FR2799139A1 - Microfluidics device with continuous flow system, useful e.g. for nucleic acid amplification or sequencing, providing temperature cycling of sample - Google Patents

Abstract

A device (A) comprising a microfluidics substrate with at least one pathway for sample flow and at least one heat transfer member (HTM) that can cycle at least part of the sample between at least two temperatures while it flows continuously along the pathway, is new. Independent claims are also included for the following: (1) performing a (bio)chemical process by cycling a flowing sample, containing reagents, between at least two temperatures; (2) performing a biochemical process by injecting at least one reagent, from a reservoir, into a channel carrying a continuous flow of solution containing a sample, and transferring heat between a thermal support and a temperature-regulated part of the channel; (3) performing a temperature cycle on a continuous flow of solution containing a sample; (4) amplifying nucleic acids, comprising: (a) mixing at least one sample comprising the nucleic acids with reagents; (b) feeding at least one channel with a continuous flow of the mixture; (c) running at least one mixture through at least one temperature regulated zone; and (d) cycling the zone through a temperature cycle of at least two temperatures in a predetermined series, so that the nucleic acid undergoes denaturation-hybridization-elongation cycle; and (5) detecting at least one nucleotide in target nucleic acid, comprising: (a) feeding a channel with a continuous flow of a target nucleic acid solution; (b) injecting a reagent for amplifying a region of the nucleic acid which carries the nucleotide to be detected into a channel from a reagent reservoir; (c) running the solution through at least one temperature regulated zone so that the nucleic acid is amplified; (d) injecting the reagent for purifying the amplification product into the channel from a second reagent reservoir; (e) running the solution through at least one temperature regulated zone, to carry out purification; (f) injecting the microsequencing reagent comprising buffer, at least one microsequencing primer, at least on ddNTP and a polymerase into the channel from a third reagent reservoir; (g) running the reaction mixture through at least one temperature regulated zone to produce at least one cycle comprising the denaturation of the target nucleic acid, the hybridization of the nucleic acid with the primer, and the incorporation of the ddNTP which is complementary to the nucleotide to be detected at the 3' end of the primer; and (h) detecting the incorporated ddNTP.

Description

Microfluidics consists of using microchannels instead of microplate test tubes to perform analyzes and reactions. These microchannels or microcircuits are etched in silicon, quartz, glass, ceramics, plastic. The size of these channels is of the order of a micrometer while the reaction volumes are of the order of nanoliter or microliter. The principle is to route the reaction media containing reagents and samples on areas corresponding to the different steps of the protocol. The integration of reactors, chromatographic columns, capillary electrophoresis systems and miniature sensing systems in these microfluidic systems allows the automation of complex protocols integrating them into a single system. These on-chip laboratories have made it possible to obtain efficient results in terms of speed of reaction, in terms of product savings and in terms of miniaturization allowing the development of portable devices. Remarkable results have also been achieved in the integration and automation of complex protocols such as biochemical or molecular biology protocols that often require many manipulations. These manipulations comprise in particular the mixing of reagents and samples, the control of the reaction temperature, the carrying out of thermal cycling, the separation by electrophoresis and the detection.

 Wolley et al. <I> (Anal. </ I> Chenz., 68, 408l-4086, <B> 1996) </ B> thus describe the integration of a PCR microreactor, a Capillary electrophoresis and a detector in a single device. The PCR reaction, the separation of the products by electrophoresis and their detection are carried out automatically. However, this device does not integrate the mixture of reagents and it does not allow the implementation of large-scale protocols.

A device-on-a-chip device for integrating a step of mixing the reagents and an enzymatic reaction has been described by Hadd et al., (Anal. </ I> Chem., 69, 3407-3412, 1997). ). This device provides a microcircuit of channels and tanks etched in a glass substrate. The displacement and the mixing of the fluids is carried out by electrokinetics.

Numerous microfluidic systems for the integration of protocols and analyzes have thus been described in particular in the international patent application WO 98/45481. One of the major difficulties in implementing these devices lies in the movement of fluids. The fluids are usually moved by electroosmosis or electrokinetics which requires an array of electrodes. Other systems use micropumps and microvalves integrated into the microfluidic substrate. In the majority of the cases, the reactions are carried out at a standstill in a microreactor and the fluids are thus displaced from one reactor to another at each stage of the protocol. These systems incorporating electrodes, microvalves or micropumps are very expensive and their complexity allows large-scale applications to simultaneously process a very large number of samples. One of the major difficulties is the distribution, mixing and transport of a very large number of products in parallel or in series.

The present invention relates to a device comprising a microfluidic substrate comprising at least one microchannel in which reactions or successions of reactions comprising a protocol are carried out. The channel is fed with continuous flow and reagents are injected successively at different stages of the protocol. The device is based on a fluid distribution and displacement system by hydrostatic pressure. All the steps of the protocol are carried out in continuous flow, sequential injections of samples and reagents make it possible to carry out a large number of reactions in a single channel. By arranging several channels in parallel it is possible to perform the same protocol in series in the same channel in parallel in different channels. The synchronization of the reactions in the channels arranged in parallel makes it possible to distribute the reagents simultaneously in the different channels. This arrangement finds a particularly advantageous application in rationalizing and reducing the number of distributions to be made. The combination of the microfluidic substrate with a thermal support makes it possible to control the reaction temperature in the different zones of the channel corresponding to the different steps of the protocol. The invention also relates to advantageous devices and methods for carrying out continuous flow thermal cycling.

The microfluidic substrate is preferably semi-disposable (used for a few hundred reactions or a few tens of hours) and removably attached to the thermal support, the fluid supply devices and the detection means. Temperature control, fluid displacement, reagent injection, continuous flow mixing and detection are fully automated. In addition the fixed device combination and a disposable microfluidic but inexpensive substrate allows a significant reduction in costs compared to systems in which everything is integrated on the same chip. SUMMARY OF THE INVENTION The subject of the present invention is a device comprising a microfluidic substrate comprising at least one microchannel in which reactions or successions of reactions comprising a protocol are carried out. The biochemical analysis device according to the invention comprises a microfluidic substrate comprising at least one channel having a feed cup for injecting sample and an outlet cup for collecting said sample; at least one reagent reservoir connected to each channel between the feed bowl and the outlet bowl; and means for supplying said channel with a continuous flow. By feed bowl and outlet bowl is meant any type of tank, fitting or orifice at the inlet and outlet of the channel. The term microfluidic substrate refers to a solid support in which channels, cuvettes or reservoirs are micro-manufactured. The solid support may consist of a polymeric material such as polymethylmethacrylate (PPMA), polycarbonate, polytetrafluoroethylene (TeflonTM), polyvinylchloride (PVC), polydimethylsiloxane (PDMS) and polysulfone. According to another embodiment of the invention the solid support is made of glass or quartz. According to an advantageous embodiment of the invention, the microfluidic substrate is made of silicon. The techniques of microfabrication and silicon etching are well known also allowing the manufacture of channels with dimensions and well-defined geometries. These techniques for machining the silicon substrate include, in particular, chemical etching techniques. Silicon also has advantages because of thermal properties. In contact with a thermal source silicon response times are very fast. According to a particularly advantageous embodiment of the invention the microfluidic substrate comprises a plurality of channels arranged in parallel, each feed bowl being respectively connected to an outlet bowl by means of the channel. Typically several hundred channels are arranged in parallel on the same microfluidic substrate. A given protocol can therefore be performed in parallel in each channel. It is also possible to perform different protocols in each of the channels. However, to facilitate in particular the automation of the injection of reagents more advantageous to perform the same protocol in all channels.

The device according to the invention comprises means for supplying at least one channel continuously. Various methods have been described for moving fluids in microfluidic substrates. Techniques such as electroosmosis and electrophoresis are thus widely used in microfluidics. In a preferred embodiment of the invention, the means for feeding said continuous flow channels apply a pressure difference between the feed bowl and the outlet bowl. The injection of fluids (samples and reagents) by pressure force allows precise control of flow rates. The flow rate imposed by pressure is independent of the composition of the fluids. Whereas in the case of electrophoresis and electrosmosis, the salt concentration and the viscosity of the solutions complicate the control of flow rates. The use of pressure also makes it possible to use channels of variable geometry. In addition, the control of fluids by pressure does not rely on a complex electrode system unlike electrophoresis and electroosmosis. In the device according to the invention, the set of poor means for injecting fluids into the microfluidic substrate and for controlling the flow of the fluids are preferably independent of the microfluidic substrate and removably attached thereto. .

In an advantageous embodiment of the invention the flow rate and the injection of fluids into the plurality of channels arranged in parallel are synchronized. This synchronization is necessary to automate and integrate the different steps of the protocol. For example, the reagents can thus be injected simultaneously into all the channels. In a particularly advantageous embodiment, the channels are fed in series with samples separated from one another by plugs. This plug separating two samples may consist of a buffer solution or reagents containing no sample, preferably this plug consists of an immiscible inert fluid. A multitude of series (or sequential) reactions can thus be carried out in a single channel of the microfluidic substrate. By way of example, the number of samples injected in series into the channel is between 1-100. The device according to the invention thus makes it possible to carry out large-scale protocols on a large number of samples since the samples are processed both in parallel and in series. In general, the injection of the samples and reagents into the microfluidic substrate and the movement of the fluids in continuous flow are obtained either by applying a positive inlet pressure or by applying a negative pressure at the outlet. Any device for applying such pressure is usable, such devices are known elsewhere. The supply of the samples to be analyzed and the placing of the reagents in the reservoirs can take place by micro-pipetting. In a particular embodiment of the invention the fluid injections in the microfluidic substrate are made by pneumatic injection. Samples or reagents are deposited in reservoirs connected to at least one channel. The injection into the channels is effected by applying a pressure of a gas or a non-miscible incompressible liquid on the reservoir. Such a pneumatic injection system can be used to impose a precise flow all the channels.

