EP1121199B1 - Device for chemical and/or biological analysis with analysis support - Google Patents

Abstract

Chemical and/or biological analysis device comprising an analysis support (100) with at least one input bowl (102) to contain a sample, at least one output bowl (104) to output the said sample, at least one internal duct (108) passing through the support to form a connection between the input bowl and the output bowl, and at least one reagent reservoir (120a, 120b, 120c) connected to each duct (108) between the input bowl and the output bowl, in which the input bowl, the output bowl and the reservoir open up onto a first face (106) of the analysis support.

Description

Technical area

The present invention relates to a device chemical and / or biological analysis, equipped with a analysis support which may be of the single-use type.

The invention finds applications in fields of chemistry and biology. In particular, the device can be used in chemical amplification or reaction processes Polymerase Chain Reaction (PCR) polymerase for analysis of genetic material (DNA).

State of the art

Systems are currently known macroscopic chemical or biological analysis using titration plates. These plates have bowls in which are mixed samples and reagents by pipetting (at the pipette). To allow chemical reactions or biological, the plates are successively heated at set temperatures by stoving successive, then cooled.

With such systems, the supply of reagents is a long and complex operation, especially in due to a successive supply of each reagent. Of plus, the thermal inertia of heating and cooling of the titration plates turns out to be too important and lengthens the analysis time.

We also know, equipment chemical and / or biochemical analysis in the form of complete structures incorporating the heating means necessary for the analysis. of the piping connection systems allow to bring the samples and reagents into the structure.

The use of this equipment implies however complex and tedious operations of connection for the supply of fluids, analytes and reagents, as well as connection operations electric for the energy supply of the means of heating. Due to the specificity of the analyzes, it is necessary to renew the operations of log in each time you use equipment.

The equipment also has a cost of high manufacturing.

A more complete illustration of the techniques and equipment in the field of biochemical analysis is given by documents (1) and (2) whose references are specified at the end of this description.

Presentation of the invention

The object of the invention is to propose a biological and / or chemical analysis device not having the limitations mentioned above.

Another goal is to reduce the times of heating, cooling and allow a accurate and selective temperature control of components to be analyzed during the different phases of reaction.

Another object of the invention is to propose a such a device can be quickly adapted to different types of products to analyze and do requiring no complex connection operations.

The invention also aims to propose a device with an analysis support, very low cost, possibly for single use, which can be and replaced after each use, or after a limited number of uses. For example, we are considering to perform a thousand sequential analyzes with a device before throwing it away.

To achieve these goals, the invention has more precisely for purpose a chemical analysis device and / or biological comprising an analysis support with at least one inlet bowl to collect a sample, at least one outlet bowl for delivering said sample, at least one internal channel through the bracket to connect the inlet bowl to the outlet bowl, and at least one tank to reagent, connected to each channel respectively between a inlet bowl and an outlet bowl, wherein the inlet bowl, the outlet bowl and the tank open on a first side of the support analysis.

The device may in particular include a plurality of inlet bowls and a plurality corresponding outlet cuvettes, each cuvette inlet respectively being connected to a bowl of associated output, by means of a channel.

The establishment of the liquids to be analyzed in inlet cuvettes and / or reagents in the corresponding reservoirs can take place by micropipette (micropipette).

According to another mode of use, the implementation place liquids to be analyzed in the cuvettes inlet and / or reagents in the tanks can be done by means of watertight supply of fluids such as a plug deposited on the tank or bowl and connected to a syringe or a tank under pressure.

The placement of liquids and / or reagents can be obtained by a combination of the two previously described modes.

For media with a large number of cuvettes and / or tanks, the supply of liquids to analyze and / or reagents can be automated at means of a dispensing robot (dispense) at high resolution. In addition, the sequential analyzes assuming the replacement in time of at least one reactive by another can be automated by sequential introduction into the tank corresponding of several different reagents. Enter two different reagents a neutral buffer liquid can, or not, be introduced into the tank.

According to a particular aspect of the invention, the or internal channels can be planned for extend near at least one second face of the analysis support so as to be separate from said second face only by a thin wall. In particular achievement the thin wall can present a thickness of less than 100 μm.

More precisely, the wall is chosen Thin enough to allow heat exchange with thermal sources external to the support analysis.

In particular, the wall separating the channels of the second side can be chosen thinner than a wall separating the channels between them or basins.

According to another aspect of the invention, the face opposite channels to the thin wall may present a thermal barrier, this can be achieved by a layer of low thermal conductive material and / or a structuring of the substrate to locate on the channels a cavity filled with air or a little gas coolant.

