FR3003772A1 - METHOD AND SYSTEM FOR MONITORING THE KINETICS OF REACTIONS - Google Patents

Abstract

This method comprises: the generation of an ordered train of drops within a carrier fluid (fp) to form a plurality of reactors, the drop train comprising a succession of elementary streams of drops, each associated with at least one reagent (ri) of said plurality of reagents (ri), each drop of an elementary train comprising the or each reagent (ri) associated with said elementary train, - circulating at least a portion of said ordered stream of drops the measurement of at least one representative parameter of each reactor over time.

Description

The present invention relates to a process for monitoring the kinetics of reactions, in a reaction medium, between at least one component of said reaction medium and each of a plurality of distinct reagents. Such a method is particularly applicable to monitoring reactions between a culture medium and multiple reagents at various concentrations. It applies in particular for the automated production of multiple antibiograms, that is to say to determine the minimum inhibitory concentrations (MICs) of bacterial growth for different antibiotics. Such antibiograms are carried out in order to establish the resistance profiles of bacteria to different antibiotics, and thus to guide practitioners in their treatment choices. The increasing number of resistant bacteria and the increase in the number of antibiotics have required, over the last twenty years, the implementation of automation tools for these antibiograms. Several strategies exist to achieve such antibiograms. In particular, it is known to use strips or discs of polymers containing antibiotics or antibiotic gradients placed on solid culture media. Such a method is for example described in WO 2010/010582. The main limitation of this method comes from the fact that the cultures take place on solid media which require a longer time to detection than in liquid media (of the order of 24 hours, ie the time required to the appearance of the colony). In addition, automation of manipulation and analysis on solid media boxes is complex and expensive. It is also known to use dilution or micro-dilution methods according to which the antibiotics are diluted and mixed with liquid culture medium in series of tubes, microtiter plate wells or pre-filled cards. antibiotics.

In particular, culturing bacteria in micro-titration plates allows rapid growth in liquid medium and the analysis of a large number of parameters, for example many dilutions of the same antibiotic or a large number of antibiotics. However, the automation of the manipulation and reading of micro-titration plates remains confined to very large hospitals with regard to microbiology, because it is very expensive and not validated for the cultivation of anaerobic microorganisms. In addition, the formation of biofilms on the well wall degrades detection, and leads to problems of reproducibility and homogeneity of the results. Pre-filled cards of antibiotics are made of adhesive polymers that are more or less permeable to oxygen, which makes it possible to make aerobic or anaerobic cultures. Such cards include, for example, 64 wells of 301.11, with which it is possible to test about twenty antibiotics at three or four different concentrations. The kinetic study of the effect of antibiotics is carried out by measuring, for a period of between 3 and 10 hours, the biomass in each of the wells. However, this method requires the pre-filling of antibiotics in the form of dehydrated powder, which implies problems of dissolution and diffusion over the entire sample during filling. In addition, biofilms can also be formed on the walls of the wells, which poses problems of reproducibility and homogeneity of the results. To reduce these problems of inhomogeneity, it has been proposed, in particular in document US 2005/0084923, to use drops as reservoirs of bacterial cultures. However, these methods are limited to a small number of dilutions, not allowing an accurate determination of minimum inhibitory concentrations, either to a single reagent, which limits the degree of automation of the method, or does not allow monitoring. kinetics of reactions, in particular the temporal evolution of living cell cultures. It has also been proposed in document FR 2 972 198 to use drops as reservoirs of bacterial cultures. However, this method is limited to the study of a single reagent or a single mixture of reagents, so that the degree of automation of this method is limited. The present invention aims to solve the above drawbacks and to provide an automated reaction monitoring method, making it possible to vary, during the same experiment, both the type of reagent analyzed and its concentration, and making it possible to monitor the kinetics of the reactions. reactions.