In another embodiment of the invention, the syringe-shifter systems arranged in parallel make it possible to regulate the flow rates in the channels. These syringe shoots can be placed either at the entrance or at the outlet of the channel.

In a preferred embodiment of the invention, the supply of fluids is carried out with an injection device as described in the patent application Device for injecting fluids into a microfluidic microsystem No.

The supply of fluids onto the microfluidic substrate can be obtained by a combination of the modes described above. For microfluidic substrates with a large number of channels the supply of fluids can be automated by means of a dispensing robot or dispensing high resolution. Moreover, serial or sequential injections supposing the replacement in time of at least one solution by another can be automated by the sequential introduction into the corresponding reservoir of several distinct solutions. Between two solutions an immiscible neutral buffer can be introduced into the reservoir. The precise control of the reaction temperature is a determining factor in the realization of chemical, biochemical and biological protocols.

In a preferred embodiment of the invention the device comprises at least one heat carrier having a heat exchange face in contact with at least one side of the microfluidic substrate so that the sample flowing through the channel is brought to a predetermined temperature. . In an advantageous embodiment of the invention, the microfluidic substrate is removably attached to the thermal support. In the realization of complex protocols, it is often essential to carry out different steps or reactions at predetermined distinct temperatures. Control of the reaction temperature is particularly important for enzymatic reactions that must be performed at precise temperatures. The device according to the invention makes it possible to produce protocols comprising different steps at different fixed temperatures as well as protocols comprising thermal cycling steps. The ability to perform such temperature cycles is particularly important for all nucleic acid amplification reactions.

The thermal support has a heat exchange face in contact with a face of the microfluidic substrate in the vicinity of which are the channels. Preferably, the face of the microfluidic substrate in contact with the thermal support has a thin wall. More specifically, the wall is chosen to be sufficiently thin to allow thermal exchange with the thermal support external to the microfluidic substrate. In particular, the wall separating the channels of the thermal support may be chosen thinner than a wall separating the channels between them or tanks. According to another aspect of the invention, the face of the channels opposite to the thin wall may have a thermal barrier, which can be made by a layer of low thermal conductive material and / or structuring of the substrate to locate the channels cavity filled with air or a low heat-transfer gas. This thermal barrier makes it possible to standardize the temperature in the channels.

 thermal support comprises at least one thermostated zone but it can also comprise several thermostated zones brought to different temperatures. The different thermostated zones are arranged to coincide with channel portions of the analysis support. These thermostated zones define zones in which a specific step of the protocol is realized. The thermostated zones coincide for example with at least one zone of the microfluidic substrate located downstream of a connection between a reagent reservoir and a channel. By associating, for example, a thermostatically controlled zone of the thermal support with a corresponding zone of the microfluidic substrate, for example downstream of each reagent reservoir, it is possible to selectively control and adapt the temperature of the reaction mixture circulating in the channel depending on each reagent. used and depending on the reaction carried out. The term "downstream" used herein refers to the flow direction of the samples from the feed bowl to the outlet cuvette. In one embodiment of the invention the thermal support comprises at least two thermostatically controlled zones at different temperatures arranged so that the sample traversing the channel is successively carried to at least two predetermined temperatures. The temperature can be regulated by using different temperature-controlled zones at constant temperature or by varying the temperature of the thermostated zone. The reaction mixture traversing the channel is thus carried successively to the different temperatures required.

also provides according to the invention a device comprising at least one thermostatically controlled zone brought to at least two different temperatures so that the sample traversing once this zone is brought to at least two predetermined temperatures. Alternatively, the device according to the invention comprises at least one thermostated zone brought to at least two distinct temperatures in a temporal succession forming a predetermined cycle so that the sample undergoes at least one temperature cycle by traversing the zone once. thermostatically controlled. Preferably, the portion of the channel passing through the thermostated zone is rectilinear.

Different systems can be envisaged for the thermal support and the thermostated zones. In a first embodiment of the invention Peltier effect elements are used for thermal support. These Peltier effect elements are known elsewhere. Different metal junctions crossed by an electric current can cool or heat a small area. A temperature probe on the Peltier element regulates the power, which is proportional to the electrical intensity and therefore allows the temperature to be regulated. Alternatively or additionally the thermal support may also include one or more channels through which a heat transfer fluid. This fluid can be used to heat or cool the analysis medium locally.

Preferably, the heating means and the means for injecting and controlling fluids are not integrated in the microfluidic substrate. The microfluidic substrate is releasably attached to these heating and fluid injection elements. This characteristic makes it possible to reduce the cost of the microfluidic substrate to a significant extent. Thus, this support can be of the single-use or multi-use type, that is to say be discarded after one or more uses. By use is meant the realization of a large-scale serial protocol and in parallel on the channels of the microfluidic substrate.

In a particularly advantageous aspect of the invention, the device according to the invention comprises detection means for measuring a physicochemical characteristic of the samples traversing the channels. Preferably, these detection means are located at the outlet cuvettes of said channels. In another embodiment, the detection means are located directly at the level of the channels and the detection is carried out in continuous flow. Advantageously, the injection of the samples, the addition of reagents, the solution mixture, the regulation of the reaction temperature and the detection are automatable in the device according to the invention. The device according to the invention is therefore totally integrated system in which the manual interventions are reduced. Different protocols can thus be fully integrated in the continuous flow device according to the invention. The invention also relates to the integration of complex protocols in a microfluidic continuous flow device and to the production of continuous flow protocols. According to the invention, there is provided a method for carrying out biochemical protocols on at least one sample, characterized in that it comprises the following steps: a) feeds a continuous flow channel with a solution containing at least one sample by applying a difference of pressure between the feed bowl and outlet cuvette of said channel; b) injecting at least one reagent from at least one reagent reservoir into said channel so as to mix said sample and said reagent. The sample can then be taken from the outlet cuvette of said channel. In a particular embodiment of the invention the method for carrying out biochemical protocols on at least one sample is characterized in that it comprises the following steps: a) feeds a continuous flow channel with a solution containing at least one sample applying a pressure difference between the feed bowl and outlet cuvette of said channel, b) injecting at least one reagent from at least one reagent reservoir into said channel so as to mix said sample and said reagent, c) at least one physicochemical parameter of said sample is detected in said channel. The methods according to the invention may furthermore have at least one of the following characteristics: the solution travels at least one thermostatically controlled zone so that the solution is brought to a predetermined temperature as it traverses said thermostated zone; - The solution travels at least one thermostatically controlled zone carried successively to at least two predetermined temperatures so that the solution is put successively said temperatures when running once said thermostated zone; - The solution travels a thermostatically controlled zone successively to at least two determined temperatures in a time sequence forming a predetermined cycle so that the solution undergoes at least once the temperature cycle by traversing once the thermostated zone; - The solution undergoes 1-35 times the temperature cycle by browsing once the thermostated zone. The control of temperature and the carrying out of thermal cycling are essential elements for all enzymatic reactions and in particular for nucleic acid amplification reactions. The methods in accordance with the invention make it possible to integrate the mixture of samples and reagents as well as the various heat treatments and do not require any manipulation. To carry out protocols on a very large scale, the methods according to the invention will preferably comprise one of the following steps: the channel is fed sequentially with a plurality of samples separated from the others by plugs consisting of an immiscible inert fluid. The process steps are performed simultaneously on a plurality of channels arranged in parallel. The processes according to the invention are also very advantageous for any reactions involving temperature cycling. For the purpose of carrying out such protocols, it is provided according to the invention a process for the continuous flow of at least one temperature cycle on a solution containing at least one sample, characterized in that it comprises the following steps: channel in continuous flow with said solution containing at least one sample, said solution is passed through a thermostatically controlled zone carried successively at at least two determined temperatures in a time sequence forming a predetermined cycle so that the solution undergoes at least once the temperature cycle by browsing once the thermostated zone. In another embodiment of the invention there is provided a method for the continuous flow of at least one temperature cycle on a solution containing at least one sample, characterized in that it comprises the following steps: a) feeding a channel in continuous flow with said solution, b) is passed to said solution a thermostatic zone carried successively 'at least two predetermined temperatures in a time sequence forming a predetermined cycle so that the solution undergoes at least once the cycle of temperature by browsing once the thermostated zone. c) detecting at least physico-chemical parameter of said sample downstream of said channel.

Preferably, it can be provided that the channel is fed with continuous flow by applying a pressure difference between the feed bowl and the outlet bowl of said channel.

Preferably it is expected that the solution undergoes 1-3S times the temperature cycle by traversing once the thermostated zone.

In order to carry out thermal cycling protocols on a very large scale, the methods according to the invention will preferably comprise one of the following steps: the channel is fed sequentially with a plurality of samples separated from each other by plugs <B> - < The different process steps are performed simultaneously on a plurality of channels arranged in parallel.

