Microfluidic sample chip, assay system using such a chip, and PCR method for detecting DNA sequences

Abstract

A microfluidic sample chip to test biological samples, especially for a PCR-type and/or fluorescence assay. The chip being in the shape of a hollow block having at least one chamber delimited by an upper wall, a lower wall and at least one side wall, into which a sample can be introduced for testing. The lower wall of the block is made of a material with a high thermal conductivity and the upper wall is made of a material with a low thermal conductivity. Preferably, the upper wall is preferably permeable to radiation in the visible spectrum between 400 and 700 nm. The block having at least two openings through which the sample can be introduced into at least one of the chambers and through which the air present in the chamber can be evacuated when the sample is introduced.

Claims

1. A system to analyze PCR-type samples contained in at least one chamber of a micro-fluidic sample chip to test biological samples, for PCR or fluorescence analysis, in the form of a hollow block comprising said at least one chamber delimited by an upper wall, a lower wall and at least one side wall, into which a sample to be tested can be introduced; wherein the block is provided with at least a lower side and an upper side parallel to each other, the lower side being disposed onto the lower wall, the upper side being disposed on the upper wall made of a material having a thermal conductivity lower than a material of the lower side and the upper side being permeable, at least in said at least one chamber, to radiations having a wavelength between 300 nm and 900 nm; wherein the hollow block comprises at least two openings to introduce the sample into said at least one chamber and to discharge pressure in the chamber during the introduction of the sample; the system comprising: a thermalization film to increase or decrease, by a thermal cycling, a temperature of the micro-fluidic sample chip and the samples therein, in a thermal contact with the lower side of the micro-fluidic sample chip; sealers to close said at least two openings in said at least one chamber to maintain a relative internal pressure of at least 5000 Pa (50 mbar) in said at least one chamber, an increase in the temperature of the samples causing said at least one chamber to expand, thereby improving the thermal contact between the lower side of the micro-fluidic sample chip and the thermalization film; a pressure controller to maintain a relative external pressure greater than 50 mbar over the entire upper side of the micro-fluidic sample chip to provide a substantially uniform thermal contact between the lower side of the micro-fluidic sample chip and the thermalization film, wherein the upper wall of the micro-fluidic sample chip comprises a transparent portion traversed by light rays and located above said at least one chamber containing one of the samples; an optical measurement instrument, comprising a camera, to optically observe the samples with a spatial resolution; and wherein the pressure controller is formed by a plate of transparent material associated with a frame arranged at a periphery of the plate and springs configured to apply a pressure onto the frame.

2. The system of claim 1, wherein the thermalization film utilizes a heat transfer liquid to provide a change in the temperature of the samples higher than or equal to 5° C./s.

3. The system of claim 1, wherein the pressure controller is configured to maintain the relative external pressure higher than 100000 Pa (1 bar) over at least one portion of the upper side of the micro-fluidic sample chip.

4. The system of claim 1, wherein the pressure controller is further formed by a housing having outer dimensions of the micro-fluidic sample chip to house the micro-fluidic sample chip therein at a room temperature, as the temperature of a sample trapped in said at least one chamber of the micro-fluidic sample chip increases, walls of the housing exert a pressure onto the upper and lower walls of the micro-fluidic sample chip.

5. The system of claim 1, further comprising an injector to introduce a sample into said at least one chamber of the micro-fluidic sample chip positioned in the system.

6. The system of claim 1 is configured to perform a polymerase chain reaction (PCR) including a digital PCR (dPCR) or a digital droplet PCR (ddPCR).

7. The system of claim 1, wherein the lower side is disposed onto the lower wall made of the material having the thermal conductivity greater than 15 W.Math.m.sup.−1.Math.K.sup.−1.

8. The system of claim 1, wherein the sealers are configured to maintain the relative internal pressure of at least 50000 Pa (500 mbar) in said at least one chamber.

9. A system to analyze PCR-type samples contained in at least one chamber of a micro-fluidic sample chip to test biological samples, for PCR or fluorescence analysis, in the form of a hollow block comprising said at least one chamber delimited by an upper wall, a lower wall and at least one side wall, into which a sample to be tested can be introduced; wherein the block is provided with at least a lower side and an upper side parallel to each other, the lower side being disposed onto the lower wall, the upper side being disposed on the upper wall made of a material having a thermal conductivity lower than a material of the lower side and the upper side being permeable, at least in said at least one chamber, to radiations having a wavelength between 300 nm and 900 nm; wherein the hollow block comprises at least two openings to introduce the sample into said at least one chamber and to discharge pressure in the chamber during the introduction of the sample; the system comprising: a thermalization film to increase or decrease, by a thermal cycling, a temperature of the micro-fluidic sample chip and the samples therein, in a thermal contact with the lower side of the micro-fluidic sample chip; sealers to close said at least two openings in said at least one chamber to maintain a relative internal pressure of at least 5000 Pa (50 mbar) in said at least one chamber, an increase in the temperature of the samples causing said at least one chamber to expand, thereby improving the thermal contact between the lower side of the micro-fluidic sample chip and the thermalization film; a pressure controller to maintain a relative external pressure greater than 50 mbar over the entire upper side of the micro-fluidic sample chip to provide a substantially uniform thermal contact between the lower side of the micro-fluidic sample chip and the thermalization film, wherein the upper wall of the micro-fluidic sample chip comprises a transparent portion traversed by light rays and located above said at least one chamber containing one of the samples; an optical measurement instrument, comprising a camera, to optically observe the samples with a spatial resolution; and wherein the pressure controller is formed by a housing having outer dimensions of the micro-fluidic sample chip to house the micro-fluidic sample chip therein at a room temperature, as the temperature of a sample trapped in said at least one chamber of the micro-fluidic sample chip increases, said walls of the housing being configured to exert a pressure onto the upper and lower walls of the micro-fluidic sample chip.

