photodetector. The output of the photodetector is a data stream corresponding to the sequence of the analyte which can be analysed using known sequencing algorithms.

[0070] FIG. 2 shows a partial, sectional view of 3 illustrating how the various microdroplets are manipulated. 3 comprises top and bottom glass or transparent plastic plates (17a and 17b) each 500 μm thick and coated with transparent layers of conductive Indium Tin Oxide (ITO) 18a and 18b having a thickness of 130 nm. Each of 18a and 18b is connected to an AC source 19 with the ITO layer on 18b being the ground. 18b is coated with a layer of amorphous silicon 20 which is 800 nm thick. 18a and 20 are each coated with a 160 nm thick layer of high purity alumina or hafnia 21a and 21b which are in turn coated with a monolayer of poly(3-(trimethoxysilyl)propyl methacrylate) 22 to render their surfaces hydrophobic. 21a and 21b are spaced 3 μm apart using spacers (not shown) so that the microdroplets undergo a degree of compression when introduced into this space. An image of a reflective pixelated screen, illuminated by an LED light source 23 is disposed generally beneath 17b and visible light (wavelength 660 or 830 nm) at a level of 0.01 Wcm.sup.2 s emitted from each diode 24 and caused to impinge on 20 by propagation in the direction of the multiple upward arrows through 17b and 18b. At the various points of impingement, photoexcited regions of charge 26 are created in 20 which induce modified liquid-solid contact angles in 21b at corresponding electrowetting locations 25. These modified properties provide the capillary force necessary to propel the microdroplets from one point 25 to another. 23 is controlled by a microprocessor (not shown) which determines which of 26 in the array are illuminated at any given time using a pre-programmed algorithm. By this means, the various microdroplets can be moved along the various pathways shown in FIG. 1 in a synchronised way.

[0071] FIG. 3 shows a top-down plan of a typical microdroplet (here 7b) located at a location 25 on 21b with the dotted outline 7′ delimiting the extent of touching. In this example, 26 is crescent-shaped in the direction of travel of 7b.