CHEMICAL DROPLET REACTOR

Abstract: A method and apparatus for combining chemical reactants wherein the method comprises the steps of: 1) dispensing the reactants into a conduit system in the form of discrete volumes or droplets (5, 15, 21, 22, 36) separated from each other by an inert immiscible liquid (1); and 2) changing the flow of the inert immiscible liquid within the conduit system in a precise sequence in such a manner that predetermined discrete volumes coalesce with each other, thereby combining the reactants; and wherein the apparatus is formed by holding two plates (42, 44) together in face to face contact, where one or both plates possesses a set of indentations (43) on the surface to be mated, thereby forming the conduits for reactant combinations.

Description: CHEMICAL DROPLET REACTOR The invention is the subject of British Patent Application No. 8200642. This invention relates to a method for combining liquid chemical reactants and apparatus for use therein. Description of prior art Automatic analysis apparatus for analysis of liquid samples as a flowing stream w disclosed by Skeggs in U.S. Patent No. 2,797,149 issued June 25 1957 and in U.S. Pate No. 2,879,141 issued March 24 1959. Such a flowing stream is customarily segment by separating successive segments with air, but it may alternatively be segmented an inert immiscible liquid, as taught in U.S. 3,479,141 issued to John Smythe Novemb 18 1969. Such methods provide an extremely powerful means of analysing a numb of liquid samples by subjecting them to a particular chemical test. SUMMARY OF THE INVENTION In contrast with continuous flow systems, the present invention does not operate wi a continuous stream, where control is achieved by the selection of appropriate flo rates for each fluid by running a pump or cyclically operated piston at a predetermin rate, but rather the liquid components of the present invention are controlled by sequence of distinct movements in closed conduits, with each movement or set movements occurring after the previous movement is completed. For example, known volume of a reactant is moved through an opening, this movement ceases, th the volume displaced is separated from the parent volume. It should be noted that t volumes of all the reactants to be combined are contained and defined by t immiscible liquid prior to combination, and that individual single reactant samples c easily be handled by virtue of the large amounts of the immiscible liquid present, arid of the method of control. Thus each sample is treated by a sequence of chang of the movements of the liquid components causing it to combine with a reactant or reactants selected at will from a pleurality of reservoirs of different reactants. The reactants are thus metered within the conduit system. It should also be noted that the conduits which are to contain the liquid components are preferably constructed by holding two plates together in face to face contact, where one or both plates possesses a set of indentations or channels on the surface to be mated. OBJECTS OF THE INVENTION Accordingly several objects of my invention are: 1. to carry out chemical reactions using volumes of reactants that may range from more than a litre to less than 10 nanolitres, and 2. to carry out repetitive chemical procedures automatically, and by less complex means than in comparable systems, and 3. to allow greater flexibility during operation, by continuously providing a choice of reactants, and 4. to isolate workers from dangerous substances including radioisotopes and pathogens, and 5. to reduce or eliminate contamination of the reactants, and 6. to prevent accidental mixing spillage or losses of the reactants during transfer, and 7. to provide a method and a means for metering samples and reactants within a conduit system. DRAWINGS Figure 1 is a plan view of two conduits being used in accordance with the method for producing droplets of reactants. The stippled areas represent carrier phase. Figure 2 is a plan view of conduits being used in accordance with an alternative method of producing droplets of reactants. F gure s a p an v ew o con u ts e ng use n accor ance witπ a memoo ιυr coalescing droplets. Figure 4 is a plan view of an apparatus in accordance with the invention, which h been adapted to allow the separation of substances by electrophesis. Figure 5 is an exploded view of one embodiment of an apparatus in accordance wi this invention. Figure 6 is a plan view of the apparatus of figure 5. DESCRIPTION OF THE INVENTION The method and apparatus of the invention may be used for initiating and controlli a chemical reaction, or preparing mixtures of reactants, wherein the method compris steps of: 1. dispensing two or more reactants, optionally in a solvent phase, into a conduit system in the form of discrete volumes, each volume being separated from each other by an inert and immiscible liquid, and 2. moving the immiscible liquid and discrete volumes through the conduit