To this end, the subject of the invention is a process of the aforementioned type, characterized in that it comprises: the generation of an ordered train of drops within a carrier fluid to form a plurality of reactors, the train dropper comprising a succession of elementary streams of drops, each associated with at least one reagent of said plurality of reagents, each drop of an elementary train comprising the or each reagent associated with this elementary stream, - the circulation of at least a portion of said ordered stream of drops, - the measurement of at least one parameter representative of each reactor over time. The method according to the invention may comprise one or more of the following characteristics, taken separately or in any technically possible combination: each elementary stream comprises at least one reagent not included in an adjacent elementary stream; the generation of the ordered train of drops comprises the generation of a plurality of elementary streams of successive drops, each associated with at least one reagent of the said plurality of reagents, between which are interposed intermediate trains of drops, and the suppression of said trains; intermediate drops; in at least one elementary stream of drops, the concentration of at least one reagent varies, in particular according to a gradient; the generation of the ordered stream of drops comprises the following steps: (a) the generation of a flow of reaction medium, (b) the filling of a capillary reaction tube with a carrier fluid immiscible with the reaction medium, c) injecting, through a capillary injection tube, an individual drop of reaction medium into the capillary reaction tube, (d) circulating the carrier fluid so that the drop of reaction medium is displaced by relative to the capillary injection tube, (f) the repetition of steps c) and d) to create an ordered train of drops of reaction medium within the carrier fluid; the step of generating the flow of reaction medium comprises: the formation of a stream of reactants comprising a succession of elementary fluxes of reagents, each elementary stream of reagents comprising at least one reagent of the said plurality of reagents, the mixture said reagent stream to a culture medium comprising said component for forming said reaction medium stream; the formation of a flow of reagents comprises the following phases: (a) the generation of a continuous flow of a solvent in a mixing channel, ([3) concomitantly with the phase a), the injection, thanks to to a capillary tube, a reagent selected from the plurality of reagents in said mixing channel, to form an elementary stream of reagent, (y) repeating the phase (3) for each of the reagents of the plurality of reagents successively ; the formation of the flow of reagents comprises, after the formation of each elementary flow of reagent and before the phase y), a phase 8) of injection, thanks to a capillary tube, of a cleaning liquid in said mixing channel ; the generation of the ordered stream of drops further comprises, after step d) and before step f), a step e) comprising the injection, into the carrier fluid, of a drop of a separating fluid immiscible with the carrier fluid and immiscible with the reaction medium, so that at least one drop of separating fluid is interposed between two reactors and prevents their coalescence; said component is a microorganism and said culture medium is a culture medium for microorganisms, and said parameter is representative of the amount of microorganism present in each reactor; said reagents are antibiotics, and said method comprises carrying out an antibiogram to test the sensitivity of said microorganism to said antibiotics. The subject of the invention is also a system for monitoring the kinetics of reactions, in a reaction medium, between at least one component of said reaction medium and each of a plurality of distinct reagents, characterized in that it comprises: module for generating an ordered train of drops in a carrier fluid to form a plurality of reactors, the drop train comprising a succession of elementary streams of drops, each associated with a reagent of said plurality of reagents, each droplet an elementary train comprising the reagent associated with this elementary stream, a drop storage and detection module capable of circulating at least a portion of said ordered stream of drops and measuring at least one parameter representative of each reactor over time. The system according to the invention may comprise one or more of the following characteristics, taken separately or in any combination: the generation module of the ordered stream of drops comprises: means for generating a medium flow reaction, - means for filling a capillary tube of reaction with a carrier fluid immiscible with the reaction medium, from a carrier fluid reservoir, - means for injecting, through a capillary injection tube, a droplet individual reaction medium in the capillary reaction tube, means for circulating the carrier fluid so that the drop of reaction medium moves relative to the capillary injection tube. the means for generating a flow of reaction medium comprise: means for forming a flow of reagents comprising a succession of elementary fluxes of reagents, each elementary flow of reagent comprising at least one reagent of said plurality of reagents, means for mixing said reagent stream with a culture medium comprising said component to form said reaction medium stream; the means for forming the flow of reagents comprise a plurality of reagent reservoirs, and means for circulating each of the reagents successively from each of said reservoirs; said storage and drop detection module comprises a fluid circulation circuit of the drops, and means for circulating at least a portion of said ordered stream of drops in said circuit by generating a pressure difference between two points of said circuit. The invention will be better understood on reading the description which follows, given solely by way of example, and with reference to the appended drawings, among which: FIG. 1 is a schematic view of a reaction system according to the invention; Figure 2 is a schematic view of a detail of the system of Figure 1; - Figure 3 is a block diagram of the steps of a method according to the invention. In the detailed description below, the reaction system described is a culture system for living microorganisms or living biological cells, and the reagents are antibiotics. In this application, the reaction medium is a culture medium for microorganisms. Nevertheless, the method described may be used in other fields to track reactions over time between one or more reaction systems and each of a plurality of reagents. In the detailed description below, a capillary tube or a channel is a millimeter-scale fluidic tube, that is to say having an internal diameter of the order of one tenth of a millimeter to one millimeter, preferably between 0.5 and 1 mm. For example, can be used to implement the present invention, connectors and capillary tubes for chromatography. A preferred embodiment uses tubes 0.5 millimeters in diameter, to obtain drops of culture medium ranging from about 100nL to several milliliters.

The system 1 according to the invention comprises a module 3 for generating a stream of drops, a module 5 for storing and detecting drops formed by the module 3, and a module for controlling modules 3 and 5. The module 3 generating a drop train is able to generate, in a capillary reaction tube, an ordered train of drops of reaction medium in a carrier fluid. This ordered train consists of a succession of elementary trains, each associated with at least one given reagent r ,. Each drop of an elementary train thus comprises, in addition to a culture medium, the reagent (s) r, associated with this elementary train t 'at a given concentration.

In addition, to prevent the risks of coalescence of the drops, a separation fluid immiscible with the carrier fluid and with the reaction medium is interposed between two drops of the drop train. Two adjacent elementary trains are distinguished from one another in that at least one of these elementary trains comprises at least one reagent which is absent from the other elementary train. As described below, each elementary stream is formed of drops in which the concentration of at least one reagent is variable. Each elementary train indeed comprises either a single reagent, whose concentration is variable within the elementary stream, or a combination of several reagents, the concentration of at least one of these reagents being variable within the elementary stream. Preferably, the concentration of this reagent within the elementary stream of drops forms a gradient between a minimum concentration, for example zero, and a maximum concentration. The concentration of reagent r thus varies monotonically in the train between these two concentrations.