The methods according to the invention are also very advantageous for all nucleic acid amplification techniques.

In a particular embodiment of the invention, the process for the amplification of nucleic acids according to the invention is characterized in that it comprises the following steps: a) said nucleic acid is mixed with reagents suitable for the nucleic acid amplification, b) a continuous flow channel is fed with the reaction mixture, c) a thermostatically controlled zone is passed through the reaction mixture successively at predetermined temperatures in a temporal succession forming a predetermined cycle, d) one chooses the temperatures and the duration of the temperature cycle of the thermostated zone as well as the time taken for the mixture to travel through the thermostated zone so that the nucleic acid undergoes several times the denaturation-hybridization-elongation cycle. In another particular embodiment of the invention, the process for the amplification of nucleic acids in accordance with the invention is characterized in that it comprises the following steps: a) a continuous flow channel is supplied with a solution comprising said nucleic acid, b) a suitable reagent for the amplification of nucleic acids from at least one reagent reservoir in said channel is injected less so as to mix said nucleic acid and said reagent; this reaction mixture a thermostatically controlled zone carried successively to temperatures determined in a time sequence forming a predetermined cycle, d) temperatures and the duration of the thermostatically controlled zone temperature cycle are selected, as well as the time taken by the mixture to travel through the thermostatically controlled zone. such that the nucleic acid undergoes the denaturation-hybridization-elongation cycle several times. The processes according to the invention may furthermore have at least one of the following characteristics: a continuous flow channel is supplied by applying a pressure difference between the feed bowl and the outlet bowl of said channel. the channel is formed in a silicon substrate. said channel is sequentially supplied with a plurality of nucleic acids separated from each other by plugs; steps a), c) and d) are performed simultaneously on a plurality of channels arranged in parallel. The methods and devices in accordance with the invention make it possible to integrate in a continuous flow large scale genotyping protocols and in particular microsequencing protocols. For the continuous detection of at least one nucleotide in at least one target nucleic acid, there is provided a method characterized in that it comprises the following steps: a) feeding a continuous flow channel with a solution comprising said nucleic acid ; b) injecting the microsequencing reagent comprising the microsequencing buffer, the microsequencing primer, at least one ddNTP and a polymerase into said channel so as to mix said nucleic acid and said reagent; c) the solution is traversed at least one thermostated zone so as to obtain at least one cycle comprising the denaturation of the target nucleic acid, the hybridization of said nucleic acid with the microsequencing primer and the incorporation of the ddNTP, complementary to the nucleotide to be detected, at the 3 'end of said primer; d) at least one ddNTP incorporated at the 3 'end of the microsequencing primer is detected.

 Optionally, this microsequencing method according to the invention can comprise at least one of the following steps: feeds the continuous flow channel by applying a pressure difference between the feed bowl and the outlet bowl of said channel.

- Thermostated zone is carried successively to temperatures determined in a time sequence forming at least one cycle.

the ddNTPs are labeled with fluorophores and in step d) the fluorescence of the incorporated ddNTP is detected.

steps a), b), c) and d) are performed simultaneously on a plurality of channels arranged in parallel.

Advantageously, the microsequencing protocol is preceded by an amplification reaction and a purification reaction of the amplification product. All these reaction steps are integrated in the same microfluidic device according to the invention.

The method for the continuous detection of at least one nucleotide in at least one target nucleic acid is characterized in that it comprises the following steps: a continuous flow channel is supplied with a solution containing at least one target nucleic acid; the reagent is injected for amplifying the region of the target nucleic acid carrying at least one nucleotide to be detected, in said channel from a first reagent reservoir; the solution is passed through the at least one thermostated zone so that the nucleic acid undergoes several times the denaturation-hybridization-elongation cycle; the reagent is injected for purification of the amplification product in said channel from a second reagent reservoir; e) at least one thermostated zone is passed through the solution to carry out the purification reaction; f) the microsequencing reagent comprising the microsequencing buffer, the microsequencing primer, at least one ddNTP and a polymerase is injected into said channel from a third reagent reservoir; g) the reaction mixture is made to travel at least one thermostated zone so as to obtain at least one cycle comprising the denaturation of the target nucleic acid, the hybridization of said nucleic acid with the microsequencing primer and the incorporation of the ddNTP, complementary to the nucleotide to be detected at the 3 'end of said primer; h) detecting at least one ddNTP incorporated at the 3 'end the microsequencing primer. The method according to the invention may further comprise the following steps - the continuous flow channel is supplied by applying a pressure difference between the feed bowl and the outlet bowl of said channel. - In steps c) and e) the thermostated zone is carried successively at predetermined temperatures in a time sequence forming at least one cycle.

the ddNTPs are labeled with fluorophores and in which at the stage the fluorescence of the incorporated ddNTP is detected. the reagent for the purification comprises an exonuclease and an alkaline phosphatase.

steps a), b), c), d), e), i), g) and h) are performed simultaneously on a plurality of channels arranged in parallel. Another particularly advantageous aspect of the invention is the miniaturization of the reaction volumes. Typically the reaction volumes are of the order of 0.1p1 to 10p1. In a preferred embodiment of the invention the reaction volumes are of the order of 0.51 to 5p1. Preferably the volume of the initial sample is 1-2 p, 1. Other features and advantages of the invention will appear in the description of preferred embodiments given by way of example and not limiting. <U> Detailed Description of Modes of </ U> Work <U> of the Invention </ U> In the following description of identical, similar, or equivalent parts of the figures are marked with the same reference numerals in order to make it easier to read. Microfluidic Substrate FIG. 1 is a sectional view of a microfluidic substrate 100 according to the invention. In this figure is shown an inlet bowl 102 formed essentially by a through opening made in a substrate 100a, in the vicinity of one of these ends. In the same way, an outlet bowl 104 is formed near a second end. The cuvettes 102, 104 open into a first face 106 of the substrate 100. An internal channel <B> 108 </ B> connects the inlet and outlet cuvettes. The channel 108 is in the form of a groove etched in a second substrate 100b bonded to the first substrate so that the latter covers the groove.

It is observed that the depth of the groove is substantially equal to the thickness of the second substrate 100b, so that only a thin wall 110 separates the channel <B> 108 </ B> from a second face 112 of the microfluidic substrate 100. In the example shown, the substrate 100 is of parallelepipedal general shape and the first and second faces are the opposite main faces and parallel. The figure also shows in section reagent reservoirs 120a, 120b, 120c arranged between the inlet or supply bowl 102 and outlet bowl 104. The tanks also open on the first face 106 of the analysis support 100. Fittings or passages 122a are provided for connecting each of the reservoirs to the channel 108. For reasons of simplification, the passages 122a are shown in plan of the figure, so that the reservoirs are not distinguishable from the supply and outlet cuvettes on the FIG. 1B shows a microfluidic substrate according to FIG. 1A, the reservoirs 120a 120b and 120c of which are respectively associated with fluid supply means 150a, 1 and 150c. These means comprise plugs or supply caps 152a, 52b, 152c sealingly applied over the reservoirs and connected to syringe pumps 154a, 154b and 154c which contain reagents. The caps may be glued to the surface of the microfluidic substrate or pressed against the surface using a seal. The references 156a, 156b and 156c respectively designate pressure sensors on lines connecting the syringe pumps to the caps 152a, 152b and 152c so as to control the pressure and / or the flow rate of the reagents. Although not shown, a similar feeding system can also equip the feed bowls or inlet bowls. As shown in FIGS. 1A and 1B, inlet bowls and tanks are subjected to atmospheric pressure, or at a pressure set by the feed system, while a vacuum line 124 is applied to outlet bowls. . Figure 2 shows more precisely and separately the two substrates 100a and 100b which form the microfluidic substrate. It is observed that the microfluidic substrate comprises a plurality of feed bowls 102 and a plurality of outlet bowls 104.

The cuvettes have the form of through openings made in the first substrate 100a. These openings have a flared shape V, forming a funnel. Furthermore, in the example of FIG. 2, each inlet cup 102 is individually connected to an outlet cup 104 via a channel 108. The microfluidic substrate comprises three reagent reservoirs 120a, 120b, 120c. In this example, each common reservoir has several channels <B> 108 </ B> to which it is connected by means of connectors 122a, 122b. The reference 122a more specifically designates bores of the first substrate 110a connecting the reservoir to corresponding branches 122b made in the second substrate 110b and respectively connected to the channels. Of course, tanks can also be individualized for the different channels. The quantities of fluid (samples and reagents) that mix at the crossing junctions 122b and channels 108 depend on the respective size of these branches and channels 108. Thermal support Figure 3 shows a microfluidic substrate 100, in accordance with that of FIG. 2, whose substrates 100a and 100b are permanently bonded. The microfluidic substrate is shown above a corresponding heat support 200. The thermal support 200 has a heat exchange face 212 facing the second face 112 of the microfluidic substrate100, in the vicinity of which are the channels. The heat exchange face 212 has three thermostated zones 220a, 220b, 220c each equipped with one or more heat sources (not shown). The three thermostated zones 220a, 220b, 220c are arranged to coincide with channel portions of the microfluidic substrate located in the vicinity respectively of the reservoirs 120a, 120b, 120c, or more precisely the branches supplying the reagents. FIGS. 4 and 5 show two embodiments of the device making it possible to improve the uniformity of the temperature in the channels by isolating their upper face, ie the face opposite to said second face 112 of the microfluidic substrate. A first solution shown in Figure 6 is to realize in the upper part 110a of the support a cavity 160 (open or not). This cavity coincides with at least a portion of the channel 108. A second solution shown in Figure 5 consists in placing between the upper and lower portions 100a, 100b of the microfluidic substrate a layer 100c of thermal insulating material.