10. The system of claim 9, wherein the thermalization film utilizes a heat transfer liquid to provide a change in the temperature of the samples higher than or equal to 5° C./s.

11. The system of claim 9, wherein the pressure controller is configured to maintain the relative external pressure higher than 100000 Pa (1 bar) over at least one portion of the upper side of the micro-fluidic sample chip.

12. The system of claim 9, wherein the pressure controller is further formed by a plate of transparent material associated with a frame arranged at the periphery of the plate and springs to apply a pressure onto the frame.

13. The system of claim 9, further comprising an injector to introduce a sample into said at least one chamber of the micro-fluidic sample chip positioned in the system.

14. The system of claim 9 is configured to perform a polymerase chain reaction (PCR) including a digital PCR (dPCR) or a digital droplet PCR (ddPCR).

15. The system of claim 9, wherein the lower side is disposed onto the lower wall made of the material having the thermal conductivity greater than 15 W.Math.m.sup.−1.Math.K.sup.−1.

16. The system of claim 9, wherein the sealers are configured to maintain the relative internal pressure of at least 50000 Pa (500 mbar) in said at least one chamber.

17. A system to analyze PCR-type samples contained in at least one chamber of a micro-fluidic sample chip to test biological samples, for PCR or fluorescence analysis, in the form of a hollow block comprising said at least one chamber delimited by an upper wall, a lower wall and at least one side wall, into which a sample to be tested can be introduced; wherein the block is provided with at least a lower side and an upper side parallel to each other, the lower side being disposed onto the lower wall, the upper side being disposed on the upper wall made of a material having a thermal conductivity lower than a material of the lower side and the upper side being permeable, at least in said at least one chamber, to radiations having a wavelength between 300 nm and 900 nm; wherein the hollow block comprises at least two openings to introduce the sample into said at least one chamber and to discharge pressure in the chamber during the introduction of the sample; the system comprising: a thermalization film to increase or decrease, by a thermal cycling, a temperature of the micro-fluidic sample chip and the samples therein, in a thermal contact with the lower side of the micro-fluidic sample chip; sealers to close said at least two openings in said at least one chamber to maintain a relative internal pressure of at least 5000 Pa (50 mbar) in said at least one chamber, an increase in the temperature of the samples causing said at least one chamber to expand, thereby improving the thermal contact between the lower side of the micro-fluidic sample chip and the thermalization film; a pressure controller to maintain a relative external pressure greater than 50 mbar over the entire upper side of the micro-fluidic sample chip to provide a substantially uniform thermal contact between the lower side of the micro-fluidic sample chip and the thermalization film, wherein the upper wall of the micro-fluidic sample chip comprises a transparent portion traversed by light rays and located above said at least one chamber containing one of the samples; an optical measurement instrument, comprising a camera, to optically observe the samples with a spatial resolution; and wherein the sealers are configured to maintain the relative internal pressure of at least 50000 Pa (500 mbar) in said at least one chamber.

18. The system of claim 17, wherein the pressure controller is formed by a plate of transparent material associated with a frame arranged at the periphery of the plate and springs to apply a pressure onto the frame.

19. The system of claim 17, wherein the pressure controller is formed by a housing having outer dimensions of the micro-fluidic sample chip to house the micro-fluidic sample chip therein at a room temperature, as the temperature of a sample trapped in said at least one chamber of the micro-fluidic sample chip increases, walls of the housing exert a pressure onto the upper and lower walls of the micro-fluidic sample chip.

20. The system of claim 17, wherein the pressure controller is configured to maintain the relative external pressure higher than 100000 Pa (1 bar) over at least one portion of the upper side of the micro-fluidic sample chip.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The invention will be better understood with the aid of the following exemplary embodiments illustrating the first and second aspects of the invention, given in a non-limiting manner together with the figures in which:

(2) FIG. 1 shows an exemplary embodiment of a micro-fluidic chip according to the invention,

(3) FIGS. 2a, 2b and 2c show an alternative embodiment of the chip in FIG. 1, in which fluid switching valves are integrated,

(4) FIG. 3 shows, in FIGS. 3a, 3b, 3c, other variants of the chip associated with a sample holder containing a sample to be analyzed, according to the system of the invention,

(5) FIGS. 4a and 4b show two variants of the system according to the invention in which the various heat transfer liquids alternately circulate through a valve assembly (FIG. 4a) or due to pressure variations to liquids (FIG. 4b),

(6) FIG. 5 shows another variant of the system according to the invention in which all the liquids in the bypass channels are recovered in the same container,

(7) FIG. 6 show another variant with a thermalization system for heat transfer liquids by means of pumps,

(8) FIGS. 7a to 7d show another variant with a thermalization chip shown in three dimensions and equipped with miniature valves of the base-mounted type,

(9) FIGS. 8a and 8b show the realization of a PCR cycle and the resulting fluorescence signal.

(10) FIG. 9 shows a diagrammatic sectional view of a sample chip according to the second aspect of the invention,

(11) FIG. 10 shows an exemplary embodiment of a system according to the second aspect of the invention, comprising particular optical measurement means,

(12) FIG. 11 show various representations of single or multi-chamber sample chips, containing a sample or sample drops.

(13) In all the figures, the same elements have the same references.

DETAILED DESCRIPTION OF THE EMBODIMENTS

(14) FIG. 1 schematically shows a micro-fluidic 1 chip exchanging heat between the heat transfer liquids when they are injected into the chip and the sample (DNA for example), not shown in this figure, in contact with the chip. The chip 1 is formed by a block of parallelepiped-shaped material having an upper side, comprising a heat exchange zone 204 provided with a thermalization zone (heat exchange) 22 with a surface S (surrounded by a dotted line in the Figures) towards which the heat transfer liquid injection channels 4, 5 converge.