For example, an elementary train may comprise a first reagent r, whose concentration is constant within the set of drops, and a second reagent whose concentration varies within the elementary stream, in particular according to a gradient. According to another example, an elementary train may comprise a first and a second reagent r, and the concentration in each of these reagents r, and varying within the elementary stream according to a gradient, for example chosen so that the ratio between the The concentrations of these reagents are substantially constant within the drop train. To generate this stream of drops, the module 3 comprises a unit 13 for generating multiple gradients, a unit 14 for generating a flow of reaction medium, and a unit 15 for generating the drop train.

The unit 13 for generating multiple gradients is capable of generating, in a capillary injection tube 17, a stream of liquid composed of a succession of elementary streams, each consisting of at least one reagent diluted in a solvent. Advantageously, the concentration of at least one reagent in the solvent varies within each elemental stream, preferably monotonically, especially linear. In addition, two successive elementary streams are advantageously separated by an intermediate flow intended to prevent contamination of a given elementary stream by a reagent of the previous elementary stream. This intermediate stream comprises for example the solvent and a cleaning liquid, as well as traces of the reagent (s) of the previous elementary stream. The unit 13 comprises a plurality of reservoirs R, reagent, each comprising a reagent r, distinct, fluidly connected to a capillary tube 21 for injecting reagent via a T-junction, and a reservoir N of liquid cleaning element n, also fluidly connected to the capillary tube 21 for injecting reagent via a T-junction. The unit 13 further comprises a reservoir S of solvent s, fluidly connected to a capillary injection tube 27 of solvent. The capillary injection tubes of reagent 21 and of solvent 27 are mounted opening into a mixing channel 29 via a T-junction 30.

The unit 13 further comprises means for circulating the reagents r 'of the solvent s and the cleaning liquid n. These circulating means are suitable for generating a flow rate of each reagent r, and cleaning liquid n in the reagent injection capillary tube 21, from each of the reagent reservoirs R and from the reservoir N of liquid These circulation means are also suitable for generating a flow of solvent s in the capillary tube 27 for injecting solvent, from the solvent tank S to the mixing channel 29. in particular, to generate a plurality of elementary streams each formed of a set of reagents diluted in a solvent, these circulation means are capable of generating a continuous flow of solvent in the capillary tube 27 of solvent injection, and, in parallel, sequentially generating a flow rate of each of the reagent sets in the reagent injection capillary tube 21, a cleaning liquid flow rate being generated in the capillary tube 27 for injecting solvent between two successive flow rates of sets of reagents.

These circulation means comprise, for example, an HPLC sampler (for high performance liquid chromatography). Alternatively, the tanks Ri and S are wells of a plate, and the circulation means comprise an automaton able to pipet the reagents r, or the solvent s from these wells. According to another variant, the tanks Ri and S are connected to a pressure generator via a pressure multiplexer. The pressure multiplexer comprises valves that can be open or closed, and whose opening generates the injection of a selected reagent, under the effect of pressure from the pressure generator, towards the capillary injection tube. reagent 21. The mixing channel 29 comprises two liquid inlets, respectively connected to the reagent injection capillary tube 21 and the solvent injection capillary tube 27, so that each reagent r, or the cleaning liquid n is mixed with the solvent in this mixing channel 29 undergoing Taylor-Aris diffusion. Thus, the mixing channel 29 is able to deliver a liquid flow, called a flow of reagents. This stream of reactants is composed of a succession of elementary streams of reactants, two successive elementary streams of reagents being separated by an intermediate stream composed of solvent and cleaning liquid, as well as traces of reagents r, of the elementary stream preceding it. . For example, the mixing channel 29 and the circulation means are configured such that within each or at least some of these elementary streams, the concentration of at least one reagent r varies linearly between a minimum concentration and a maximum concentration. The injection of cleaning liquid makes it possible to rinse the reagent injection capillary tube 21 and the mixing channel 29 between two successive injections of reagents, and thus to prevent a reagent of a given elemental stream from contaminating the reagent. next elementary stream if the presence of this reagent is not desired in this next elementary stream. In particular, thanks to the injection of the cleaning liquid, each elementary stream comprises at least one reagent absent from the elementary stream following or preceding it. The unit 14 for generating a flow of reaction medium is capable of generating, in a capillary tube 33 for injection of a reaction medium, a flow of reaction medium, by mixing the successive elementary streams from the unit 13 and the unit. a flow of culture medium. The unit 14 thus comprises a reservoir M of culture medium m, fluidly connected to the outlet of the mixing channel 29, via a T-junction 37, so that the culture medium m can be successively mixed with each of the elementary fluxes of reagents in a capillary tube 33 for injecting a reaction medium. The mixture, of variable composition, thus obtained, is called the reaction medium.