Methods of Manufacturing a Microfluidic Substrate In a particular embodiment of the microfluidic substrate, the latter may comprise a first substrate having through apertures which respectively form the cuvettes, the connectors and the reservoirs and a second substrate, bonded to the first substrate having grooves, covered by the first substrate to form channels and coinciding respectively with the through openings. This particularly simple structure makes it possible to reduce the manufacturing costs of microfluidic substrates. The manufacture of the microfluidic substrate can take place, according to the invention, according to a method comprising the following successive steps forming, in a first substrate, openings through said openings respectively corresponding to feed bowls or outlet cuvettes, or to reagent reservoirs, - forming in a second groove substrate in a pattern for interconnecting at least two openings of the first substrate, - bonding of the first substrate to the second substrate so as to cover the grooves, - thinning of the second substrate after bonding, preserving a substrate thickness greater than a maximum depth of the grooves.

FIGS. 6 to 9, described below, give an example of a process for producing a microfluidic substrate as described above.

In a first substrate plate 110a, for example silicon, it is practiced, as shown in Figure 6, through openings. These through openings constitute the cuvettes or reservoirs 102, 104, 120a, 120b, 120c. The openings etched by chemical means are carried out with flanks inclined by anisotropic chemical etching for example (KOH) so as to give them a flared shape. The location of the openings is defined by an etching mask (not shown) in register with the pattern of the grooves. The drilling of the layer 100c of thermal insulating material, for example SiO 2 in the case of the variant proposed in FIG. 5, may be carried out, for example, by dry CHF3 etching, the size of the perforation being defined by an etching mask. or using the walls of the hole created chemically as a mask.

FIG. 7 shows the etching of grooves forming the channels 108, in a second substrate 100b, for example silicon. Etching is performed through an etching mask (not shown) having a pattern corresponding to the desired channels. This is, for example, a chemical etching (KOH). The depth of the grooves is for example of the order of 100pm for a substrate 100b with a thickness of 250 to 450gm. It can also be a SG6 dry etching for making grooves deeper than wide for example l00p.mx20gm.

A third step shown in FIG. 8 comprises 1p sealing of the first and second substrates 100a and 100b, so as to place the cuvettes or reservoirs 102, 104, 120a, 120b, 120c in communication with the channels (grooves). </ B> correspondents. Sealing takes place, for example, by direct bonding (molecular) of the two substrates.

During this operation, the grooves 108 of the second substrate 100b are covered by the first substrate 100a to form the channels.

A final step, represented in FIG. 9, comprises the thinning of the second substrate 100b so as to preserve between the channel 108 and the outer surface 112 only a thin wall 110.

This wall 110 has a thickness of the order of lOpm so as to promote heat exchange. Thinning is performed by etching and / or mechanochemical polishing.

A plurality of microfluidic substrates according to the invention can be produced simultaneously and collectively according to the above method in two silicon wafers (corresponding to the first and second substrates). In this case, the process is completed by cutting the slices with the saw to individualize the microfluidic substrates. Devices and methods for carrying out biochemical protocols by thermal cycling The devices and methods in accordance with the invention make it possible to carry out a continuous flow of biochemical protocols including a step by thermal cycling. The present invention relates inter alia to the chain polymerization reaction (or PCR), widely used in genetic analysis. The invention which can be used in genetic analysis is however also usable for many protocols in the field of biochemistry and molecular biology.

We will first make brief reminders on the physicochemical properties of DNA and then we will present an element of prior art concerning a device to perform the PCR. DNA is a macromolecule composed of a sequence of four different nucleotides (A, C, G), the four nucleotides are capable of pairing two by two (A: T and G: C). complementary strands are able to hybridize and separate successively to infinity.The double or single-stranded state depends on stringency conditions (pH, salt, temperature) .This is the ability of successive hybridization and denaturation which is used during the PCR reaction (polymerization reaction in chame).

In nature during cell division, DNA is duplicated to ensure the transmission of genetic information. The synthesis of this DNA is provided by enzymes. These are the polymerized DNAs. From one strand of template DNA, they incorporate in front of each nucleotide that which is complementary to it, thus creating a new strand of DNA. It's elongation. In vitro, to achieve this synthesis, it is necessary to provide polymerization in addition to the matrix and dNTPs, a primer that will initiate the polymerization. This primer is a short DNA fragment (about twenty nucleotides) complementary to one end of the template DNA fragment. To obtain double-stranded DNA, two primers are needed: one each strand, on either side of the fragment that one wants to amplify. In order to reproduce DNA in multiple copies, PCR exploits the denaturation and hybridization capabilities of DNA as well as elongation by polymerization. The principle is the following - denaturation by heating the solution to 94 C, so as to completely separate the two strands of DNA and eliminate secondary structures; - hybridization of the primer on the single strand by lowering the temperature to allow specific matching; and elongation of a strand of DNA by placing itself at the optimum temperature of activity of the thermostable polymer, ie 72 C. After these three steps which constitute a cycle, we denature again: the neosynthesized DNA will be used to his matrix turn. The cycles are thus repeated twenty to thirty times. The increase in the amount of matrix is exponential. There exist, in genetic analysis, other protocols requiring temperature cycling: thus known PCR (Polymerase Chain Reaction) -based amplification techniques such as RT-PCR, specific allele PCR, and Taq Man PCR. (White B, 1997). Also known are LCR (Ligase Chain Reaction) techniques such as LCR, LCR Gap, Asymmetric Gap LCR, RT-LCR, Oligonucleotide Ligation Assay (OLA), and OLA-PCR (Bouma, SR, 1993). , (Segev, D., 1990), (Nikiforov, T., 1995), (Marshall, RL, 1994), Nickerson, DA et al., 1990). Cyclic sequencing reactions are known from clones or PCR reactions. Cyclic microsequencing (single nucleotide primer extension) reactions are known (Cohen, D., l996).

All these techniques are concerned by the invention insofar as they involve a temperature cycling.

Various devices have been designed to implement biological protocols including thermal cycling steps.

In a known type of device, the biological sample is placed in a reservoir which is brought successively to the temperatures required to obtain the desired heat treatments. Some devices use a Peltier thermostating system. The thermal response time of these devices is of the order of 3 C / s. The method used by these devices is simple to implement but results in very long cycle times to achieve the entire protocol. This solution does not increase the reaction rates.

Other techniques use a reservoir etched in a silicon substrate. The heating of the tank is obtained by an electrical resistance formed platinum deposit. The cooling of the tank is obtained by permanent conduction on a cold plate. To improve the thermal response time during temperature cycles, the reservoir (or reaction chamber) is suspended by silicon beams produced by chemical etching in the substrate. The thermal response time is greater than ten degrees per second. The technique used, however, hardly compatible protocol integration in a laboratory chip that is to say in a microfluidic substrate where the principle is to circulate liquids between different biological or biochemical reactions. In particular, it is possible to work continuously and therefore to integrate a large-scale protocol.

In another type of known device micro-channels for circulating biological fluids are micro-machined on a substrate of silicon, glass or polymeric material. These micro-channels or capillaries make it possible to circulate samples continuously. The fluid successively passes through zones at fixed temperatures according to the necessary heat treatments. This solution leads to realize the microchannels in the form of coils. Figure 10 illustrates a device of this type. This is a top view of a substrate in which a micro-channel 1, shown figuratively, has been formed. The micro-channel 1 forms a coil between an inlet orifice 2 and an outlet orifice 3. The references 4, 5 and 6 represent zones subjected to temperatures T1, T2 and T3 respectively. The coil comprises active sections in which the fluid to be treated undergoes the denaturation-hybridization-elongation cycle, these active sections alternate with passive sections serving to bring the fluid back to the zone at denaturation temperature.

A major disadvantage of this provision is that it imposes unacceptable limits to miniaturization. Indeed, it is necessary to have as many thermal zones as there are different temperatures in a thermal cycle. In addition, each thermal zone must be separated by a sufficient distance from another thermal zone to ensure a uniform temperature in the isothermal zones. Another obstacle to miniaturization is due to the number of loops of the coil which corresponds to the number of thermal cycles desired. Miniaturization implies a decrease in the width of the channels, which poses fluidic problems (risk of clogging, pressure losses). In addition, at imposed liquid flow, flow velocities become important, which forces to increase the size of the thermal zones. Indeed, the length of a heating zone must be equal to the reaction time multiplied by the speed of the liquid.

GB-A-2,325,464 discloses yet another type of device. In this device, the reaction chamber has a plurality of parallel microchannels, having a common input and output. The fluid to be treated passes into the reaction chamber and is subjected to three different temperatures, either in three separate zones or in the same zone. device is not designed to work in continuous flow since the fluid is kept at a standstill during the process steps. At best fluid flow can be resumed during the elongation step. This solution involves sequential means for circulating the fluid that are quite complex and that must be perfectly coordinated with the temperature cycles.