system i such a sequence that predetermined discrete volumes individually coalesce with eac other, thereby combining the reactants. The system is particularly suited to the m mpliation of microscopic quantities reactant with volumes of less than 10 nanolitres, but larger quantities of more than litre could also be used. The system is capable of treating single volumes of reactan individually. The discrete volumes may be separated from their parent volumes by passing a sma volume of reactant from a reservoir, through a constriction and into a conduit meetin the reservoir at right angles. The discrete volume is now separated from the pare volume in the reservoir by passing carrier phase along the conduit. The discrete volumes of reactants are coalesced with each other by pressing volume together by gravity, or by withdrawing the carrier phase from between a pleur-ality o discrete volumes, thereby causing them to move together until they collide, or b causing one volume to remain in a position partially blocking the path of a secon discrete volume, then moving this second discrete volume through the opening thu formed, causing the two discrete volumes to be pressed together and to coalesce * . The reactants can be liquids, or gases capable of being condensed in the apparatus, or solids or gases dissolved in a suitable solvent. This reactant liquid, hereafter referred to as a reactant, will be supported, defined and moved by another, immiscible liquid, referred to hereafter as the carrier phase. Suitable carrier phases include mineral oils, light silicon oils, water, and fluorinated hydrocarbons. The carrier phase may be a mixture of several substances, and preferably has approximately .the same density as the reactants. Surface acting agents may be included in the carrier and/or reactant phase to produce suitable surface properties, e.g. to allow efficient merging. Suitable surface acting agents include cholesterol, gelatine, sodium dioxyl, succinate Teepol, and Triton-x-100. The cross-section of the conduits is generally of the same order of magnitude as the cross-section of the droplets which are to be used in them, but smaller conduits, or larger droplets or discrete volumes may be used, causing the droplets to become elongated. The carrier phase may be moved by any suitable means, including the distortion of flexible tubing or sheeting, blowing or sucking by mouth, pumps or pistons. Pistons can be moved by screw threads turned by electric motors, (which may optionally be controlled electronically using microprocessor technology). Figure 1 of the accompanying drawings illustra.es one method of separating droplets of a predetermined volume from a larger reservoir of reactant. The reactant is introduced into a conduit containing carrier phase 1 from a side arm 2, by being sucked or pushed through a small opening 3. When the correct volume has passed through into the conduit a flow of carrier phase separates it from the reactant in the reservoir, and carries it down the conduit. The volume of the droplet produced can be determined by at least three methods: 1. The piston responsible for pushing the reactant through the opening is moved a known distance, corresponding to the volume of the reagent displaced. When this movement is complete the droplet is broken off by a relatively fast, shortlived current of carrier phase. 2. If large numbers of droplets are required, a continuous flow of carrier phase flows down the conduit. When reactant is simultaneously passed through the opening 3 series of droplets will be formed, each broken off just before it spans the conduit The exact size of the droplets thus produced will depend on the magnitudes of th two currents. 3. The volume of the droplet before separation 5, is estimated visually, optionally usi a telescope or microscope with a graduated eye piece. When the correct volu is achieved it is separated by a current of carrier phase. Figure 2. illustrates another method of forming droplets. In this case the reactant is passed into a conduit of considerably narrower width 11 than the droplets to produced. The conduit is graduated relative to the opening 12 of a side arm 13. T reactant is passed into the narrow conduit to a certain graduation 14, whereup carrier phase is introduced from the side arm, thus separating a droplet of the requir size 15. Figure 3. illustrates a method of coalescing droplets, which can be of different siz The coalescence is effected at a site where two conduits, 23 and 25 in figure 3, mer to form a single conduit 24, preferably in a Y-configuration as shown. A current fro conduit 23 to conduit 24 is produced which carries a droplet 21 to the site f coalescence. When the surface of this droplet protrudes into the space where condui 23 and 25 converge, the valves feeding conduit 23 are closed. A current from cond 25 to conduit 24 now carries a second droplet past the first, and in doing so the t droplets are pressed together, and coalesce to form a single