The culture medium m comprises microorganisms such as cells or bacteria. The unit 14 further comprises means 34 for circulating the culture medium m, able to control the flow of culture medium m towards the T-junction 37, with respect to the flow of liquid from the unit 13, so as to obtain in the capillary tube 33 a reaction medium of the desired composition, in particular for controlling the final concentration of reagents in the reaction medium. The unit 15 for generating the drop train is able to generate, in a capillary reaction tube 39, a set of drops, in particular from the reaction medium stream coming from the unit 14, from a carrier fluid stream. fp and a separation fluid stream f '. The unit 15 thus comprises a reservoir Fp of carrier fluid fp and a reservoir Fs of separation fluid f '. The separation fluid f is a fluid immiscible with the carrier fluid fp and immiscible with the culture medium m. The carrier fluid fp and the separation fluid f, are preferably immiscible oils between them, such as for example the fluorinated oil as the carrier fluid and the mineral oil for the separation fluid, the culture medium m being an immiscible aqueous medium with the above-mentioned oils. The reservoir Fp, Fs carrier fluid fp and separating fluid f, and the capillary tube 33 are mounted opening into the capillary reaction tube 39 via a cross junction 36 equipped with suitable valves. The unit 15 further comprises means 41 for circulating the carrier fluid fp and the separating fluid fs. These circulation means 41 are able to control the flow rate of the carrier fluid fp and of the separation fluid f, from the reservoir tanks Fp, Fs of the carrier fluid fp and of the separation fluid f5 to the reaction capillary tube 39. the reservoirs Fp and Fs of carrier fluid fp and separating fluid f comprise syringes, and the circulation means 41 comprise syringe pumps. Alternatively, the circulation means 41 are adapted to apply a controlled pressure on tanks Fp and Fs to generate a fluid flow of these tanks. Controlling the respective flow rates of the reaction medium, the carrier fluid and the separation fluid makes it possible to form dispersed mono-dispersed (oil-in-water) emulsion drops. By imposing a speed and a duration of circulation of the reaction medium, of the carrier fluid and of the separation fluid, it is possible to precisely inject a determined volume of reaction medium and, alternatively, of separation fluid into the carrier fluid in the form of individual drops. Each drop of reaction medium thus formed constitutes a microorganism culture reactor within the carrier fluid fp.

The unit 15 for generating the drop train thus makes it possible to form a train of drops of reaction medium within the carrier fluid, in which two successive drops of reaction medium are advantageously separated by a drop of separating fluid. Due to the composition of the reaction medium formed by the unit 14, the train of drops comprises a succession of elementary streams of drops each associated with at least one of the reactants r 'two successive elementary trains being separated by an intermediate train of drops composed solvent and cleaning liquid but none of the reactants except in the form of traces. In addition, within each elementary stream of drops, the concentration of at least one reagent r varies from one drop to another according to a gradient, for example linearly. The length of the capillary reaction tube 39 in which the drops and the imposed flow rates are formed define the amount of drops that can be used experimentally and the time step between each measurement. It is thus possible to work on several thousand drops in parallel. This one-dimensional drop manipulation method allows to preserve the identity of each drop during an experiment, and to control perfectly their composition by avoiding any loss by evaporation or reversal. The module 5 for storing and detecting drops of reaction medium comprises a circulation circuit 48 for drops and a unit 50 for storing and detecting these drops. The circulation circuit 48 is isolated from the generation module 3 of the drop train by a pinch valve 52. The capillary reaction tube 39 opens into the circuit 48 on a T-junction 54, followed by a two-way valve 56 , for orienting the stream of drops either to the storage and detection unit 50 or to a waste tank W1. In particular, such an arrangement makes it possible to eliminate from the drop train the drops containing cleaning liquid, i.e. drops of the intermediate trains. The drop train received by the storage unit 50 is therefore composed of a succession of elementary streams of reaction medium drops all comprising at least one reagent. Furthermore, each elementary stream comprises at least one reagent not included in an adjacent elementary stream.

The T junction 54 opens into the circuit 48 on a cross junction 60 also connected to three branches 62, 64, 66 of the circuit 48. A first branch 62 opens into a channel 52 of the storage unit 50. As described in more detail with reference to FIG. 2, the storage unit 50 in fact comprises a linear detection channel 52, which extends between two ends, hereinafter referred to as "input" 52a and "output" 52b. , between which the fluid can flow in two opposite directions of circulation. The inlet 52a of the detection channel 52 is fluidly connected to the cross junction 60, while its outlet 52b is fluidly connected to a T-junction 68.

The other two branches 64, 66 also extend between the cross-shaped junction 60 and the T-junction 68. These two limbs 64, 66 comprise means for putting into circulation the drift train, making it possible to generate movements of the train of drops in the linear channel 52 of detection in both directions of circulation, and thus allowing the realization of multiple measurements over time for each of the drops of the train. In particular, the branch 64 comprises means for generating a positive or negative pressure difference between the input 52a and the output 52b of the detection channel 52, and thus causing the drop train contained in this channel 52 to move in one direction or in the other.

The branch 64 comprises for this purpose two reservoirs 70, 72 of carrier fluid fp, respectively connected fluidically to the cross junction 60 and to the T-junction 68 via two-way valves 74, 76. pressurizing means are adapted to apply a pressure Po on each of the tanks 70, 72, via valves 82, 84. Thus, when the valve 82 (respectively 84) is open, the carrier fluid fp contained in the tank 70 (respectively 72 ) is maintained at the pressure Po by the first pressurizing means. Second pressurizing means 86 are adapted to apply a pressure P, greater than Po, on one or the other of the tanks 70 or 72 selectively, through a valve 88. The application of the pressure P on the reservoir 70 (respectively 72) is able to generate a flow of carrier fluid fp from this reservoir to the detection channel 52 via the cross junction 60 (respectively via the T-junction 68), thus a displacement of the drop train in this channel 52 from the input 52a (respectively the output 52b) to the output 52b (respectively the input 52a).