(15) The fluid injection zone 201 comprises a pipe 15 with a first heat transfer liquid, connected to the chip 1 via a first connection port 2, while a second pipe 14 is connected to the chip 1 by via a second connection port 3. The input ports 2 and 3 are respectively connected to the supply channels 4 and 5 respectively extending up to the junctions 8 and 9, to which the bypass channels 6 and 7 are also respectively connected, which respectively extend to the output ports 16 and 17 for discharging the heat transfer liquid into the bypass pipes 18 and 19 respectively. (The supply channels can be the bypass channels and vice versa).

(16) Each junction 8, 9 extends by means a supply channel portion 20, 21 respectively, which meet at their other ends at the inlet 10 of the cavity 202 for introducing the heat transfer liquid into the inlet homogenization zone 203 which comprises a homogenization tree for the liquid 29a (in order to give it a good flow rate homogeneity at the inlet of the thermalization zone 22). The heat exchange zone 204 comprises the thermalization zone 22 itself, preferably formed by a plurality of parallel channels 11, preferably uniformly distributed over substantially the entire width of the chip, in the zone 22 for contacting the sample to be analyzed. At the other end of these channels 11, the heat transfer liquid is collected at the outlet 30bis of the thermalization zone 22 (which is part of the heat exchange zone 204) and then, after passing through the outlet homogenization zone 205 comprising a homogenizing tree 29b preferably similar to that 29a disposed in the inlet homogenization zone 203, is collected at the fluid outlet zone 206 via the outlet 30 of the cavity 202 connected to the connection port 12 of the chip 1 in the outlet pipe 13 (the pipes 13, 14, 15, 18 and 19 are not part of the chip 1 in this example).

(17) According to an alternative embodiment, an independent output is provided for liquids at different temperatures, for example by means of one or more valves after the connection port 12 for orienting the liquid into different tanks in order to limit the mixing of liquids of different temperatures). Each injection channel 4, 5 comprises a junction 8, 9 towards an outlet 16, 17 for additional liquid for circulating the heat transfer liquid continuously in the pipe 18, 19 upstream of the chip 1 and thereby stabilizing the temperature of the liquid in order to avoid to perturbation due to change in temperature of the heat-transfer liquid.

(18) The distance L between the junctions 8 and 9 and the thermalization zone 22 depends on the thermal characteristics of the chip and must be as follows:
L<S/a

(19) S being the surface of the upper side of the cavity (202) in m2, a being a correction coefficient equal to 0.005 m.

(20) In this way, the transient effect of the materials surrounding the thermalizing liquid upstream of the thermalization zone is not sufficient to prevent a reproducible change in temperature as previously defined.

(21) Thus, for a heat transfer fluid flow rate of about 10 ml/min (1.6e-7 m.sup.3/s), this distance L between the junctions 20 and 21 and the fluid inlet 10bis of the thermalization zone 22 will preferably be less than 2 cm.

(22) FIG. 2A represents an alternative embodiment of the chip in FIG. 1, in which liquid switching valves 23, 24, 25 and 26 are integrated for allowing each liquid from the pipes 14 and 15 to pass either into the channels 11 or in the bypass channel 6, 7 provided for this purpose.

(23) Thus, when closing the switching valve 23 and simultaneously opening the valve 24, the liquid from the pipe 15 is then sent into the bypass pipe 18. Simultaneously (if desired) the liquid from the pipe 14, if the valve 25 is opened and the valve 26 is closed, will enter the chip and, after homogenization, the channels 11 to carry out the thermalization of the DNA sample which will contact the chip.

(24) FIGS. 2B and 2C respectively represent an enlarged detail of an exemplary embodiment of a pneumatically-controlled valve integrated to the chip in an open position (FIG. 2B) and a closed position (FIG. 2C) under the action of a control signal.

(25) In a manner known per se (Unger et al., Science 288: 7, 113, 2000), each valve is formed for example by a membrane 28 which is open in the rest position (P=0, FIG. 2B) and which is closed when it is activated by injection of a pressurized control gas (FIG. 2C) which causes this membrane 28 to stick onto the opposite portion 27 of the pipe on which it is fixed.

(26) FIG. 3A represents a top view of the chip in FIG. 1, on which a few additional embodiment details are represented notably at homogenization trees 29a and b. Each tree comprises a first junction close to the inlet 10 or outlet 30 dividing the fluid inlet of output channel into two side channels 31 and 32 which divide a second time at the junctions 34 and 35, allowing thereby the homogenization of the liquid flow rate along the large section of the inlet 10bis and the outlet 30bis of the thermalization zone 22. This homogenization is due to the fact that each end of the junctions of the trees formed by the side channel is equidistant between the liquid inlet of outlet, giving thereby a equivalent flow resistance to these different paths.

(27) FIGS. 3b and 3c show sectional views along A-A of the chip 1 surmounted by a sample holder (not shown in FIG. 3a).

(28) In the first variant in FIG. 3b, the chip 1 is represented in a parallelepiped-shaped block 40 of polymeric material (here 5 mm high) such as polydimethylsiloxane (PDMS) in the upper part of which there is a plurality (seven in the figure) of parallel channels 11 (of rectangular section) opening onto the surface of the chip 1, with a depth of 100 microns in this embodiment, these channels having a width preferably between 1 and 2 mm, each channel 11 being separated from the neighboring channel by a distance preferably lower than the distance from the surface of the chip to the sample (i.e. about 170 microns in this example, corresponding to the thickness of the glass slide 41). The glass slide 41 (or any other material allowing a good heat transfer between the heat transfer liquid circulating in the channels 11 in use) supporting the sample is applied to the channels 11 in order to close them preferably in a watertight manner, while the upper side of this slide 41 is locally treated by using a polyethylene glycol (PEG) based treatment to prevent the adsorption of the DNA on the glass surface, for example more particularly with the aid of a polylysine-polyethylene glycol copolymer which has a good adsorption capacity on glass. All around the thus treated zone 42 extends a silicone crown forming a sample holder 43, which is closed, after introduction of the sample, by a film 44, for example of plastic (here a polypropylene film 100 microns thick). In this version, the chip and sample holder assembly is preferably sealed, the assembly being discarded after use.