droplet. If either droplet is smaller than the other, then the conduit through which it emerg should be correspondingly narrower. All conduits should have diameters approximate the same as the droplets to be used in them. The various movements of the fluids are produced by connecting the conduits pistons, pumps or to reservoirs containing compressed gas or partial vacuum. Contr is achieved by conventional valves. Conduits can be heated by electric circuits or coils or by electromagnetic radiatio allowing coalesced droplets to be incubated at the desired temperature. Solid sta heat pumps can be used to cool conduits both for incubation at low temperatures a to allow the storage of unstable reactants. Heating can be achieved by reversing t heat pumps. Heating and cooling can also be achieved by circulating a fluid throu separate ducts. After coalescence the reactants can be thoroughly mixed by moving the droplet up a down a conduit, or if required by passing it through a constriction several times. Ports can be provided to allow droplets to be removed or introduced with a syring needle. The droplet can be passed into a tube and transferred to any standard instrument o chemical or biochemical analysis. Alternatively parts of the device itself can b adapted to form the sample chambers of such instruments. For example conduits ca be formed with two plain transparent walls to form the sample chambers o photometers. A single transparent wall opposite a reflecting surface could also b used. Conduits can also be packed with the materials used for electrophoresis and chro matography. Figure 4. shows a conduit which is adapted for electrophoresis. The electrophoreti conduit 31, is packed with electrophoretic material. The sample to be separated b electrophoresis is introduced as a droplet from conduit 32, and this droplet is merge on one side with the surface of the electrophoretic material (e.g. agarose, poly acrylamide or cellulose) and on the other side with a semipermeable membrane. Thi semipermeable membrane 33 encloses a chamber filled with buffer, containing a electrode 34 and provided with a gas vent 35 if required. Another droplet 36 is merge on to the other end of the electrophoretic material and to another semipermeabl membrane and electrode assembly 37. A current is passed between the electrode which causes the constituents to migrate at various speeds. The fastest fraction migrate into the receiving droplet first. This droplet is now exchanged for another 3 after a period, allowing the collection of a series of droplets containing differen fractions. The ability to handle very small samples makes the system suited to use with Hig Performance Liquid Chromatography. The system can be connected to a conventional H.P.L.C. system by fine bore tubing to a mass spectroscopy system. The progress of droplets can be automatically monitored by detecting fluctuations i the light propagated through the conduit as the droplets pass. Similarly, when tw droplets merge the light scattered from a beam passing through both droplets sudden decreases. Windows in an opaque coating of the conduits can be used, and in cas where the monitoring of many sites is required, optie fibres could carry light to or fro a central emitter or detector. The position of droplets could also be detected wh they pass between an infra-~red em miter and detector, or when they pass between t plates of a conductance cell, or by the resultant change in the refractive index of t contents of the conduits. In any automatic embodiment of the device, all pressu generating and monitoring means can be controlled by microprocessors or compute The preferred method of construction of the apparatus is by producing indentations channels of the appropriate configuration in the surface of a plate, and to clamp hold this plate against another, producing closed ducts or conduits. The second pla may have a planar surface, or it may have indentations, either in .the mirror image the first plate's or in a different configuration. Also it is possible to produce thr dimensional configurations, where conduits cross over each other, by using three more plates. Preferably at least one of the plates is transparent, e.g. glass or perspe allowing visual inspection of the procedures. The other plate may be Kel-F or Tefl (materials suited for use with aqueous reactants) or glass, perspex, metal, polyviny chloride, polypropylene etc. Indentations in glass can be etched. Plastics and metals can be etched, moulded machined. Electrical contacts can be produced by depositing a metallic layer of t desired configuration on either plate. Micro-organisms can be cultured in droplets containing the appropriate nutrients a incubated at the correct temperature. In place of droplets, larger segments occupying a considerable length of conduit can separated from reservoirs of reactants and coalesced with other segments in the sam way as droplets. Contamination of the walls can be reduced by: 1. constructing conduits with highly polished walls, 2. constructing conduits with walls coated with or constructed from a material which repels the reactant phase but attracts the carrier phase, 3. washing the conduits with segments of a cleaning liquid prior to the passage of eac «; volume of reactant. 