This thermal barrier makes it possible to standardize the temperature in the channels.

According to another aspect of the invention, the device may further comprise a thermal support independent of the analysis support, thermal support having a heat exchange face with at least a thermal source, and said thermal medium can be removably attached to the support of analysis in order to put in contact the exchange face with the second face of the analysis support.

The separate character of the analysis medium and the thermal support allows to design media analysis without own means of heating or cooling. This characteristic allows therefore reduce in significant proportions the cost of the analysis support. So, this support can be of the single or multiple use type uses, that is, to be thrown away after one or several uses. We mean by use the sequential realization of a number of neighboring analyzes for example 1000.

The heat exchange face may include a or several thermostatically controlled zones, each equipped with minus a heat source. Thermostatic zones coincide with at least one area of the analysis support located downstream of a connection between a reagent and a channel.

By associating a thermostated area of the support thermal with a corresponding area of the support located in the vicinity, by example downstream of each reagent tank it is possible to selectively control and adapt the temperature of the liquid to be analyzed according to each reagent used.

The term downstream used here refers to to a flow direction of the liquid to be analyzed from the entrance bowls to the bowls of exit.

Thermal sources may include one or several electric heating resistors thermostatically controlled.

Alternatively or in addition thermal sources may also include one or several channels crossed by a heat transfer fluid. This fluid can be used to heat or cool locally the analysis support.

In a particular embodiment of the support analysis, it may comprise a first substrate having through openings which form respectively the bowls and reservoirs, and a second substrate, glued to the first substrate, the second substrate having grooves, covered by the first substrate to form channels, and coinciding respectively with the openings therethrough.

This particularly simple structure allows reduce media manufacturing costs analysis.

• formation, dans un premier substrat, d'ouvertures traversantes, lesdites ouvertures correspondant respectivement à des cuvettes d'entrée ou de sortie, ou à des réservoirs de réactif,
• formation dans un deuxième substrat de rainures selon un motif permettant de relier entre elles au moins deux ouvertures du premier substrat,
• collage du premier substrat sur le deuxième substrat de façon à recouvrir les rainures,
• amincissement du deuxième substrat, après le collage, en préservant une épaisseur de substrat supérieure à une profondeur maximale des rainures.

The production of the support can take place, in accordance with the invention, according to a process comprising the following successive steps:

• forming, in a first substrate, through openings, said openings corresponding respectively to inlet or outlet cuvettes, or to reagent reservoirs,
• formation in a second substrate of grooves according to a pattern making it possible to connect 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, while preserving a thickness of substrate greater than a maximum depth of the grooves.

According to a particular embodiment, the first substrate can have two layers thermally weakly conductive, eg thick a few microns.

According to a second particular mode of realization, the first substrate can present at minus a non-through opening so as to create at minus a thermal insulation cavity.

The invention also relates to a method of implementation of an analysis device as described above, according to which the support is brought into contact with analysis with thermal support during a phase fixed-term analysis, at least one sample to be analyzed and at least one reagent being introduced into analysis support prior to the analysis phase or during the analysis phase, and then after the analysis, we remove the support analysis support thermal.

At the end of the analysis, the analysis support can be reused.

Other features and benefits of the present invention will emerge more clearly from the description which will follow, with reference to the figures of the drawings attached. This description is given purely illustrative and not limiting.

Brief description of the figures

• Figure 1A is a schematic section simplified version of an analysis the invention.
• Figure 1B shows the analysis support for Figure 1A equipped with filling means reagent tanks.
• Figure 2 is an exploded perspective showing more precisely the structure of the support analysis.
• Figure 3 is a perspective view simplified analysis support according to the figure 2 and a thermal support.
• Figure 4 is a longitudinal section schematic of the analysis support set up on the thermal support.
• Figure 5 is a plan view of part an analysis support in accordance with the invention, constituting a variant with respect to FIGS. 1 to 4.
• Figure 6 is a cross section simplified analysis support including part as shown in Figure 5.
• Figure 7 is a cross section simplified analysis support including part in accordance with FIG. 5 and constituting a variant by compared to figure 6.
• Figures 8, 9, 10 and 11 are sections longitudinal diagrams of substrates during steps successive manufacturing of an analysis support according to the invention.
• Figure 12 is a cross section of a analysis support and thermal support in accordance with the invention and illustrating a particular embodiment thermal support.

Detailed description of modes of implementation of the invention

In the following description, parts identical, similar or equivalent figures are marked with the same numerical references in order to facilitate reading.

Figure 1 is a section of a support 100 according to the invention.