(29) It should be noted that, in all the embodiments according to the invention, the film or the wall 44 (generally transparent) and the side wall 43 delimiting the cavity 45 may be formed by a single piece, according to a variant of the invention, for example molded, of transparent plastic material.

(30) In the second variant of FIG. 3c, the channels 11 are closed by means of an aluminum sheet 41 of 300 microns in thickness, on which is applied the sample holder formed by a clamping piece 48 in the form of a crown to maintain the film 41 on the channels in a sealed manner, at the bottom of which is placed an aluminum film 42 (in this example identical to the film 41) supporting a sample holder piece 43 made of polycarbonate provided with a cavity having a height of 200 microns, whose bottom is formed by the film 42 and filling ports 47 which are closed by a polyester/silicone adhesive film 46 in this example. The set 42, 43, 46, after filling and testing the sample, can be discarded, the rest can be reused.

(31) The separation films 41 and 42 between the heat transfer liquid and the sample are generally carried out from a heat conducting material whose thermal conductivity/thickness ratio (lambda/e) is higher than 1000 Wm.sup.−2K.sup.−1 and whose thermal diffusivity/squared thickness ratio (D/e.sup.2) is greater than 2 s.sup.−1 [For example, a glass slide of 500 microns meets these criteria, which corresponds to the reasonable limit in terms of conductivity and diffusivity to obtain a change in temperature in a few seconds].

(32) The heat transfer liquid flow rate per unit area to be thermalized (surface of the exchange zone) required for the thermalization of the sample will preferably be less than 30 mL.Math.min.sup.−1.Math.cm.sup.−2.

(33) FIG. 4 describes two variants of use of the chip and its system described in FIGS. 1 to 3, to carry out the thermal “cycling” required in a PCR-type analysis by means of heat transfer liquids of different temperatures successively circulated in the channels 11 of the chip, in thermal contact with the sample. For this purpose, the system in FIG. 4 comprises means for switching the path followed by the heat transfer liquid so that, for each heat transfer liquid, it passes either through the channels 11 of the thermalization zone 22, or by a bypass channel. Several configurations are possible to perform this switching process.

(34) For example, according to the variant in FIG. 4a, pneumatic switching valves are used, for example integrated in the chip (as shown in FIG. 3), arranged upstream of the thermalization zone 22 with the sample and on the two circulation junction, for directing the liquid leaving the heat transfer liquid source 60, flowing in the channel 61, the thermalization film 62 for the heat transfer liquid (to bring the liquid to a good temperature), the channel 63, either to the exchange zone 67 through the open valve 64 (and the closed valve 65) and the channel 66, i.e. to the bypass channel 68, through the open valve 65 (and the closed valve 64 connected to the junction 69 at the valve 65). When a valve 64 is open allowing the heat transfer liquid of the branch to circulate, all the other valves 64 are closed (except exceptions) while the valve 65 (of the branch whose valve 64 is open) is closed, all the others valves 65 being open to allow the bypass of the chip 1. These pneumatic valves will close the micro-fluidic channels concerned with a gas under pressure applied on a deformable membrane positioned above the channel (see FIGS. 2B and 2C) as it is commonly used in micro-fluidic chips made of elastomer such as PDMS.

(35) According to the variant in FIG. 4b, independent and variable-pressure heat transfer liquid sources are used. For this purpose, the pressure of heat transfer liquid transferred to the exchange zone 22 must be higher than the pressure of the other heat transfer liquids. The pressure of the other sources must be dimensioned, in a manner known per se, as a function of the flow resistance of the different branches of the circuit so that this pressure is sufficiently low to avoid any transfer of liquid from these sources into the exchange zone. This solution however requires a fine adjustment of the pressures of the different heat transfer liquids to obtain a proper operation.

(36) Advantageously, whatever the variant used, the heat transfer liquid can be re-circulated for example independently for each source by means of pumps. This makes it possible to limit the energy consumption necessary for controlling the temperature of each source by reusing the previously thermalized heat transfer liquid. For this purpose, it is possible, for example, to use a piston or gear positive displacement pump which makes it possible to ensure a constant heat transfer liquid flow rate for each source, which makes it easier to control the temperature accurately (for example, ceramic pumps which can tolerate high temperatures). Generally speaking, all the materials and equipment used in the context of the present invention cab generally withstand (and operate at) temperatures of at least 100° Celsius when it is desired to perform PCR-type analyzes.

(37) To do this, each circulation junction is redirected preferably to the original heat transfer fluid source and the outlet of the exchange zone can be distributed to all the sources. The outlet of the chip can also be redirected to its original source, but in this case it is necessary to add a valve to redirect the liquid to the tank. (See FIG. 6).

(38) A tank can also be inserted into the circuit upstream of the pump to ensure good filling of the circuit with the heat transfer liquid. In some configurations, the flow rates cannot be balanced in the different channels of the circuit and some tanks can be filled faster than others. It may then be advantageous to connect the tanks to each other by the pipe 121 (FIG. 6) so that their levels equilibrate, which also has the advantage of filling all the tanks from a single opening. Advantageously, these tanks can have a volume lower than 20 ml, allowing a small space requirement, a reduced thermal capacity and low thermal losses.