4. preventing the droplets or segments from remaining stationary by oscillating th flow of carrier phase thus maintaining a film of carrier phase around the reactan An apparatus can be constructed in accordance with the method of the invention o joining tubes and pipes in the desired configuration. This invention has applications in many branches of medicine, chemistry, biochemistry geology, etc., notably in procedures which utilize very small volumes, such as forensi and molecular biology work. The invention could be used to perform biological o chemical assays of sub-samples during purification procedures, and to produce chemica derivatives of samples prior to analysis by chromatography or mass spectroscopy. I could also be used for chemical analysis or synthesis in outer space, in conditions o very low gravity and pressure. OPERATION OF INVENTION Figure 5 is an exploded view of one embodiment of an apparatus in accordance wit the method of the invention. This embodiment is capable of mixing predetermine volumes of two aqueous reactants under sterile conditions. The device comprises a steel base 41 supporting a Teflon block 42 in which U sectioned indentations 43, of the configuration shown, have been machined. A shee of siliconized glass 44 is pressed against this block by a clamp 45 which is tightene by nuts 46 and bolts 47. The five branches of the U-sectioned conduits thus forme lead to five stop-cock valves 48. The middle and outer pair of these conduits ar connected by Teflon tubing 49 to reservoirs of the carrier phase, which is miner spirit. The remaining two branches are connected to glass syringes, each containin one of the two reactants to be mixed. Figure 6 is a plan view of the device of figure 5 and uses the same reference numeral Reservoirs 55 and 58 are half-filled with mineral spirit with the ends of the tuDing 5 opening below the surface of the mineral spirit, while the end of an air vent 57 ope above the surface in each. A syringe containing air 60 is connected to reservoir 5 above the surface of the mineral spirit. The ports serving the valves 51 and 53 lead to two syringes 61 and 62 containing t two reactants. The conduits are initially filled with white spirit. An operating sequence for one cycle is as follows: 1. Valves 48, 51, and 52 are opened. Valves 53 and 54 are closed. 2. Syringe 61 is moved in until reactant reaches constriction 3. 3. Syringe 61 is moved further by the required amount, pushing a known volum reactant through the constriction 3. 4. Valve 51 is closed. 5. Syringe 60 is moved out until the volume of reactant protruding through constric 3 is broken off, and the resultant droplet moves towards the Y-junction 64. V 48 is closed. 6. Valves 53 and 54 are opened and a similar sequence of events produces a drople the reactant from syringe 62. 7. Syringe 60 moves out until the droplet originating from syringe 62 protrudes into Y-junction. Valves 53 and 54 are closed. The positions of the droplets illustrated at this stage in Figure 6. 8. Valve 48 is opened and syringe 60 moves out until the droplet of the reactant f syringe 61 moves into the Y-junction 64, where it is pressed against the o droplet causing the droplets to coalesce. 9. If a colour change reaction is involved, such a colour change can be recor in- mediately, or after incubation using a thermostatically controlled coil 57. droplet can be discarded by passing it into a reservoir 59 of mineral spirit. If the droplet is to be analysed using external instrumentation, it can be move a site opposite port 65, all valves closed, plug 66 removed and the droplet imp and removed by a needle and syringe and transferred to the instrument. The is then replaced. 10. Syringe 60 is disconnected, emptied of air, and reconnected, leaving the de primed for another cycle of operation. It is emphasised that Figure 5 shows a very simple embodiment of the invent Embodiments are envisaged having reservoirs for the introduction of more than twe different reactants. It is also proposed to operate the valves automatically, and and cool regions of the conduits to allow incubation of reacting mixtures, and storage of reactants for long periods. While the above description contains many specificities, these should not be constr as limitations on the scope of the invention, but rather as an exemplification of a preferred embodiments thereof. Many other variations are pos-sium, ιut uαm^c i% ,„ possible to press together and coalesce droplets by gravity, and to use water as the carrier phase for oil soluable reactants. Accordingly, the scope of the invention should not be determined by the embodiments illustrated but by the following claims and their legal equivalents.