US Pat. No. 5,736,314 discloses a device for carrying out PCR. The solution travels through a tube surrounded by annular heating elements. Each element heats at a given temperature the tube section it surrounds. As it travels through the tube, the solution is therefore subjected to different successive temperatures which, suitably chosen, make it possible to implement the PCR. This device has the disadvantage that it requires having as many thermal zones as it has different temperatures in a cycle and there are cycles. This makes the circuit of the solution particularly long. In addition, each thermal zone should be isolated as much as possible from adjacent areas to ensure as much as possible a uniform temperature in each zone. However, this is difficult to achieve in practice and / or very expensive. In addition, it also lengthens the circuit.

None of the devices of the prior art makes it possible to process a sample by a continuous flow thermal protocol and having the best compromise between the following parameters: - minimum active surface area for the device, - maximum processed substance flow rate (reaction volume per unit time), - maximum channel section for a minimum internal channel area.

An object of the invention is to provide a thermal cycling device to reduce the length of the circuit of the solution and to obtain at low cost exactly required temperatures for thermal cycling.

With a view to achieving this object, provision is made according to the invention for a device for carrying out chemical or biochemical reactions by thermal cycling, comprising at least one channel, means for continuously supplying the channel with a solution and means for giving the channel at least two predetermined temperatures so that the solution is set at these temperatures as it travels once the channel, the device having means for passing the channel from one to the other of the temperatures during time during the course of the channel by the solution.

Thus, all the solution circulating in the channel (or in the channel section considered) is brought successively to the different temperatures required by the thermal cycling. By choosing the travel speed, the number of times the solution undergoes this cycling in the channel is thus determined. The number of cycles will be even higher than the speed of travel of the solution will be low.

The invention makes it possible to reduce the dimensions of the device, which reduces the cost of production. The use of a single thermal zone for the channel eliminates the difficulties appearing in the prior art with neighboring thermal zones at different temperatures. The invention makes it possible to give the channel a linear configuration, which is easy, and therefore inexpensive, to manufacture. It is very advantageous to eliminate bends and to reduce the length of the channels, especially when the fluid is circulating by electroosmotic effect. The invention limits the risks of contamination between different successive protocols and facilitates rinsing and cleaning. The times of the different stages of the cycles can be easily modified without changing either the microstructure or the thermostatization mode, but only the flow rate of the fluid to be treated. Very slow rates can be used since the length of the circuit can be small. This brings a lot of advantages, especially when the liquid is displaced by pressure because the pressure drops are much smaller. The invention makes it possible to rapidly treat a large amount of solution since the thermal cycling takes place at a continuous flow of liquid. It ensures a high flow of liquid.

The invention makes it possible to carry out numerous protocols such as all types of PCRs such as PCR, RT-PCR, specific allele PCR, all types of LCR (Ligase Chain Reaction) such as LCR, LCR Gap. , Asymmetric Gap LCR, RT-LCR, Oligonucleotide Ligation Assay (OLA), and OLA-PCR; cyclic sequencing reactions from clones or PCR reactions; cyclic microsequencing (single nucleotide primer extension) reactions; . - any other biological operation requiring thermal cycling.

The invention may furthermore exhibit at least one of the following features - the device is arranged so that the solution is brought to temperatures in a time sequence forming a predetermined cycle and so that the solution undergoes at least twice the cycle when traversing the channel once, the device is arranged so that the solution is set to at least three different temperatures when it runs once the channel; the device comprises at least two channels in mutual communication and means for giving each channel at least two predetermined respective temperatures and for passing each channel over time from one to the other of the associated temperatures so that the solution is put to temperature when it runs once each channel; the channel being a first channel and the means for modifying the temperature being able to modify it during a predetermined period, the device comprises at least one constant temperature channel during the predetermined period and communicating with the first channel; the device comprises several channels arranged in parallel with each other, the channels having identical temperatures with each other at any given instant; - The device comprises a plate in which is formed the or each channel; and the plate is made of silicon.

According to the invention there is also provided a method of carrying out biochemical chemical reactions by thermal cycling, in which a channel is continuously fed with a solution and the solution is given at least two predetermined temperatures so that the solution is put into the temperatures as it travels through the channel, in which the channel is passed from one to another temperature over time during the course of the channel by the solution.

The method may furthermore have at least one of the following features - the reaction is for DNA or DNA fragments; <BR> <BR> <BR> <BR> <BR> <BR> B> DNA, and the reaction comprises a polymerase chain reaction.

Further provided according to the invention a product having undergone a chemical or biochemical reaction carried out according to a process according to the invention.

Other features and advantages of the invention will become apparent in the following description of five preferred embodiments given by way of limiting examples. In the accompanying drawings - Figures 11 to 15 are schematic views of devices according to five embodiments respectively; FIG. 16 is a plan view of a substrate that can be used for these five modes; and - Figures 17 and 18 are cross sectional views along the respective planes VII-VII and VIII-VIII of Figure 16.

Five embodiments of the device of the invention applied here to the implementation of a PCR protocol by thermal cycling will be described in their principles with reference to FIGS. 11 to 15.

In the first embodiment illustrated in FIG. 11, the device 101 comprises a thermal cycling unit 102 comprising a substrate 103. The substrate will be described below in greater detail with reference to FIGS. 16 to 18. The substrate 103 presents a channel 104 in fluid communication by its upstream end with an upstream supply duct 106 and its downstream end with a downstream outlet duct 108.

The device 101 comprises means 105 for continuously traversing the channel 104 by a solution supplied by the conduit 106 and exiting through the conduit 108, the solution thus traversing once the channel 104 along its length throughout the protocol. The solution contains the necessary reagents for carrying out the PCR. The unit 102 comprises means for heating or cooling the substrate 103 at will, these means being conventional and known per se. In the following and for simplicity, these means will be called heating means being understood that they serve for heating and cooling and thus allow to raise or lower the temperature of the substrate and the channel. The heating means are arranged to heat or cool the substrate 103 in its entirety so whatever the temperature they give at a given instant to the channel 104, all sections of the channel 104 have the same temperature between them.

The unit 102 comprises means for controlling the heating means so that they give the channel 104 different temperatures successively over time. These temperatures are here three in number and are those known, used during the PCR. Thus, the channel 104 is first placed at a temperature T1, then cooled to a temperature T2, then warmed to an intermediate temperature T3. This temporal succession forms a temperature cycle. This cycle is repeated many times over time, as illustrated by the graph of FIG. 11. Thus, after a period at temperature T3, channel 104 'again placed at temperature T1 for the beginning of a new cycle and so on.

This cycling is carried out while the solution travels the channel from the supply duct 106 to the outlet duct 108. The channel section 104 and the speed of the solution are chosen here so that the solution is brought to the different temperatures T1 T2. and T3, between its entry and its exit of the channel, undergoes the cycle of twenty to thirty times for example. Therefore, the PCR reactions succeed one another. The different sections of the channel which upstream to downstream have at a given instant the same temperature, differ only in the fact that they are crossed by respective fractions of solution which have already undergone a number of different thermal cycling and as much as these solution fractions approach the output duct.

The second embodiment, illustrated in FIG. 12 with references increased by 100, differs from the preceding one only by the fact that several channels 204 extending in parallel are formed in the substrate 103. It occurs in each channel 204 of this mode what was happening in channel 104 of the first mode. In particular, the heating means perform the same thermal cycling on the entire substrate. In this second mode, at any time, all the sections of all the channels 204 have the same temperature between them. The channels 204 here each have their supply and output ducts in their own right. It is thus possible to treat different solutions at the same time. Alternatively, the channels 204 could be associated with common supply and output conduits, for example if the same solution runs through the different channels.