In this figure, a bowl is represented 102 mainly formed by an opening crossing made in a substrate 100a of the support, near one of its ends. Of the same way, an outlet bowl 104 is made at the neighborhood of a second end. The bowls 102, 104 open into a first face 106 of the support 100. An internal channel 108 connects the inlet and outlet bowls exit.

Channel 108 is in the form of a groove etched in a second substrate 100b glued to first substrate so that the latter covers the groove.

We observe that the depth of the groove is practically equal to the thickness of the second substrate 100b, so that only a thin wall 110 separates the channel 108 of a second face 112 of the analysis support 100.

In the example illustrated, the support 100 is general parallelepiped shape and the first and second faces are the opposite major faces and parallel.

The figure also shows in section 120a, 120b, 120c reagent reservoirs arranged between inlet and outlet bowls 102 and 102. tanks also open on the first side 106 of the analysis support 100. Fittings or passages 122a are provided to connect each of the tanks to channel 108.

For reasons of simplification, passages 122a are shown in the plane of the figure, so that the tanks do stand out no entry and exit cuvettes in Figure 1.

The liquid to be analyzed can be introduced into the entry bowls using a pipette.

Reagent reservoirs can be filled in the same way.

In the case of sequential analysis sequentially implement different reagents, in the case also where the reagents must be kept at a controlled temperature before use, it is best to use small tanks volumes powered by a syringe pump type system as shown in Figure 1B.

Figure 1B shows an analysis support according to FIG. 1A whose reservoirs 120a, 120b and 120c are respectively associated with means fluid supply 150a, 150b and 150c.

These means comprise plugs, or supply caps 152a, 152b, 152c applied from tightly above the tanks and connected to Syringe pusher 154a, 154b and 154c which contain reagents. The caps can be glued to the surface of the analysis stand or tight against the surface, using a seal.

The references 156a, 156b and 156c denote pressure sensors respectively provided on pipes connecting the syringe pumps to caps 152a, 152b and 152c, so as to control the pressure and / or flow of reagents.

Although not shown, a system of similar power can also equip entry bowls.

As shown in Figures 1A and 1B, the inlet bowls and tanks are subject to the atmospheric pressure, or at a pressure set by the feeding system, while a vacuum line 124 is applied to the outlet cuvettes.

A first spontaneous filling of the support analysis can be performed with a polar solvent (such as alcohol) followed by a nominal solvent to avoid the formation of bubbles. This filling puts benefit from a capillary effect in the channels.

After this first filling, the analytes and reagents are added.

The analytical product arriving at the cuvettes of output can be taken there also by means of pipettes.

Figure 2 shows more precisely and separately the two substrates 100a and 100b which form the analytical support.

It is observed that the analysis support comprises a plurality of inlet cuvettes 102 and a plurality outlet cuvettes 104.

The bowls are shaped like openings through openings in the first substrate 100a. These openings have a flared shape in V, forming a funnel.

Also, in the example in Figure 2, each inlet bowl 102 is individually connected to an outlet basin 104 through a channel 108.

The analysis support includes three reservoirs reagent 120a, 120b, 120c.

In this example, each tank is common to several channels 108 to which it is connected by means of connectors 122a, 122b. Reference 122a designates more precisely bores of the first substrate 110a connecting a tank to branches 122b corresponding, practiced in the second substrate 110b and respectively connected to the channels. (Good heard, tanks can also be individualized for the different channels).

Quantities of liquids (liquid to be analyzed, and reagents) that mix at the crossroads of branches 122b and channels 108 depend on the respective size of these branches and canals 108.

FIG. 3 shows an analysis support 100, conforms to that of FIG. 2, the substrates of which 100a and 100b are permanently glued.

The analysis support is represented above a corresponding thermal support 200.

The thermal support 200 has a face heat exchange 212 facing the second side 112 of the analysis support 100, in the vicinity of which are the channels. The heat exchange face 212 of the thermal support 200 and the second face 112 of the analysis support are intended to be put into contact.

The heat exchange face 212 has three thermostatic zones 220a, 220b, 220c each equipped one or more heat sources (no shown).

The three thermostated zones 220a, 220b, 220c are arranged to coincide with portions analysis support channels located in the vicinity respectively reservoirs 120a, 120b, 120c, or more precisely branches supplying the reagents.

The fluid in the channel 122b can pass through a only once each thermal zone or several times thanks to suitable channel patterns as shown Figure 5 described later.

Figure 4 is a schematic section of analysis support reported on the thermal support to represent in more detail the thermostated zones.