(39) Two embodiments of the invention will now be described with reference to FIGS. 5 and 6:

Example 1

(40) In FIG. 5, a first pressurized gas generator 80 generates a compressed gas (air and/or inert gas such as nitrogen and/or argon) which flows via the line 84 into the gas sky 89a of the tank 87 of a first heat transfer liquid 89b. A second pressurized gas generator 81 generates a compressed gas (preferably the same as the first generator) which flows via the line 85 in the gas sky 90a of the tank 88 of a second heat transfer liquid 90b. The two liquid 89b and 90b are respectively injected by the pressure exerted by the respective gaseous skies, respectively in the pipes 91 and 92 up to the respective inlet ports 93 and 94 of the chip 1, of the type described in FIGS. 1 to 3. The liquid flows meet at the junction 98 substantially located at the inlet of the exchange zone 95 in which one or the other heat transfer liquid alternately circulates. When the pressure of one liquid is greater than that of the other (at least 40%, preferably at least 42% but less than 55% so as not to create a reflux of the liquid into the other way. These minimum and maximum values depend on the geometry of the chip and the temperature of the liquid to be injected. They are determined experimentally by thermal imaging or modeling so as to obtain the desired flows as described below), it is this liquid that will enter the exchange zone as well as the bypass channel associated therewith, while the other liquid continues to circulate in the bypass channel associated therewith (96 for the first liquid and 89b, 97 for the second liquid 90b). At the junction 99 to the output of the chip where the pipes 96 and 97 converge, the liquids are directed to the outlet port 100 and flow through the pipe 101 towards the recovery container 102 which contains a liquid mixture 103b. The alternation of the liquids in the exchange zone 95 and the temperature variation in this zone are controlled by the control system 83. The pipes 96 and 97 enable the liquids to circulate continuously. In this way, the distance between the junction 98 and the inlet of the chamber 95 can remain, according to the invention, lower than the value defined above for L. In the present example of the system according to the invention, the gas generators whose pressure is controlled by a computer (for example the systems of the company ELVESYS sold under the trade name “Elveflow OB1 mk3”) are used as a means of circulation that pressurize the two tanks 87 and 88 controlled in temperature with a thermoelectric module. The pressures of the delivered gases are set according to two configurations so as to obtain a temperature control of the DNA sample (or other) at two different temperatures. In the first configuration, the pressure of the gas delivered by the second generator 81 is at least 1.5 times higher than the pressure of the gas delivered by the first generator 80 (determined experimentally by thermal imaging or modeling so as to obtain the desired flows as described below), so that the liquid 89b contained in the tank 87 at a first temperature circulates only in the bypass channel 96 while the liquid 90b contained in the tank 88 at a second temperature circulates in the bypass channel 97 and in the exchange zone 95. In this configuration, the sample is thus very quickly brought to the second temperature by indirect heat exchange with the second heat transfer fluid 90b. The precise ratio between the pressures of each generator depends on the precise geometry of the chip, the temperatures of the heat transfer liquids that affect their viscosity and the selected way so as to circulate in the exchange zone. The precise values of these pressures can be determined experimentally by thermal imaging of the heat conducting side of the chip which makes it possible to image the temperature of the circulating liquids respectively in the channels 4, 5, 95, 96 and 97 through the heat conducting layer. For this purpose, the pressure values of the generators must be adjusted for each liquid source (each temperature) that can circulate in the exchange zone (two, in this case). For each source of circulating liquid, the good pressure balance is achieved when the thermal imaging shows that the entire surface of the exchange zone 95 is at the desired temperature and the bypass way 96 or 97 is at the temperature of the liquid that must pass in this one. It is also possible to predict these pressures by a hydrodynamic modeling taking into account the geometrical parameters and dependence parameters of the viscosity of the heat transfer liquid temperature.

(41) Conversely, in the second configuration, the pressure of the gas delivered by the first generator 80 is higher (under the same conditions as explained above) than the pressure of the gas delivered by the second generator 81 so that the liquid 90b contained in the tank 88 at a second temperature circulates only in the bypass channel 97 while the liquid 89b contained in the tank 87 at a first temperature circulates in the bypass channel 96 and in the exchange zone 95. In this configuration, the sample is very quickly brought to the first temperature by indirect heat exchange with the first heat transfer liquid 89b.

(42) At any time, the heat transfer liquid circulate in the pipes, in particular 91, 92, 96, 97, so that the change in temperature in the heat exchange zone is rapid (less than 5 s), reproducible and the sample temperature can be precisely controlled, even when low heat transfer fluid flow rates are used, for example flow rates of less than or equal to 10 ml/min.

(43) Such a system can be used to perform PCR reactions, but also observations on live biological samples. Advantageously, the use of a thermoelectric module makes it possible to control the temperature of the sample at temperatures below the room temperature. This possibility may be useful to study the physical, chemical or biological phenomena such as the polymerization dynamics for the microtubules within living cells, which requires the thermalization of the cells at temperatures below 5° C.

(44) According to another alternative embodiment, the injection channels 63 can meet into one single channel before the junctions 69 (see FIG. 4), as it is the case in FIG. 5. As the transport of the liquid in the micro-fluidic chip is laminar (non-turbulent), the liquids in the single channel 63 do not mix and keep their respective temperatures up to the junction 69 or they can be separated again between the bypass channel 68 and the channel 66 guiding the liquid to the thermalization zone 67.

(45) Generally, the height of the thermalization zone 22 will be inferior to one millimeter, preferably to 400 microns, which allows a high convection coefficient and a low time of renewal of the heat transfer liquid in the chip for low flow rates of injection into the chip.