In the third embodiment illustrated in FIG. 13, and for which the references of the analogous elements are increased by 200, the device again comprises several channels. In addition, it has this time more than one thermal cycling unit 302 (substrate, heating means and control means), but several units 302a, 302b of this type, for example two in number. The two units 302a 302b are arranged in series so that the channels 304a, 304b of the upstream unit 302a communicate via their downstream end and via respective transfer conduits 309 with the upstream ends of the channels 304b of the downstream unit 302b. Each set of channels in mutual communication forms a circuit. The upstream unit 302a is in itself identical to that of the second mode and subjects the solution to the thermal cycling already described associated with the temperatures T1, T2 and T3. The downstream unit 303b performs in this case a thermal cycling whose parameters are different from those of the cycling of the upstream unit. Thus, this downstream cycling is associated with three temperatures T4, and T6, different from the temperatures T1, T2 and T3. Moreover, the duration and the succession of temperatures, illustrated in the lowest graph in FIG. 3, are different from those of the first cycling. Consequently, the solution traversing each circuit undergoes several times the cycling of the upstream unit 302a as it traverses this unit, then passes through the transfer conduit 309 and arrives on the downstream unit 302b where it undergoes the cycling specific to this unit, and this several times provided that the speed of the solution in this unit is sufficiently slow. The solution finally leaves the device <B> 301 </ B> through the outlet duct 308. In the device according to the fourth embodiment illustrated in FIG. 14, the numerical references of the analogous elements have been increased by 300. The device 401 has a thermal cycling unit 402 similar to that of the second mode. It further comprises two units 410a and 410b each comprising a substrate 403 and means for ensuring the channels 412 passing through these units a temperature, respectively T7 and T8, temporally constant. The three units 410a, 402 and 410b are arranged in series and in this order with their respective channels in upstream-downstream communication. Thus, the solution introduced into a supply duct 406 traverses a channel 412 of the upstream unit 410a where it is placed at the constant temperature T7 for a predetermined period, in particular greater than the duration of one cycle of the unit 402. It then crosses the transfer duct 409 and then arrives in the thermal cycling unit 402 where it undergoes the thermal cycle several times. Then, passing through the second transfer conduit 409, it opens in the downstream unit 410b at constant temperature T5 where it remains at this temperature for another predetermined period. It is then evacuated from the device via the outlet duct 408. With reference to FIG. 15, in which the similar elements carry increased references of 400, a fifth embodiment is illustrated in which a secondary duct 514 opens into the duct. Thus, the solution to be treated, for example by a mixture of reagents, is formed just before arrival on the thermal cycling unit which is identical to that of the second mode 502. An upstream mixer is thus formed. . Alternatively or cumulatively, a downstream separator can be provided by putting the outlet duct in lateral communication with a bypass duct. Naturally, these different embodiments can be combined with each other, for example by adding at least one fixed temperature unit to the device of FIG. 3. It is important to note that these various embodiments make the protocol lean with a flow solution. continuous uninterrupted. FIGS. 16 to 18 show a multi-channel thermal cycling unit 2 that can be used in the embodiments shown. Substrate 3 comprises <B> ", </ B> a silicon plate 16, but this plate could be made of another material, for example glass, quartz, polymer material or plastic. by chemical etching of micro-grooves (in a manner known per se) in an upper face of the plate, each channel 4 is rectilinear and has an isosceles triangle profile in V) one side of which extends in the plane of the face of the plate. An inclined ramp connects the bottom of the channel to the face of the plate at each end of the channel At this stage, each channel 4 is open at the top of the plate The substrate 3 comprises a second plate 18 having orifices 20 This plate extends over the plate 16. It thus closes the upper face of the channels and gives access thereto, the solution circulates in one of the orifices and then in the the can al 4 associated, then in other orifice. The second plate 18 can be made in the same type of materials as the first 16. The two plates are sealed or glued to one another for example. An advantage of silicon is that silicon is a good thermal conductor, which gives very short temperature response times. In addition, microtechnologies on silicon are widely known and it is easy to perfectly control the dimensions of the channels. An example of channel dimensions is channel width: 100 μm (from a few microns to a few hundred microns is also possible); - length of the channels: up to several centimeters.

The heating means 20 are placed under the plate 16 against its lower face opposite to that carrying the upper channels. They provide heating and cooling of the plate in a manner known per se, for example by Pelletier effect, Joule effect, radiation or convection.

Note also that it is possible to integrate the heating means 20 directly on the silicon, for example by machining heating resistors' the surface of one of the two plates 16, 18, and placing the assembly on a cold source to evacuate the heat.

During the passage of the solution on the unit 2, the number of cycles is a function of the time spent by the solution on the unit. It is therefore important to control the flow rate of the solution. This control can be carried out by means of a syringe pump or by pumping (pressure force or electro-osmosis for example).

The different channels of the same device can for example be used for the path of solutions comprising different DNAs.

The channel may also be formed by a capillary tube, provided that the control of the temperature of the solution remains possible.

The temperatures T1 to T8 will generally be different from the ambient temperature and the device comprises in each embodiment automated means for controlling the temperature of the thermal cycling unit for carrying out the cycling.

 Typing nucleic acids in a continuous flow device Many techniques have been developed to detect typing mutations and polymorphisms. The so-called microsequencing technique makes it possible to identify the nucleotide present at a given position in a nucleic acid. The microsequencing technique has been developed for the typing of SNPs (single nucleotide polymorphism) for the detection of point mutations, but this technique can also be used for the detection of more complex polymorphisms. This technique is more accurate than SNP typing techniques based on hybridization because it combines the specificity of hybridization with the specificity of enzymatic recognition of the nucleotide by a polymerase. The nucleotide of interest is detected by extension of a primer with a ddNTP (nucleotide blocking base). A primer complementary to the region directly upstream of the nucleotide of interest is used. This primer is paired with the target nucleic acid so that the 3 'end of the primer is adjacent to the specific nucleotide base to be detected. This primer initiates the complementary strand synthesis by a polymerase in the presence of ddNTPs and allows the elongation of this primer with a single ddNTP which is the complement of the nucleotide present in the target nucleic acid. The embedded ddNTP is then detected. Preferably the ddNTPs are labeled to facilitate their detection.

For the detection of a specific nucleotide in a sample such as genomic DNA, for example, this microsequencing technique must be preceded by a step to amplify the region carrying the polymorphism to be analyzed. After the amplification reaction, it is usually a PCR reaction, DNA sample purification is required to remove excess dNTPs and PCR primers. After the microsequencing reaction a second purification step is used to remove excess labeled ddNTPs prior to detection of ddNTP incorporated at the 3 'end of the microsequencing primer. This latter purification generally involves gel separation. The microsequencing genotyping protocol is therefore complex, it involves different steps each comprising the mixture of reagents and long reaction times. The various enzymatic reactions of the protocol must be carried out at precise temperatures, the PCR and the microsequencing reaction are carried out by thermal cycling.

The genotyping of a large number of polymorphisms such as SNPs on families or case / control groups makes it possible to find associations between certain polymorphisms and certain phenotypes. These association studies thus lead to the identification of genes involved in pathologies, in response to therapeutic products and in other complex phenotypes.

One of the limiting factors in these association studies is the need to genotype several hundred individuals for several thousand or tens of thousands of polymorphisms.

A great deal of research is now being conducted to develop genotyping techniques, including large scale microsequencing techniques. A homogeneous phase protocol has been developed in which PCR, purification, microsequencing reaction and detection are performed in solution in the well of a microplate (Chen et al., Proc.Natl.Acad.Sci. <I> USA, </ i> 94/20: 10756-10761, 1997). In this homogeneous phase protocol, the incorporated ddNTP is detected by fluorescence by energy transfer, the microsequencing primer is therefore marked. Despite the integration of all the steps in a microplate, this method does not allow genotyping on a very large scale. Indeed, the successive addition of the reagents in the microplate imposes a very large number of distributions at each stage of the protocol. These distributions can be automated using a dispensing robot but the distributions are extremely slow and the risk of evaporation becomes important. The reaction volumes therefore remain substantial (10-100 μl) involving a large consumption of sample and often very expensive reagents. In addition, manual manipulation of the microplates is necessary to perform the various heat treatments and the distributions between each step.

An object of the invention is to provide a device and methods for genotyping on a very large scale. In order to achieve the goal, the genotyping protocol is integrated in a continuous flow in a microfluidic device according to the invention. The reagent distribution is fully automated and the heat treatments are integrated into the device. The microfluidic substrate constitutes a closed system and the risks of evaporation of the reaction mixture are thus eliminated. This results in a miniaturization of the reaction volumes which is accompanied by a very low consumption of sample and reagent. The protocol is performed in parallel and in series on hundreds of channels to obtain a genotype very large scale. In addition, the combination of a semi-disposable and removable microfluidic substrate with fixed external elements (fluid supply systems, thermal support) is the guarantee of a reduction in costs. Figure 19 is a sectional view of a microfluidic substrate 100 for continuous flow genotyping. In this figure are represented input cuvettes 102 and outlet cuvettes 104. Internal channels 08 arranged in parallel respectively connect a feed bowl and outlet cuvette. The feed bowls and outlet bowls are formed essentially by a through opening in a substrate 100a. The channels 108 are in the form of grooves etched in a substrate 100b. The figure also shows reagent reservoirs 122a, 122b, 122c arranged between feed cups 102 and outlet cuvettes 104. Connectors connect each of the reagent reservoirs to all channels 108. It is observed that the microfluidic substrate has a plurality of channels in parallel. Typically at least <100> channels are arranged in parallel.

The substrate 102b is thinned so as to promote heat exchange when the microfluidic substrate is attached to the thermal medium.

Although not shown in the figure the feed cups the tanks are respectively associated with fluid supply systems for continuous supply channels with samples and for the distribution of reagents.

The thermal support on which the microfluidic substrate is reported is not shown. This thermal support comprises several thermostated zones including cycling zones. These thermostated zones coincide with the sections of the channels 108 situated between the reagent reservoirs 122a and 122b as well as with the sections of the channels 108 situated between the reagent reservoir 122c and the outlet cuvettes 104.

The device according to the invention also comprises a detection system at the outlet cuvettes. The detection is carried out in parallel and in series on all the channels 108. Alternatively the detection takes place at the outlet cuvettes but slightly upstream at the outlet of the last thermostated zone. In this a plate of transparent material (glass or quartz example) can locally replace the substrate 100a to directly detect the samples traversing channel.

Particularly advantageously, the injection of samples and reagents, the regulation of reaction temperatures and temperature cycles and the detection are fully automated.

The samples are injected in continuous flow into the channels 108 through the feed bowls <B> 1 </ B> 02. Preferably the same DNA is injected in series into a channel 108, the DNA injections are separated from each other by plugs of an immiscible inert fluid. The injections are synchronous on all the channels 108 arranged in parallel. The samples injected at a time t travel the channels synchronously so that the injections of reagents and the detection are carried out simultaneously in parallel on all the channels.