For the sake of clarity of the figure, the analysis support and thermal support are represented with a slight spacing. These supports are however in contact.

As indicated above, the zones thermostats may include several sources thermal. This is the case of the thermostated zone 220a. This includes a first heat source 230 formed of electrical resistors, such as by example of platinum microresistances. It involves also two sources 232 and 234 in the form of channels crossed by heat transfer fluids.

In the case of a PCR-type analysis, the electrical resistors of the first source 230 can be brought to a temperature of 94 ° C, the heat transfer fluid of the second heat source 232 at a temperature of 55 ° C and the heat transfer fluid of the third heat source 234 at a temperature of 72 ° C.

These temperatures correspond respectively to steps of denaturation, hybridization and of DNA elongation (see document (1))

Thermal sources can be miniaturized so that thermal support has a submillimetric thermal resolution (less than one millimeter).

FIG. 5 is a top view of a portion of a first substrate 100a of an analysis support and shows an alternative embodiment of a channel 108.

Channel 108 is folded in a pattern repeated geometric.

In the figure, there is also shown in dashed line the position of thermal sources a thermostatically controlled area 220, a thermal support can be associated with the analysis support. we observe that, thanks to the geometric pattern of the channel, a liquid analyze can, by traversing different sections of the pattern, be brought into thermal contact so sequential with different heat sources of the thermostatically controlled area.

Figures 6 and 7 show two variants of realization of the device making it possible to improve uniformity of temperature in the channels in insulating their upper face, i.e. the face opposite to said second face 112 of the support analysis. A first solution shown in Figure 6 is to perform in the upper part 100a of the hybridization support a cavity 160 (opening or not). This cavity coincides with at minus part of channel 108. A second solution, shown in Figure 7, is to set up between the upper and lower parts 100a, 100b of the analysis support a layer 100c of material poor conductor of heat. It is possible also to use an upper substrate equipped with a layer 100c of a thermal insulating material.

Figures 8 to 11, described below, give an example of a method of producing a support analysis as described above.

In a first substrate plate 100a, for example made of silicon, through openings are made, as shown in FIG. 8. These openings constitute the bowls or tanks 102, 104, 120a, 120b, 120c. The chemically etched openings are made with inclined sides 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) coincident 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. 7, can be carried out, for example, by etching CHF ₃ by dry process, the dimension of the perforation being defined by a mask or using the walls of the chemically created hole as a mask.

Figure 9 shows the etching of grooves, forming the channels 108, in a second substrate 100b for example silicon. The engraving is done at 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 100μm for a substrate 100b with a thickness of 250 to 450μm.

It can also be an SG6 dry etching allowing to make grooves deeper than wide for example 100µmx20µm.

A third step shown in Figure 10 includes sealing of the first and second substrates 100a and 100b, so as to communicating the cuvettes or reservoirs 102, 104, 120a, 120b, 120c, with channels (grooves) 108 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, shown in Figure 11, comprises thinning the second substrate 100b of way to preserve between the channel 108 and the surface 112 exterior that a thin wall 110.

This wall 110 has a thickness of around 10µm so as to encourage exchanges thermal.

Thinning is achieved by etching and / or by mechanochemical polishing.

A plurality of analysis supports conforming to the invention can be manufactured 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 a cutting slices with the saw to individualize the analysis supports.

Figure 12 shows an embodiment particular of the thermal support 200 of a device analysis according to the invention.

The thermal support 200 comprises for essentially a base 202 on which are arranged one or several thermostatic bars. In the example of the figure, the thermal support includes three bars thermostatically controlled 320a, 320b, 320c which form respectively three thermostatically controlled zones.

All or some of the bars can be drowned in a thermal insulating material. In the example of the figure two bars 320b and 320c are surrounded by a solid thermal insulating material, while the first bar 320a is left in free contact with ambient air on its side faces.

Each bar is equipped with heating and / or cooling.

The first bar 320a is equipped with a channel 322a which traverses it and which makes it possible to thermostate it by circulation of a heat transfer fluid.

The other bars 320b and 320c are also equipped with such channels 322b and 322c. The channels are respectively connected to thermostatic baths with pumping systems (not shown) for circulate the coolant.

The connection between the baths and the bars can take place by means of hydraulic connections not represented.

The channels can be of circular type, as the figures show, but can also be equipped with fin systems to optimize the heat exchange.

Additional heating elements can to be integrated in the bars. As illustration, the third bar 320c is equipped an electrical resistance 330. The resistance electric is used here as "hot spring" while the coolant is used as "cold source".