Claims: CLAIMS 1. A method of combining chemical reactants comprising the steps of: dispensing two or more liquid reactants into a conduit system in the form discrete volumes of liquid, each volume being separated from each other and fro all undispensed reactants by an inert immiscible liquid, thereby metering t reactants within the conduit system; and changing the flow of said immiscible liquid and said discrete volumes through sa conduit system in a precise sequence, in such a manner that predetermined discre volumes are individually coalesced with each other, thereby combining t reactants. 2. A method as claimed in claim 1 in which said discrete volumes of chemical reactants are sufficiently small in relation to said conduits to form subst ntial spherical droplets with diameters less than the conduits. 3. A method as claimed in claim 1 in which said discrete volumes of chemic reactants are sufficiently large in relation to said conduits to be elongated by t conduits. 4. A method as claimed in claim 1 in which each of said discrete volumes of reactan is formed by passing each chemical reactant from a reservoir, through an openin into a conduit initially containing said carrier phase, then moving said carrier pha causing a volume of said reactant to become separated from the reactant in t reservoir, said opening being optionally constricted, thereby metering the reacta within the conduit system. 5. A method as claimed in claim 4 in which the volume of reactant passed out of th reservoir is estimated by assuming it to be spherical, and by measuring the diamet t il of such a sphere optically, after which said volume is adjusted and separated. 6. A method as claimed in claim 4 in which the volume of reactant passed out of th reservoir is estimated before separation by measuring the length of conduit whic it occupies, after which said volume is adjusted and separated. 7. A method as claimed in claim 4 in which the volume of reactant passed out of th reservoir is determined by displacing said volume by moving & piston a knowi distance, or by means of a well calibrated pump, prior to the separation of sai volume. 8. A method as claimed in claim 1 in which the immiscible liquid is moved until discrete volume of reactant partially blocks the opening of a conduit, whereupo another discrete volume of reactant is moved out of or into said opening causin these two discrete volumes to be preyed together, causing them to coalesce. 9. A method as claimed in claim 1 in which said discrete volumes of reactants contai suspended micro-organisms. 10. A method as claimed in claim 1 in which surface acting chemical agents ar dissolved in said chemical reagents or in said immiscible liquid, or in both. 11. An apparatus for combining chemical reactants comprising two or more plates hel in face to face contact, in which one or more plates possesses a set of indentation or channels, and bores, on the surface to be mated, such that closed conduits ar formed, these conduits being in communication with reservoirs to contain two o more reactants and an inert immiscible liquid and with means for producing an controlling movements of the liquid components, said plates having surfaces wit low affinity for the reactant to be used, which apparatus includes at least one zon where at least three conduits meet, which zones allow the formation an coalescence of chemical reactants. 12. An apparatus as claimed in claim 8 but in whieh said conduits are constructed b joining a set of tubular elements in the appropriate conformation. 13. An apparatus as claimed in claim 11 or 12, comprising three conduits which join i a T or Y configuration, these three conduits leading to reservoirs for an immiscibl liquid, each reservoir possessing means for moving said immiscible liquid, in whic one or more of said conduits communicates with reservoirs for a pleurality reactants. OMPI

Inventors: SHAW STEWART PATRICK DOUGLAS, SHAW STEWART, PATRICK, DOUGLAS
Applicants: SHAW STEWART P D, SHAW STEWART, PATRICK, DOUGLAS
Application year: 1982
Main classification: G01N1
IPC codes: B01F3, B01F5, B01F13, B01J14, B01J19, G01N1, G01N30, G01N31, G01N35