The PCR reagents are injected in parallel into all the channels at the reagent reservoir 122a. One and the same reagent is injected at a time and in all the channels in parallel. By cons, in order to type the same DNA for 100 polymorphic sites, 100 different reagents are injected in series in a given channel. Since <B> 100 </ B> channels are arranged in parallel on the microfluidic substrate, 100 DNAs are typed in parallel on the same substrate. The injections in the same channel are separated from each other by cork injections.

The reaction mixture then travels through a thermostatically controlled cycle zone where the amplification reaction is carried out. Note that the channel is rectilinear and that the reaction mixture travels only one thermal zone. The advantages of this particular arrangement according to the invention are described above. It should also be noted that, given the characteristics of the PCR reaction, it does not matter what cycle temperature the thermostated zone is at when a fraction of the sample enters this zone. Finally, it should be noted that this arrangement makes it possible to vary at will the number of thermal cycles performed (typically between 15 and 35 cycles for PCR) by regulating the cycling times and the flow rate of the solution flowing through the channel.

The second step of the protocol, the PCR product purification reaction, begins with the injection of the purification reagent at the reservoir 122b. The same reagent is injected in parallel and in series into all <B> 108 channels. </ B>

The reaction mixture then goes through a first thermostatically controlled zone at 37 ° C. for a purification reaction and a thermostated zone at 94 ° C. for the inactivation of the purification enzymes.

The microsequencing reaction begins with the injection of microsequencing reagents. As previously for PCR reagents, a different reagent is used for typing each polymorphism. For the typing of <B> 100 </ B> polymorphisms 100 different reactants are injected in series in the same channel 108.

The reaction mixture then travels through a second thermostatically controlled cycle zone where the microsequencing reaction is carried out.

At the exit of this thermostated zone is the detection in continuous flow, in parallel and in series of ddNTPs incorporated at the 3 'end of the microsequencing primer. Typically ddNTPS are labeled with fluorophores.

The detection is carried out in solution by measuring the fluorescence of the fluorescence temporal correlation or by spectropolarimetry. These detection means are known elsewhere.

<U> Example: </ U> <U> Intersting of a <U> <U> Genotyping Protocol in a Continuous Flow Microfluidic Device </ U> The reaction mixture traverses a channel in which all the steps are performed necessary for a genotyping protocol: PCR, purification, microsequencing reaction and detection.

The microfluidic substrate has <B> 100 </ B> channels in parallel. These channels are rectilinear and have a diameter of the order of 600 μm in the reaction zones. The area of the section of a channel is of the order of 0.25 mmz.

The genotyping protocol is performed in parallel in the 100 channels. In addition, 100 samples in succession are injected into each channel. The microfluidic substrate therefore makes it possible, thanks to a cross-distribution of 100 samples per 100 polymorphisms, to carry out 10,000 genotyping reactions on a microfluidic substrate.

The injection of the reagents is carried out at different points of the channel, it is synchronized so as to be able to carry out the reactions in series, in parallel in continuous flow.

To avoid any contamination, a given channel is fed in series with the same DNA. However this DNA is analyzed for 100 different polymorphisms.

The microfluidic substrate is silanized according to the protocol described by Schoffner et al. (Chip PCR.1 Passivation Surface of Microfabricated Silicon-Glass Chip for PCR, Mann A. Schoffner, J. Cheng, GE Hvichia, LJ Kricka, P. Wilding.Nucleic Acid Research, <B> 1996 </ B> Vol 24 , No. 2, pp 375-379). The silanized substrates can be stored for several months. The silanization step of the substrates can be carried out during the manufacture of the substrate. Before using the substrate, all the channels are filled with previously degassed filtered water. A high flow rate of about 25 μl / min is applied for 15 minutes to drive out all the air bubbles present in the circuits. A solution 10 mM Tris HCl and mM KCl pH 8.3 previously degassed and filtered is then injected at a rate of 5 g / min for 15 min, taking care not to form air bubbles.

 This operation is preferably done on a device outside the genotyping chain that can process several substrates in parallel.

Each DNA sample is diluted in a 1 mM Tris-HCl buffer, 1 M pH 8.3 EDTA previously degassed and filtered in order to obtain 30 μl of a final DNA solution. A DNA preparation per channel is necessary. Each genotyping reaction uses 0.25u1 of this solution, 30 u1 is the amount needed to make 1 successive genotyping on the same individual. This solution is placed in the injection device in parallel and in series samples. For each pair of PCR primers, 30 μl of a solution must be prepared: 0.6 μM of each unpurified PCR primer, 20 mM Tris-HCl and 100 mM KCl pH 8, previously degassed and filtered, 4 mM MgCl 2, 0.2 mM each. dNTP (dATP, dCTP dGTP, dTTP) and 1U of Taq Gold. A preparation by genotype site is necessary. Each genotyping reaction uses 0.25 μl of this solution for a final reaction volume of 0.5 μl, 30 μ1 is the amount needed to make <B> 100 </ B> parallel genotyping over 100 different channels. These solutions are placed in the serial injection device of the PCR primers and are stored at 4 ° C. The PCR purification reaction mixture is common to all genotyping reactions. A volume of 5500 μl of a solution: 20 mM Tris-HCl pH 8.0, 10 MgCl 2 pre-degassed and filtered, 550 U shrimp alkaline phosphatase (SAP) and 50 U exonuclease 1 , is prepared and stored at 4 C in the distribution device of the PCR purification reaction mixture. Each genotyping reaction uses 0.5 μl of this solution for a final reaction volume of 1 μl. 5500 p1 is the amount needed to do 10,000 genotypes in parallel.

For each microsequencing oligonucleotide, 120 μl of a solution must be prepared: 1 μM microsequencing oligonucleotide, 40 mm Tris-HCl pH 9.5 degassed beforehand and filtered, 8 mM MgCl 2, 10 nM of a ddNTP-Tamra and 10 nM ddNTP-Fam, each ddNTP corresponding to one site allele, and 6 U of thermosequenase. A preparation by genotype site is necessary. Each genotyping reaction uses 1 μl of this solution for a final reaction volume of 2 μl. 120 μl corresponds to the amount necessary to make 100 genotypes in parallel on 100 different channels. These solutions are placed in the serial injection device of the microsequencing primers and are stored at 4 C.

The flow rates at the outlet of the substrate are 40 μl / hour, which makes it possible for reactions of 2 μm to carry out 10 reactions / channel / hour separated by 10 plugs of 2. The separating plugs are composed of liquid immiscible with the reaction mediums, such as paraffin oil for example. They physically separate reactions from each other throughout the path in the channel and have a volume equivalent to the reaction volume.

The DNA sample is distributed in a volume of 0.25 μl per genotyping reaction. It is injected with a flow rate of 5 pl / hour which represents a time of 3 minutes and a length of lmm. Each sample injection is separated by 0.25 μl of immiscible cap. 0.25 μl of PCR reagent are injected at a flow rate of 5 μl / hour and mix with the DNA samples. Each injection of PCR reagent is separated by injecting 0.25 μl of immiscible cap. The PCR reaction therefore represents a volume of 0.5 μl a length in the channel of 2 mm and moves with a flow rate of 10 μl / hour.

This reaction mixture travels through a thermostatically controlled zone at 94 ° C for 10 minutes to activate the polymerase. It then goes through a cycle zone, one cycle of which consists of a 10 sec stage. at 94 C then 10 sec. at 55 C 10 sec. at 72 C for a duration corresponding to 35 cycles.

After the 0.5 μl of reagent for PCR purification are injected with a flow rate of 10 μl / hour. Each injection of reagent is separated by the injection of 0.5 μl of stopper. The PCR purification reaction therefore represents a volume of 1 μl a length in the 4 mm channel moves with a flow rate of 20 μl / hour.

This reaction mixture then travels through a thermostatically controlled zone at 37 ° C. for 20 minutes for a purification reaction and then a thermostatically controlled zone at 94 ° C. for 10 minutes for the inactivation of the purification enzymes (SAP EXO 1).

Then 1 μl of miocrosequencing reagent is injected at a rate of 20 μl / hour. Each injection of microsequencing reagent is separated by the injection of 1 μl of immiscible cap. The microsequencing reaction therefore represents a volume of 2 μl and a length in the 8 mm channel and moves with a flow rate of 40 μl / hour.

This microsequencing reaction mixture (2 μl) travels through a cycle zone of which one cycle is composed of a step of 10 sec. at 94 C then 10 sec. at 55 C and 10 sec. at 72 C for a duration corresponding to 25 cycles.

The detection is performed online and continuously after the microsequencing zone. Detection is performed by measuring polarized fluorescence for the Fam and Tamra fluorophores. The excitation wavelengths are respectively 488 nm and 520 nm while the emission wavelengths are 520 nm and 575 nm. Two fluorescence measurements are made simultaneously, one in the plane of polarization of the excitation beam and the other in the plane perpendicular to it. The ratio of these two measurements makes it possible to differentiate the presence of a labeled oligonucleotide (microsequencing oligonucleotide elongated during the reaction) from two to five times more free markers.