The branch 66 includes means for discharging liquid leaving the channel 52 to a waste tank W2. Branch 66 comprises for this purpose a waste tank W2 connected, via a T junction 90, on the one hand to an upstream capillary 92 opening into the cross junction 60, and on the other hand to a downstream capillary 94 opening into the T-junction 68. The upstream and downstream capillaries 92 92 are each provided with a two-way valve 96, 98 respectively. Each valve 96 or 98, when closed, makes it possible to prevent a flow of liquid in the capillary upstream 92 or in the downstream capillary 94. In particular, the valves 96, 98 guide a flow of carrier liquid fp from the reservoir 70 or 72 to the channel 52 of detection, preventing this flow to be driven to the waste tank W2, and guide a flow of liquid from the detection channel 52 to the tank 70 or 72, preventing this flow to be driven to the waste tank W2. Conversely, when this valve 96 or 98 is open, it allows a flow of liquid in the upstream capillary 92 or in the downstream capillary 94. In particular, the valve 96 or 98 allows a flow of liquid leaving the channel 52, during the displacement of the train of drops in this channel 52, to the waste tank W2. The branch 66 makes it possible in particular to empty and rinse the detection circuit 48 at the end of the measurements, by injecting carrier fluid into the channel 52 through one of the tanks 70 or 72 and evacuating the contents of this channel. 52 by the upstream capillary 92 or downstream 94 in the waste tank W2. The storage and detection unit 50 will now be described in greater detail with reference to FIG. 2. The storage and detection unit 50 comprises means for referencing the drops to identify them in the drop train, and means for monitoring reactions occurring in each of the drops. The storage and detection unit 50 comprises for this purpose the linear detection channel 52, and the drop detection and counting means, comprising a light emission source 100 and at least one detector 102, 104.

The detection channel 52 is for example formed of a capillary tube comprising two upstream portions 106 and downstream 108 coiled in two coils, and a measurement capillary section 110 interposed between the upstream 106 and downstream portions 108. The measuring capillary section 110 defines a central axis A-A '. The upstream portions 106 and downstream 108, coiled in two coils, can store the stream of drops while minimizing the space required for this storage. The two upstream portions 106 and downstream 108 are respectively connected fluidically to the inlet 52a and the outlet 52b of the channel 52. The lengths of the upstream portion 106 and downstream 108 are chosen greater than the length of the stream of drops analyzed, so that all the drops of this train can pass into the measuring capillary section 110 without any drop leaving the channel 52. Advantageously, the upstream 106 and downstream portions 108 are placed in a thermostatically controlled chamber, in which the concentrations in gas and lighting are controlled.

The wall of the measuring capillary section 110 is at least partially transparent to the light radiation emitted by the light emitting source 100 and to the light radiation picked up by the detector (s) 102, 104. The emission source light 100 is placed transversely on one side of the central axis A-A '. It preferably comprises a laser.

The source 100 is able to emit a coherent incident beam 111 directed towards the measurement capillary section 110, after reflection on at least one mirror 112, and after focusing by an objective 114. Advantageously, the incident light beam 111 is a laser beam of wavelength between 400 nm and 800 nm Preferably, the mirror 112 is a dichroic mirror capable of reflecting the light rays from the source 100 and transmitting light rays whose wavelength is within a predetermined range for example between 400 nm and 800 nm. A first detector 102, disposed on another side of the central axis A-A ', is adapted to receive an output beam 116 resulting from the crossing by the incident beam of the measurement capillary section 110. incidence of the incident beam 111 on the measurement capillary section 110 is for example equal to 0 °. The first detector is then able to determine from the output beam 116 the optical density of the fluid passing through the measuring capillary section 110, and therefore from each of the droplets of the drop train. The determination of the optical density makes it possible to determine the content of each drop, in particular its biomass. In particular, in the context of carrying out multiple antibiograms, it is thus possible to measure, each time a given drop passes through the measurement capillary beam, the concentration of bacteria in this drop.

The detector 102 may also be arranged at an angle of up to 90 ° with respect to the incident beam 111, for the measurement of the light scattered by each drop.