The second bar 320b has an element 340 for measuring the temperature, such as a resistance, used to control, for example, the temperature of the associated thermostated bath.

Reference 100 generally designates a removable analysis support arranged on the support thermal so as to be in contact with the bars Circulators. The detailed description of such support is not repeated here. We can refer to this subject explanations given with reference to figures preceding. The analysis support 100 can be simply placed on the thermal support 200. It can also be pressed against thermal support at means of a flange or a suction system not represented.

As the means for the control of temperature, that is to say in particular the baths thermostats and the thermostated bars, are integral with the thermal support or in connection fluidic with the thermal support, and as the analysis support is removable, it is possible to realize the latter in a simple and inexpensive way.

CITED DOCUMENTS

(1) Martin U. Kopp, et al., "Chemical Amplification: Continuous Flow PCR on a Chip", Science, Vol. 280, 15 May 1998, pages 1046-1048

(2) "Chip advance but cost constraints forced remain" in Nature Biotechnology, vol. 16, June 1998, page 509.

Claims (16)

1. Chemical and/or biological analysis device comprising:

   an analysis support (100) with at least one input bowl (102) to collect a sample, at least one output bowl (104) to output the said sample, at least one internal duct (108) passing through the support to connect the input bowl and the output bowl, and at least one reagent reservoir (120a, 120b, 120c) connected to each duct (108) between the input bowl and the output bowl, in which the input bowl, the output bowl and the reservoir open up onto a first face (106) of the analysis support, in which the internal duct (100) extends close to at least one second face (112) of the analysis support so that it is separated from the said second face by a thin wall (110) and

   a thermal support (200) independent of the analysis support (100), the thermal support having one heat exchange face (212) with at least one thermostat controlled area (220a, 220b, 220c), provided with at least one thermal source;

      the analysis support (100) possibly being added removably onto the thermal support (200) to put the heat exchange face (212) of the thermal support into contact with the second face (112) of the analysis support (100).
2. Device according to claim 1, characterized in that a thermal barrier is placed on one side of the ducts opposite a second face of the support.
3. Device according to claim 2, in which the thermal barrier comprises a thermal insulation layer-(100c) above the ducts.
4. Device according to claim 2, in which the thermal barrier comprises a thermal insulation cavity (160) above the ducts.
5. Device according to claim 1, in which the thin wall (110) is less than 100 µm thick.
6. Analysis device according to claim 1, in which the thermostat controlled area coincides with at least one analysis support area (100) located close to a connector (122b) between a reagent reservoir and a duct, when the analysis support is transferred onto the thermal support.
7. Device according to claim 1, in which the thermal support comprises cooling means and/or heating means.
8. Device according to claim 7, in which the heating means comprise at least one electrical resistance (230) .
9. Device according to claim 7, in which the cooling means and/or the heating means comprise at least one heat transporting fluid duct.
10. Device according to claim 1, comprising several input bowls (102) and several corresponding output bowls (104), each input bowl being connected to a corresponding output bowl through a duct (108).
11. Device according to claim 10, comprising several reagent reservoirs (120a, 120b, 120c), each reservoir being connected to each of the ducts (108).
12. Device according to claim 11, with external means (150a, 150b, 150c) of filling the reservoirs, comprising at least one push syringe (154a, 154b, 154c) with or without a reagent mixer, connected in a leak tight manner to at least one reservoir.
13. Device according to claim 12, in which the filling means comprise feed caps (152a, 152b, 152c) covering the reservoirs in a leak tight manner and each equipped with at least one duct connected to a corresponding push-syringe.
14. Device according to claim 1, in which the analysis support (100) comprises a first substrate (100a) with through openings that form bowls and reservoirs respectively, and a second substrate (100b) glued to the first substrate, the second substrate having grooves (108) covered by the first substrate to form ducts and coinciding with through openings.
15. Process for manufacturing an analysis support according to claim 1, comprising the following steps:

    formation of through openings in a first substrate (100a), each of the said openings corresponding to an input or output bowl, or a reagent reservoir,

    formation of grooves in a second substrate (100b), according to a pattern that joins at least two openings in the first substrate to each other,

    gluing of the first substrate to the second substrate in order to cover the grooves,

    thinning of the second substrate after gluing, in which the thickness of the substrate is greater than the maximum depth of the grooves.
16. Process for use of an analysis device according to claim 1, in which the analysis support is put into contact with the thermal support for a determined analysis time, at least one sample to be analysed and at least one reagent being added into the analysis support before the analysis phase starts, or during the analysis phase, and then the analysis support is removed from the thermal support after the analysis phase.

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