Claims (33)

A biochemical analysis device comprising a) a microfluidic substrate having at least one channel having a feed cup for injecting a sample, an outlet cup for collecting said sample, and at least one reagent reservoir connected to each channel between the feed bowl and outlet bowl; b) means for supplying said channel with a continuous flow. 2. Device according to claim 1 characterized in that the microfluidic substrate comprises a plurality of channels arranged in parallel. 3. Device according to claim 2 wherein the means for supplying said continuous flow channel apply a pressure difference between the feed bowl and the outlet bowl. 4. Device according to any one of claims 1-3 wherein said microfluidic substrate is silicon. 5. Device according to any one of claims 1-4 comprising a thermal medium having a heat exchange face in contact with a surface of the microfluidic substrate so that the sample flowing through the channel is heated to a predetermined temperature. 6. Device according to claim 5 wherein the face of the microfluidic substrate in contact with the heat exchange face of the heat support has a thin wall. 7. Device according to claim 5 or 6 wherein the microfluidic substrate is removably attached to the thermal medium. 8. Device according to any one of claims 5-7 wherein the thermal support comprises at least two thermostatically controlled zones at different temperatures arranged so that the sample flowing through the channel is successively at least two predetermined temperatures. 9. Device according to any one of claims 5-8 wherein at least one thermostated zone is brought to at least two distinct temperatures so that the sample traversing once this zone is brought to at least two predetermined temperatures. 10. Device according to any one of claims 5-8 wherein at least one thermostatically controlled zone is brought to at least two distinct temperatures in a time sequence forming a predetermined cycle so that the sample undergoes at least one cycle. temperature by browsing once the thermostated zone. 11. Device according to one of claims 8 or 9 characterized in that the portion of the channel through the thermostated zone is rectilinear. 12. Device according to any one of claims 1-11 comprising detection means for measuring a physico-chemical characteristic of the samples traversing the channels. 13. Device according to claim 12 wherein said detecting means are located at the outlet cuvettes of said channels. 14. Process for the production of biochemical protocols on at least one sample, characterized in that it comprises the following steps: a) feeds a continuous flow channel with a solution containing at least one sample by applying a pressure difference between the cuvette supply and outlet cuvette of said channel, b) at least one reagent is injected from at least one reagent reservoir into said channel so as to mix said sample and said reagent. 15. A method for carrying out biochemical protocols on at least one sample, characterized in that it comprises the following steps: a) feeding a continuous flow channel with a solution containing at least one sample by applying a pressure difference between the bowl; and the outlet cuvette of said channel, b) injecting at least one reagent from at least one reagent reservoir into said channel so as to change said sample and said reagent, c) detecting at least one Physico-chemical parameter of said sample in said channel. 16. Method according to one of claims 14 or 15 wherein the solution travels at least one thermostatically controlled zone so that the solution is set at a predetermined temperature as it traverses said thermostated zone. 17. A method according to any one of claims 14-16 wherein the solution travels at least one thermostatically controlled zone carried successively at at least two determined temperatures so that the solution is successively put said temperatures when it runs once said thermostated zone. . 18. A method according to any one of claims 14-17 wherein the solution travels through a thermostatically controlled zone carried successively at at least two predetermined temperatures in a temporal succession forming a predetermined cycle so that the solution undergoes at least one cycle. temperature by browsing once the thermostated zone. 19. The method of claim 18 wherein the solution undergoes 1 to 35 times the temperature cycle by traversing once the thermostated zone. 20. The method according to claim 14, wherein said channel is fed sequentially with a plurality of samples separated from one another by plugs. 21. Method according to any one of claims 14-20 wherein steps a), b) and optionally c) are performed simultaneously on a plurality of channels arranged in parallel. 22. Process for the continuous flow of at least one temperature cycle on a solution containing at least one sample, characterized in that it comprises the following steps: a) a continuous flow channel is fed with said solution, b) said solution is passed through a thermostatically controlled zone carried successively at at least two predetermined temperatures in a time sequence forming a predetermined cycle so that the solution undergoes at least once the temperature cycle by traversing once the thermostated zone. 23. Proce "for the continuous flow of at least one temperature cycle on a solution containing at least one sample, characterized in that it comprises the following steps: a) feeding a continuous flow channel with said solution, b) said solution is passed through a thermostatically controlled zone carried successively at at least two predetermined temperatures in a time sequence forming a predetermined cycle so that the solution undergoes at least one temperature cycle by traversing once the thermostated zone, c) at least one physicochemical parameter of said sample is detected in said channel. 24. Method according to one of claims 22 or 23 wherein is fed to the continuous flow channel by applying a pressure difference between the feed bowl and the outlet bowl of said channel. 25. A method according to any one of claims 22-24 wherein said channel is fed sequentially with a plurality of samples separated from one another by plugs. 26. A method according to any one of claims 22-25 wherein steps a), b) and optionally c) are performed simultaneously on a plurality of channels arranged in parallel. 27. Process for the amplification of nucleic acids, characterized in that it comprises the following steps: a) said nucleic acid is mixed with appropriate reagents for the amplification of nucleic acids; b) a channel is fed into a continuous flow; with the reaction mixture, c) is passed through the reaction mixture a thermostatically controlled zone successively 'temperatures determined in a time sequence forming a predetermined cycle, d) selects the temperatures and the duration of the temperature cycle of the thermostated zone and the time taken by the mixture to travel through the thermostated zone so that the nucleic acid undergoes the denaturation-hybridization-elongation cycle several times. 28. Process for the amplification of nucleic acids, characterized in that it comprises the following steps: a) feeding a continuous flow channel with a solution comprising said nucleic acid; b) injecting at least one reagent suitable for amplification. of nucleic acids from at least one reagent reservoir in said channel so as to mix said nucleic acid and said reagent; c) the reaction mixture is passed through a thermostatically controlled zone carried successively at predetermined temperatures in a temporal succession; forming a predetermined cycle, d) selects the temperatures and the duration of the temperature cycle of the thermostated zone as well as the time taken by the mixture to travel through the thermostated zone so that the nucleic acid undergoes several times the denaturation-hybridization cycle. elongation. 29. The method of claim 27 or 28 wherein is fed continuous channel by applying a pressure difference between the feed bowl and the outlet bowl of said channel. 30. Method according to one of claims 27-29 wherein said channel is formed in a silicon substrate. 31. The method of any of claims 27-30 wherein said channel is sequentially fed with a plurality of nucleic acids separated from one another by plugs. 32. The method according to claim 27, wherein steps c) and d) are performed simultaneously on a plurality of channels arranged in parallel. 33. Method for the continuous detection of at least one nucleotide in at least one target nucleic acid, characterized in that it comprises the following steps: a continuous flow channel is supplied with a solution comprising said nucleic acid; injecting the microsequencing reagent comprising the microsequencing buffer, the microsequencing primer, at least one ddNTP and a polymerase into said channel so as to mix said nucleic acid and said reagent; the solution is passed through the at least one thermostated zone so as to obtain at least one cycle comprising the denaturation of the target nucleic acid, the hybridization of said nucleic acid with the microsequencing primer and the incorporation of the ddNTP, complementary to the nucleotide detect at the 3 'end of said primer; at least one ddNTP incorporated at the 3 'end of the microsequencing primer is detected. The method of claim 33 wherein the continuous flow channel is applied applying a pressure difference between the feed bowl and the outlet bowl of said channel. 35. Method according to one of claims 33 or 34 wherein in step c) thermostated zone is carried successively to temperatures determined in a time sequence forming at least one cycle. 36. Method according to one of claims 33-35 wherein the ddNTPs are labeled with fluorophores and wherein in step d) the fluorescence of the incorporated ddNTP is detected. 37. A method according to any one of claims 33-36 wherein steps a), b), c) and d) are performed simultaneously on a plurality of channels arranged in parallel. A method for the continuous detection of at least one nucleotide in at least one target nucleic acid, characterized in that it comprises the following steps: a continuous flow channel is fed with a solution containing at least one target nucleic acid; the reagent is injected for amplifying the region of the target nucleic acid carrying at least one nucleotide to be detected, in said channel from a first reagent reservoir; the solution is passed through the at least one thermostated zone so that the nucleic acid undergoes several times the denaturation-hybridization-elongation cycle; the reagent is injected for purification of the amplification product in said channel from a second reagent reservoir; e) at least one thermostated zone is passed through the solution to carry out the purification reaction; f) the microsequencing reagent comprising the microsequencing buffer, the microsequencing primer, at least one ddNTP and a polymerase is injected into said channel from a third reagent reservoir; g) the reaction mixture is passed through at least one thermostated zone so as to obtain at least one cycle comprising the denaturation of the target nucleic acid, the hybridization of said nucleic acid with the microsequencing primer and the incorporation of the ddNTP, complementary to the nucleotide to be detected at the 3 'end of said primer; h) detecting at least one ddNTP incorporated at the 3 'end of the microsequencing primer. 39. The method of claim 38 wherein the continuous flow channel is supplied by applying a pressure difference between the feed bowl and the outlet bowl of said channel. 40. Method according to one of claims 38-39 wherein in steps c) and e) the thermostatically controlled zone is carried successively to temperatures determined in a time sequence forming at least one cycle. 41. Method according to one of claims 38-40 wherein the ddNTPs are labeled with fluorophores and wherein in step h) the fluorescence of the incorporated ddNTP is detected. 42. Method according to one of claims 38-41 wherein the reagent for purification comprises an exonuclease and an alkaline phosphatase. 43. The method of any of claims 38-42 wherein steps a), c), d), f), g) and h) are performed simultaneously on a plurality of parallel arranged channels.