Advantageously, the reaction medium forming each drop is loaded with an optically active product, for example fluorescent. Thus, each drop receiving the incident beam is able to emit a reflected beam 118 of wavelength advantageously between 400 nm and 800 nm. This reflected beam 118 is therefore able to be transmitted through the mirror 112. A second detector 104, placed facing the mirror 112 on the same side of the central axis AA 'as the mirror 112, is adapted to receive the reflected beam 118. , and thus to detect the passage of each drop in the measuring capillary section 110. The detectors 102 and 104 comprise, for example, a photodiode or a photon multiplier tube. The storage and detection unit 50 thus makes it possible both to detect and to reference each drop during its multiple passages in the channel 52, and to follow the reactions taking place in each drop over time. The control module is able to control the modules 3 and 5, in particular the means for circulating the different fluids, in particular the valves and the syringes, and to receive and process the information coming from the detectors 102 and 104 in order to return the results of the measurements. A reaction tracking method according to an embodiment of the invention, implemented by means of the tracking system will now be described with reference to FIG. 3. This method comprises the generation by the module 3 of a train ordered by drops of reaction medium within the carrier fluid fp, in the capillary reaction tube 39 For this, during a step 120, the module 3 generates a flow of reaction medium in the capillary tube 33. 25 This step 120 comprises a phase 122 of generation by the unit 13 of a stream of reagents, formed of a succession of gradients of reagents, in the injection capillary tube 17. During this phase 122, the circulation means generate successively a flow rate of each of the reactants n from each of the reservoirs RI in the capillary tube 21 for injecting reagent to the mixing channel 29, by inserting a flow of cleaning liquid n from the reservoir N into the capillary tube reagent injection area 21 to the mixing channel 29 after each flow of a TI reagent. Concomitantly, the circulation means generate a flow of solvent s in the capillary tube 27 of solvent injection, from the tank S of solvent 35 to the mixing channel 29.

The reagent streams r, and the cleaning liquid n successively arrive at the mixing channel 29, in which they mix with the solvent s undergoing Taylor-Aris diffusion. Thus, the mixing channel 29 provides at its output a liquid stream of reagents, composed of a succession of reactant gradients r, between which are intercalated liquid streams composed of a mixture of solvent and cleaning liquid, not containing none of the reagents r ,. For example, the concentration of each of the reactants r varies linearly within a gradient between a minimum concentration and a maximum concentration.

During the phase 122, the relative flow rates of the various reagents with respect to the solvent flow rate can be adjusted independently of each other, so as to precisely adjust the minimum and maximum concentration of each reagent r, in each drop of the train. elementary associated. The phase 122 is followed by a generation phase 124 by the unit 14, in the capillary tube 33, of the flow of the reaction medium, by mixing the flow of reagents with a flow of culture medium m coming from the reservoir M. When of this phase 124, the circulation means 34 generate a flow rate of culture medium m via the T-junction 37 in the capillary tube 33, in which the flow of culture medium m mixes with the flow of reagents, therefore mixing successively with each of the reagent gradients. Then, during a step 126, the unit 15 generates a stream of drops from the reaction medium stream from the capillary tube 33, a carrier fluid flow fp and a separation fluid stream fs. During this step 126, the circulation means 41 generate a flow of carrier fluid fp towards the capillary reaction tube 39, and cause an injection of individual drops of reaction medium from the capillary tube 33 into the capillary reaction tube 39 , within the carrier fluid fp immiscible with the reaction medium. In addition, after each injection of a drop of reaction medium, the circulation means 41 cause an injection of at least one individual drop of separation fluid fs into the capillary reaction tube 39, within the carrier fluid fp . Thus, at least one drop of separation fluid is interposed between two drops of reaction medium, preventing their coalescence. Thus, during step 126, a train of drops of reaction medium composed of a succession of elementary trains each associated with a reagent r is formed in the reaction capillary tube 39, each elementary train being formed of a succession of drops containing the reagent r, at a given concentration. Within each elementary stream of drops, the concentration of reagent r varies from one drop to another according to a gradient, for example linearly.

Two successive drops of reaction medium are separated by a drop of separating fluid fp. In addition, two successive elementary trains are separated in the capillary reaction tube 39 by an intermediate train comprising several drops formed by mixing culture medium m, solvent s and cleaning liquid n, and traces of the reagent or reagents. (s) of the preceding elementary train.

Then, during a sorting step 132, the drop train is transferred from the drop train generation module 3 to the drop circulation circuit 48 through the pinch valve 52 and the T-junction 54. their passage through the junction 54, the drops are sorted either to the waste tank Wl, or to the circuit 48 of circulation of drops and detection, depending on their content, determined from the position of these drops in the train . In particular, when drops of an elementary stream of drops arrive at the junction 54, the valve 56 is closed, so that the drops are oriented towards the circulation circuit 48 of the drops. On the contrary, when drops of an intermediate gear arrive at the junction 54, the valve 56 is open, so that these drops are oriented towards the waste tank W1 to be removed. Thus, the train of drops received by the storage unit 50 is composed of a succession of elementary streams of drops of reaction medium comprising at least one reagent. During a step 134, the train of drops is transferred into the channel 52. For this purpose, the valves 74, 76 and 96 are closed, and the valve 98 is open, which forces the train of drops to pass, to through the cross junction 60, in the detection channel 52. The entire flow circulation circuit 48 is preferably initially loaded with carrier fluid. Thus, during step 134, the carrier fluid initially contained in the channel 52 is driven by the train of drops through the junction 68, the valve 98 and the junction 90, to the waste tank W2. Once the entire set of drops has been transferred into the detection channel 52, the drop train is subjected in a measuring step 136 to a reciprocating movement in this channel 52, so that each drop makes multiple passes in the measuring capillary section 110.

During this movement back and forth, the valves 52, 96 and 98 are closed.

This step 136 comprises a plurality of sequences composed of a forward phase 136a, during which the train of drops flows in the channel 52 towards the output 52b, and a return phase 136b, during which the train of drops flows in the channel 52 towards the entry 52a.