Example 2

(46) In this example corresponding to FIG. 6, the micro-fluidic chip 1 for temperature control comprises a substantially parallelepiped-shaped cavity whose upper side corresponding to the thermalization zone 22 has a surface S of 1 cm.sup.2 and a height of 300 μm. It comprises five ports 2, 3, 16, 17, 12 (as in FIG. 1) and is used to switch two heat transfer liquids 112 and 114 at different temperatures between the thermal exchange zone 22 and two circulation junction by means of four integrated valves 23, 24, 25 and 26 as shown in FIGS. 1 to 3. It is made by molding PDMS and bonded on an aluminum sheet of 300 μm in thickness by means of a light-activatable adhesive (e.g., glue sold under the trade name “Loctite 3922”) on which the sample holder is placed in thermal contact. The chip is supplied by two flow tanks 110 and 111 of respectively heat transfer liquids 112 and 114, each of them being connected to a positive displacement pump 116, 117 providing a flow rate of 10 ml/min, whatever the pressure in the circuit, and an online thermalization device for the heat transfer liquid comprising an aluminum body for a significant thermal exchange between this body and the liquid, a Joule effect heating ceramic element in contact with the body (such as those marketed by the company Thorlabs), a miniature temperature sensor (such as marketed by the company Radiospares under the name “PT100”) and an electronic card for control temperature equipped with a system control PID for controlling the temperature of the body by means of the temperature sensor.

(47) The two tanks 110 and 111 are arranged respectively upstream of the pumps 116, 117 so as to serve as a liquid supply. The tank levels can be adjusted relative to one another by a system of communicating vessels. In addition, a “3/2”-type valve 118 makes it possible to redirect the liquid leaving the chip via the pipe 13 to the tank 110 or 111 supplying the contents of the thermalization zone 22, under the control of a control system, non shown in the figure, controlled by a computer sequencing the different valves according to the liquid and the desired injection duration.

(48) To carry out a PCR analysis with a system as described in FIG. 6, it is preferable to use a cartridge composed of a parallelepiped-shaped micro-fluidic chamber of 20 μl, having a surface of 1 cm.sup.2 and a height of 200 microns, for example molded in a polycarbonate piece glued (at the micro-channel 11) on an aluminum sheet of 200 μm in thickness: this chamber is filled with the PCR reagent mixture and the sample to be analyzed (for more details concerning the procedure described, see the article of Houssin et al. cited above). This cartridge is pressed against the aluminum sheet of the thermalization chip to achieve a good thermal contact. It is also possible to carry out a Real-time PCR analysis in the same conditions as in the article of Houssin et al. by placing under the chip a chamber for receiving the reagent while measuring the fluorescence. The sample is first thermalized at 95° C. for 30 s by circulating the heat transfer liquid thermalized at 95° C. by the online temperature controller while the heat transfer liquid thermalized at 65° C. is redirected to the circulation junction. To do this, the valve 24 positioned on the circulation junction of the heat transfer liquid source at 95° C. and the valve 25 for transmitting the liquid to the exchange zone from the source 111 at 65° C. are closed. On the other hand, the valve 26 positioned on the circulation junction of the heat transfer liquid source at 65° C. and the valve 23 for transmitting the liquid to the exchange zone from the source at 95° C. are open. The valve 118 for redirecting the liquid leaving the exchange zone is positioned so as to redirect the liquid leaving the chamber to the pipe 120 and the tank 110 located upstream of the thermalization system at 95° C.

(49) Then, 40 cycles of temperature variation between 95° C. and 65° C., with an alternation of 5 s are performed in order to amplify the DNA contained in the sample by the PCR reaction. For the, the state of the valves 23, 24, 25, 26 and 118 is reversed every 5 s.

Example 3

(50) In this example corresponding to FIGS. 7a to 7d, the micro-fluidic microchip 1 for temperature control comprises a cavity of the same geometry as in Example 2. It comprises 4 ports 2, 3, 16, 17 and makes it possible to switch two heat transfer liquids 112 and 114 at different temperatures between the heat exchange zone 22 and two circulation junctions by means of four integrated valves 23, 24, 36 and 37. It is made out of a polycarbonate piece formed from a sandwich of two micro-machined (CNC) polycarbonate pieces, then glued by hot melting or assisted by a solvent by well-known methods in the plastics industry, which makes it possible to create channels inside the polycarbonate piece, while avoiding their contact with the aluminum layer, which limits the heat exchange parasites with the thermalization zone (22). On the surface of this polycarbonate piece on the cavity 202 is fixed (preferably glued) an aluminum sheet 41 of 500 μm in thickness by pressing, which enables to seal the cavity and to ensure the heat exchange with the sample. Advantageously, this aluminum sheet preferably does not cover the entire surface of the chip, but only the thermalization zone 22, (slightly protruding from it) in order to limit thermal losses by conduction along the sheet. The valves 24, 26, 36, 37 used are base-mounted-type miniature valves directly fixed on the chip to prevent any channeling out of the chip. The chip is supplied by two tanks and two pumps according to a pattern identical to that in Example 2 except that the valve 118 in Example 2 is replaced by a valve 37 integrated in the chip and the recirculation channels 119 and 120 are partially integrated in the chip, which has the advantage of being less bulky, cheaper to achieve, of limiting heat loss and of increasing the reliability of the system by reducing the number of fluid connectors.

(51) In addition, a “3/2” valve 36, which replaces the valves 23 and 25 in Example 2, makes it possible to switch the source of liquid entering the chip through the inlets 2 and 3 towards the thermalization zone 22, which makes it possible to minimize the distance L by the use of a single space-saving valve positioned closest to the inlet orifice for the fluid 10. The assembly is controlled by a computer sequencing the different valves according to the liquid and the desired injection duration.

(52) To carry out a PCR analysis with a system as described in FIG. 7, a cartridge is preferably used as described in Example 2. The sample is first thermalized at 95° C. for 30 seconds by circulating the heat transfer liquid thermalized at 95° C. by the on-line temperature controller while the heat transfer liquid thermalized at 65° C. is redirected to the circulation junction. To do this, the valve 36 positioned so as to circulate the liquid from the heat transfer liquid source at 95° C. entering through the inlet 2, while the valve 24 is in the closed position so as to block the recirculation of the liquid at 95° C. though its bypass way. At the same time, the valve 26 is opened for recirculating the liquid at 65° C. through the bypass way and the valve 37 is positioned so that the liquid from the thermalization zone 22 is redirected to the pipe 120 and the tank 110 located upstream of the thermalization system at 95° C.