In the forward phase 136a, the valve 84 is open, so that the carrier fluid fp contained in the reservoir 72 is maintained at the pressure Po by the pressurizing means 80. Furthermore, the valve 82 is closed, and the second pressurizing means 86 are controlled to apply a pressure P, greater than Po, on the tank 70. The valves 74 and 76 are also open, so that it is created a positive pressure difference between the reservoir 70 and the reservoir 72, driving the carrier fluid from the reservoir 70 to the reservoir 72 through the channel 52. Indeed, the application of the pressure P on the reservoir 70 thus generates a flow of carrier fluid fp from this reservoir 70 to the cross junction 60. This flow of carrier liquid fp is guided to the detection channel 52, the valve 96 preventing this flow to be driven to the waste tank W2. This flow thus causes the drop train to move in the channel 52 from the inlet 52a to the outlet 52b, the whole train of drops being held in the channel 52. During this movement, the drop train expels fluid carrier, the valves 76 and 98 guiding this flow of carrier liquid leaving the channel 52 to the reservoir 72. During the return phase 136b, the valve 82 is open, so that the carrier fluid fp contained in the reservoir 70 is maintained at the pressure Po by the means 78 of pressurization. Furthermore, the valve 84 is closed, and the second pressurizing means 86 are controlled to apply a pressure P, greater than Po, on the reservoir 72. The valves 74 and 76 are also open, so that it is created a positive pressure difference between the reservoir 72 and the reservoir 70, driving the carrier fluid from the reservoir 72 to the reservoir 70 through the channel 52. The application of the pressure P on the reservoir 72 generates a flow of carrier fluid fp from this reservoir 72 to the T-junction 68. This flow of carrier liquid fp is guided towards the detection channel 52, the valve 98 preventing this flow from being driven towards the waste tank W2. This flow thus causes the drop train to move in the channel 52 from the output 52b to the input 52a, the entire drop train being maintained in the channel 52.

During this movement, the drop train expels the carrier fluid from the channel 52, the valves 96, 52 and 74 guiding this flow of carrier liquid exiting the channel 52 to the reservoir 70. In each of the forward phases 136a and 136b return, each of the drops passes into the measurement capillary section 110. The source 100 continuously emits a coherent incident beam 111 directed, after reflection on at least one mirror 112, and after focusing by an objective 114, towards the measuring capillary section 110. The detector 102 receives the output beam 116 resulting from the crossing by the incident beam of the measurement capillary section 110, and determines, from this output beam 116, the optical density of the fluid passing through the measuring capillary section 110. in particular, when a drop passes through the measurement capillary section 110, the detector thus determines the content of this drop, in particular its biomass.

Moreover, when a drop passes through the measurement capillary section 110, it fluoresces a reflected beam 118, which is transmitted through the mirror 112 and received by the detector 104. The detector 104 thus detects the passage of each drop in the measuring capillary section 110. During these round trips, each drop is labeled by its position in the train. In step 136, the drop train remains entirely in the channel 52, so that it is theoretically possible to perform as many measurements in time as necessary on each of the drops. The storage and detection unit 50 thus makes it possible both to detect and to reference each drop during its multiple passages in the channel 52, and to follow the reactions taking place in each drop over time. For example, in performing multiple antibiograms, the concentration of bacteria and antibiotics in each drop is determined at least every 15 minutes.

Thus, for each concentration of each antibiotic, there is available at the end of the experiment a set of kinetic data reflecting the effect of the antibiotic at this concentration on the bacterium. Such measurements make it possible to determine the minimum inhibitory concentration of each antibiotic, corresponding to the minimum concentration from which the bacteria do not develop in the culture medium.

At the end of the experiment, during a step 140, the train of drops is discharged from the channel 52 by the branch 66, to the waste tank W2. For this purpose, the valves 96, 82 and 84 are closed while the valves 74 and 98 are open.

The second pressurizing means 86 are controlled to apply a pressure P on the reservoir 70, generating a flow of carrier fluid fp from this reservoir 70 to the cross junction 60, then through the channel 52 from the inlet 52a to the outlet 52b, until the entire set of drops flows via the valve 98 into the waste tank W2.

During this movement, the drop train expels carrier fluid, the valves 76 and 98 guiding this flow of carrier liquid leaving the channel 52 to the reservoir 72. The method according to the invention thus allows to study accurately, reproducibly and automatically growth kinetics of aerobic and anaerobic bacteria based on one to several thousand parameters in parallel.

It should be understood that the exemplary embodiment presented above is not limiting. In addition, the process according to the invention is not limited to the production of antibiograms.

Claims (16)

REVENDICATIONS1. A process for monitoring the kinetics of reactions, in a reaction medium, between at least one component of said reaction medium and each of a plurality of distinct reactants (r 1), characterized in that it comprises: - generation (120, 126) of an ordered train of drops in a carrier fluid (fp) to form a plurality of reactors, the drop train comprising a succession of elementary streams of drops, each associated with at least one reagent (r,) of said plurality of reagents (r,), each drop of an elementary stream comprising the or each reagent (r,) associated with that elementary stream, - circulating (136) at least a portion of said ordered stream of drops - the measurement of at least one parameter representative of each reactor over time. 2. Method according to claim 1, characterized in that each elementary stream comprises at least one reagent (r,) not included in an adjacent elementary stream. 3. A method according to claim 1 or 2, characterized in that the generation (120, 126) of the ordered train of drops comprises the generation (126) of a plurality of elementary streams of successive drops, each associated with at least one reagent (r) of said plurality of reagents (r,), between which intermediate drip trains are interposed, and deletion (132) of said intermediate drip trains. 4. A process according to claim 1 or 2, characterized in that within at least one elementary stream of drops, the concentration of at least one reagent (r) varies, in particular according to a gradient. 5. Method according to any one of the preceding claims, characterized in that the generation (120, 126) of the ordered stream of drops comprises the following steps: (a) the generation (120) of a flow of reaction medium, (b) filling a capillary reaction tube (39) with a carrier fluid (fp) immiscible with the reaction medium, (c) injecting (126), by means of a capillary injection tube (33) , an individual drop of reaction medium in the reaction capillary tube (39), (d) circulating the carrier fluid (fp) so that the drop of reaction medium moves relative to the capillary injection tube ( 33), (f) repeating steps c) and d) to create an ordered train of drops of reaction medium within the carrier fluid (fp). 6. A process according to claim 5, characterized in that the step of generating (120) the reaction medium stream comprises: the formation (122) of a stream of reagents comprising a succession of elementary fluxes of reagents (r ,), each elemental stream of reagents comprising at least one reagent (r,) of said plurality of reagents (r,), - mixing (124) of said reagent stream with a culture medium (m) comprising said component for forming said flow of reaction medium. 7. A process according to claim 6, characterized in that the formation (122) of a stream of reagents comprises the following phases: (a) the generation of a continuous flow of a solvent (s) in a channel of mixture (29), ([3) concomitantly with the phase a), the injection, thanks to a capillary tube (21), of a reagent (r,) chosen from the plurality of reagents (r,) in said channel mixing (29), to form an elementary stream of reagent, (y) repeating the phase (3) for each of the reagents (r,) of the plurality of reagents (r,) successively. 8. A process according to claim 7, characterized in that the formation (122) of the reagent stream comprises, after the formation of each elemental stream of reagent and before the phase y), a phase 8) of injection, thanks to a capillary tube (21), a cleaning liquid (n) in said mixing channel (29). 9. A method according to any one of claims 5 to 8, characterized in that the generation (120, 126) of the ordered train of drops further comprises, after step d) and before step f), step e) comprising injecting, in the carrier fluid (fp), a drop of a separation fluid (f,), immiscible with the carrier fluid (fp) and immiscible with the reaction medium, so at least one drop of separation fluid (f5) is interposed between two reactors and prevents their coalescence. 10. A process according to any one of the preceding claims, characterized in that said component is a microorganism and said culture medium (m) is a culture medium of microorganisms, and in that said parameter is representative of the amount of microorganism present in each reactor. 11. The method of claim 10, characterized in that said reagents are antibiotics, and in that said method comprises performing an antibiogram to test the sensitivity of said microorganism to said antibiotics. 12. System (1) for monitoring the kinetics of reactions, in a reaction medium, between at least one component of said reaction medium and each of a plurality of distinct reagents (r 1), characterized in that it comprises: a generation module (3) for an ordered train of drops in a carrier fluid (fp) for forming a plurality of reactors, the drop train comprising a succession of elementary streams of drops, each associated with a reagent (r,) of said plurality of reactants (r,), each drop of an elementary train comprising the reagent (r,) associated with this elementary stream, - a module (5) for storage and detection of drops, suitable for circulating at least a portion of said ordered stream of drops and measuring at least one parameter representative of each reactor over time. 13. System according to claim 12, characterized in that the generation module (3) of the ordered train of drops comprises: - means (13, 14) for generating a flow of reaction medium, - means for filling a capillary reaction tube (39) with a carrier fluid (fp) immiscible with the reaction medium, from a reservoir (Fp) of fluid carrier, - means (15) for injecting, by means of a capillary injection tube (33), an individual drop of reaction medium into the capillary reaction tube (39), - means for circulating the carrier fluid ( fa) so that the drop of reaction medium moves relative to the capillary injection tube (33). 14.- System according to claim 13, characterized in that the means (14) for generating a reaction medium stream comprise: - means (13) for forming a stream of reagents comprising a succession of elementary fluxes of reagents ( r)), each elemental reagent stream comprising at least one reagent (r) of said plurality of reagents (r); - means (14) for mixing said reagent stream with a culture medium (m) comprising said reagent stream; component for forming said reaction medium stream. 15. The system of claim 14, characterized in that the means (13) for forming the flow of reagents comprise a plurality of reservoirs (R) of reagent, and means for circulating each reagent (r, ) successively from each of said reservoirs (R,). 16.- System according to any one of claims 12 to 14, characterized in that said module (5) for storing and detecting drops comprises a fluid circulation circuit drops (48, 50), and means for placing circulating at least a portion of said ordered stream of drops in said circuit (48, 50) by generating a pressure difference (P-P0) between two points of said circuit (48, 50).