(53) Then, 40 cycles of temperature variation between 95° C. and 65° C., with an alternation of 5 s, are performed in order to amplify the DNA contained in the sample by the PCR reaction. For this, the state of the valves 23, 24, 25, 26 and 118 in FIG. 6 (24, 26, 36, 37 in FIG. 7a) is reversed every 5 s.

(54) FIG. 8a shows the results measured with a thermal imaging camera and expressed as % of total change in temperature: it is found that the sample temperature reached 95% of the set temperature value after about 1.5 s.

(55) After 40 cycles, the system according to the invention is configured so as to continuously circulate the heat transfer liquid 114 at 65° C. in the thermalization zone 22, then the temperature of the liquid 114 of the source is gradually increased (until 85° C.) linearly over time so as to achieve what is commonly called by those skilled in this type of analysis, “a melting curve”, i.e. a curve establishing the correspondence between the temperature and fluorescence level of the sample. This curve makes it possible to check the hybridization temperature of the amplified sequence, this information being used as a quality control of the PCR reaction. The fluorescence signal obtained is shown in FIG. 8b in which the progressive amplification over time of the fluorescence signal is clearly visible, followed by the melting curve.

(56) The system according to the second aspect of the invention includes, as diagrammatically represented in FIG. 9 a consumable container or a micro-fluidic sample chip for performing rapid real-time PCR reactions. The sample chip can contain one or more chambers (FIG. 11) in which real-time PCR reactions are carried out. It comprises two walls 42 and 44 with parallel outer sides, one of which 42 (lower side) is intended to allow the control of the temperature of a sample and its eventual reagent placed in the reaction chamber 45 and the other 44 (upper side) is intended to the optical measurement, including fluorescence. To allow a good temperature transfer between the thermalization film 41 and the sample and the reagent, it is preferable that at least one of the following conditions (preferably several and more preferably all of them) is fulfilled:

(57) 1. the consumable contain is maintained in contact with the thermalization film at a pressure greater than or equal to 5000 Pa (50 mBar), but preferably greater than or equal to 100000 Pa (1 Bar) (average pressure on the contact surface);

(58) 2. the reaction chamber is sealed so as to withstand a pressure at least equal to 50000 Pa (500 mbar), preferably greater than or equal to 100 000 Pa (1 bar) or is maintained pressurized artificially (outside) with means for pressurizing at a pressure greater than or equal to 5000 Pa (50 mBar), preferably greater than or equal to 50000 Pa (500 mBar). In this way, the heat transfer between the sample and the thermalization film can be done in good conditions;

(59) 3. the heat conducting layer between the reagent and the thermalization film is sufficiently conductive, that is to say greater than or equal to 15 w.Math.m.sup.−1.Math.K.sup.−1, preferably greater than or equal to 100 w.Math.m.sup.−1.Math.K.sup.−1 and is not made of a PCR inhibiting material, such as for example aluminum or its derivatives;

(60) 4. the wall and the upper side of the sample chip intended to enable the optical measurement is made out of a material having a thermal conductivity preferably less than or equal to 1 w.Math.m.sup.−1.Math.K.sup.−1 and preferably an effusivity lower than or equal to 1000 J.Math.m.sup.−2.Math.K.sup.−1.Math.s.sup.−0.5, preferably transparent for the visible wavelengths, preferably withstanding temperatures greater than or equal to 95° C. without deforming and preferably not being a PCR inhibitor, which may be for example a plastics material selected from polycarbonates and/or polymers and/or cyclic olefin copolymers COP, cyclic olefin copolymer COC and their derivatives. All these materials are well known to those skilled in the art of micro-fluidics (see, for example, the article of Rajeeb K. Jena et al.: “Cyclic olefin copolymer based micro-fluidic devices for biochip applications: Ultraviolet surface grafting using 2-methacryloyloxyethyl phosphorycholine”); and

(61) 5. The heat conducting layer between the reagent and the thermalization film is sufficiently thin (<=500 μm, preferably <=300 μm) so that its surface can conform to the surface of the thermalization film under the effect pressure, in particular, in the thermalization chamber.

(62) Advantageously, the thermalization film 41 can use a heat transfer liquid allowing a rapid temperature transfer (less than or equal to 5 s.) As described, in particular, in the first aspect of the invention.

(63) The pressurizing means 213 for the chip on the thermalization film 41 may be formed for example by a transparent glass piece (293) which is pressed on the chip by means of springs supported by a frame (294, 295, 296) and applying sufficient pressure on the chip (see FIG. 10). A slide mechanism (not shown) is for example provided to lift the frame and thus provide access to the space provided for the chip in order to place it before the implementation of the reaction or after implementation thereof.

(64) But the pressurizing means can now also be a frame maintaining a pressure on the periphery of the chip (if it is sufficiently rigid) in order to avoid the deformation thereof under the effect of the pressure in the reaction chambers.

(65) The sample chip can comprise a single chamber 45 (FIG. 11a): in this implementation mode, the optical measurement using the means 210 and the light source 211 can be made with a simple avalanche-diode-type sensor on which the light from the chamber 45 is refocused. This configuration has the advantage of allowing a measurement with an equal sensitivity on all the surface of the chamber, the signal generated by the sensor being proportional to the increase in fluorescence in the chamber, even if it is not the case for the distribution of the fluorescence in the chamber, for example when a low copy number of target DNA is initially present. A camera can also be used as a sensor, which measures the fluorescence homogeneity in the chamber for focusing purposes or for controlling the reaction homogeneity on the surface of the chamber. In this case, the sensor used will advantageously be of sCMOS technology, which provides a high sensitivity and a low signal-to-noise ratio for low exposure times, so as to follow, if necessary, in real time, the fluorescence signal. The introduction of the sample and the reagent into the reaction chamber 45 is carried out via the openings 47 shown here in the transparent upper wall 44: but at least one of these openings can be made through the side walls 43 of the sample chip which can have a rectangular or square or cylindrical parallelepiped shape. After introducing the sample, the openings 47 are preferably closed with a sealing adhesive.

(66) FIG. 10 schematically shows the device and the chip forming the system according to the second aspect of the invention and described in Example 4 below.

(67) The sample chip in FIG. 11b includes four chambers (or more if necessary); each chamber can, in particular, contain a different PCR reagent, the various test conditions in the different chambers can be compared under the same temperature conditions. In this case, the detection can be carried out with a sensor matrix having the same spatial organization as the chambers and on which the image of the chambers (four sensors in FIG. 11b) or a camera sensor can be refocused, as previously described.

(68) FIG. 11c shows another mode of implementation with a single chamber for performing a PCR on droplets of sample for performing a so-called “digital” PCR. A camera is then used to film the reaction in the droplets.

(69) In all the FIGS. 11a to 11c, black zones indicate the presence of fluorescence, indicative of a positive PCR reaction.

(70) The following exemplary embodiments make it possible to illustrate, in particular, the second aspect of the invention described above:

Example 4

(71) In this fourth example, the temperature control means for the samples contained in the micro-fluidic sample chip is a micro-fluidic thermalization chip in which two heat transfer liquids having two different temperatures (typically 65° C. and 95° C.) are caused to circulate alternately, as described above and in the manner shown in FIG. 4a.

(72) In FIG. 10, the sample chip 289 comprises for example a single chamber 45, which can be filled through two openings (an inlet port 290 for the sample and reagents and an air outlet port 291—or vice versa—see FIGS. 3b, 3c and 7d) by means for example of a pipette. It is formed by an aluminum film 41 to 200 μm in thickness for its lower wall and its heat conducting lower side and a transparent polycarbonate piece 44 in which the ports 290 and 291 (47 in FIG. 3c) for filling are drilled.

(73) After filling, the openings 290, 291 of the sample chip are sealed with a silicone/polyester adhesive in order to maintain a pressure therein. The sample chip is then placed (FIG. 10) in a housing delimited laterally by a fastening frame 48 above the thermalization interface 41 (metal film) disposed above the thermalization chip 1 according to the first aspect of the invention and as described with reference to FIG. 3c. A lever system (not shown) used for example to lower a frame 296 on which a glass piece 293 mounted on four springs 294, 295 is fixed, which will apply a controlled and uniformly distributed pressure of 20N on the surface of the sample chip 289 once the system is engaged (equivalent to 100000 Pa (1 bar)). A thin layer 292 of transparent elastomer (so-called soft layer) is fixed under the glass piece 293 in order to homogenize the pressure on the surface of the chip and to avoid the detachment of the sealing adhesive in the openings 290 and 291.

(74) An optical detector is mounted on the frame 296, comprising a LED diode 297 shifted to the right in the figure, in which the wavelength is adapted to the fluorescence excitation wavelength of the intercalating element Cybergreen commonly used (and added into the sample) for the measurement of real-time PCR. This LED 297 is directed to the reaction chamber 45 of the chip. a lens 298 for collimating the light emitted by the LED and producing a homogeneous excitation across the surface of the chamber 45. an excitation filter 299 for restricting to the desired value the spectrum of the light emitted by the LED. an optical sensor 300 placed above the chamber 45, having a square shape, of the MPPC type (of the company Hamamatsu) of 3×3 mm on which the image of the chamber is focused by means of two plano-convex lenses 301 and 302, positioned so that the projected image of the chamber does not extend beyond the surface of the sensor 300. an emission filter 303 adapted to measure the fluorescence of the intercalating element Cybergreen and compatible with the light spectrum delivered by the excitation filter 299, this emission filter 303 being positioned between the two lenses 301 and 302.

(75) A data acquisition system (not shown in the figure) makes it possible to measure in real time the fluorescence signal delivered by the sensor 300. The system is implemented to perform 40 temperature cycles with an alternation of 5 s to amplify the DNA contained in the sample by PCR reaction.

(76) After 40 cycles, the system is configured to gradually increase the temperature in a linear manner over time. This produces what is called in the PCR jargon a “melting curve” (FIG. 8), that is to say the correspondence between the temperature and the fluorescence level for the sample. This curve makes it possible to check the hybridization temperature of the amplified sequence, this information being used by those skilled in the art to control the quality of the PCR reaction. The fluorescence signal obtained is shown in FIG. 8b.

Example 5

(77) This exemplary embodiment is in all aspects identical to Example 4, the chip sample comprises four chambers while the sensor is replaced with a sensor array 2×2 of the same type.

Example 6

(78) In this example, the chip comprises a single chamber 45 and the sensor is a Hamamatsu C13770-50U sCMOS camera for observing the PCR chamber with high spatial resolution. The PCR is carried out in micro-droplets of 10 nL of reagents in Fluorinert FC-40 oil (Sigma-aldrich) which are produced by means of a suitable micro-fluidic device (for example the Droplet Generator Pack Elveflow) and are injected into the chamber 45. The amplification in each droplet can be observed in real time by the camera. The results obtained are similar to those obtained in FIG. 8.

(79) These various examples show that the pressure applied simultaneously on the chip and in the chip (passively by the pressure naturally induced by the increase in temperature of the reagent or actively by the pressurization of the reagent) allows a good thermal contact between the aluminum sheet of the chip containing the sample and the aluminum sheet of the thermalization chip. Thanks, in particular to this good contact, it is possible to carry out rapid PCRs.