FR2889591A1 - Immobilized phase biosensor for receiving a signal emitted by bioelements and to convert the received signal into electrical signal by transduction unit, comprises a case and a removable immobilization support of bioelements - Google Patents

Abstract

An immobilized phase biosensor for receiving a signal emitted by bioelements and to convert the received signal into electrical signal by transduction unit, comprising a case and a removable immobilization support (16) of bioelements (18). The signal emitted by bioelements is converted into electrical signal, when the support is assembled on the case to isolate the bioelements and the transduction unit. The support: is fixed on a frame (30) of plug-in card in a slit of a case by thermal adhesive; is transparent to the signal; and extends vertically. An immobilized phase biosensor for receiving a signal emitted by bioelements and to convert the received signal into electrical signal by transduction unit, comprising a case and a removable immobilization support (16) of bioelements (18). The signal emitted by bioelements is converted into electrical signal, when the support is assembled on the case to isolate the bioelements and the transduction unit. The support: is fixed on a frame (30) in a slit of a case of plug-in card by thermal adhesive; is transparent to the signal; and extends vertically. The communication channel connects the bioelements and a source of external fluid with the biosensor, in which the fluid circulates from the bottom to top of the support. An independent claim is included for a plug-in card in a slit of a case.

Description

The invention relates to a biosensor comprising a housing, a support

  immobilization of bioelements and transduction means, the transducing means being adapted, when the support is mounted on the housing in a service position, to receive a signal emitted by said bioelements, and to convert it into a

  electrical signal. The invention also relates to a plug-in card in this case and on which said support is fixed.

  Various chemical or physical methods are used to detect polluting chemical constituents in sectors as diverse as the environment, health, or agri-food. In particular, it is known to detect polluting constituents by means of bioelements, in particular microorganisms, chosen to react in a specific manner to a target agent, that is to say to a specific chemical or to a physical agent present in their environment. A target agent may be, for example, ammonia, cadmium, chromium, cobalt, copper, mercury, zinc, a particular protein, a PCB, etc. In response to a target agent, some microorganisms consume oxygen for metabolism, produce CO2 or light. On the contrary, contacting with a target agent may also result in a decrease in the light produced by certain microorganisms.

  These microorganisms are conventionally incorporated in disposable measuring kits or in more durable and reusable analysis systems called biosensors.

  Some biosensors include a set of luminescent bacteria suspended in a liquid or immobilized on a support. The set of bacteria is optically coupled, for example by means of optical fibers, to a photodetector, for example a luminometer or a photodiode. These biosensors function in the manner of an optoelectronic probe: The bacteria are brought into contact with a fluid that may contain the target agent to be detected and / or measured, under conditions allowing the bacteria to react. The eventual reaction of the bacteria modifies the amount of light received by the photodetector. These modifications are interpreted by a microprocessor, for example to provide a measure of the content of the target agent or trigger an alert. Such biosensors are for example described in WO 02/023168 (EP 1 317 666) or in WO 00/33065.

  Biosensors that need to fix bacteria on a support, called immobilized phase biosensors, can be used in the laboratory or on site. However, if the bacteria die while using the biosensor, the biosensor is out of order and needs to be replaced unless new bacteria are established. The use of such biosensors can therefore generate high costs and requires a high qualification of the operators.

  There is therefore a need for an immobilized phase biosensor simpler and less expensive to use.

  The object of the invention is to satisfy this need.

  According to the invention, this object is achieved by means of a biosensor comprising a housing, an immobilization support for bioelements and transduction means, the transduction means being suitable, when the support is mounted on the housing in a position service, to receive a signal, preferably electromagnetic radiation, visible or not, emitted by said bioelements and to convert it into information on its nature and / or its intensity. The biosensor according to the invention is remarkable in that the support is removable from the housing independently of the transduction means.

  As will be seen in more detail in the following description, the disassembly allows a quick and simple replacement of the support, especially in case of destruction of bioelements. High operator qualification is not required for this purpose.

  In addition, a change of bioelements is possible by simply replacing the support. The biosensor according to the invention can thus easily adapt to a particular target agent. It is not necessarily specialized to detect one or more predetermined target agents.

  Preferably, the biosensor according to the invention also comprises one or more of the following preferred characteristics: the support is manually removable, that is to say that it can be mounted and removed from the housing without a particular instrument.

  - The support is arranged to isolate physically, in the service position, bioelements and transduction means.

  - The support is transparent to the signal.

  - The bracket is attached to a frame of a plug-in card in a slot of the housing.

  - The support is glued to the frame by means of a thermal glue.

  - The frame is made of ERTACETAL plastic.

  The biosensor comprises communication means capable of bringing into fluid connection, in the operating position, the bioelements and a source of fluid external to the biosensor, the fluid being capable of containing the target agent.

  In the service position, the support extends substantially vertically.

  The biosensor comprises means for circulating a fluid upwardly along the support.

  The signal is electromagnetic radiation, preferably visible light.

  The invention also relates to a plug-in card in a slot of a housing of a biosensor according to the invention, comprising an immobilization support of bioelements fixed on a frame.

  Other features and advantages of the present invention will appear on reading the following description and on examining the appended drawing in which: FIG. 1 represents a schematic perspective view of a biosensor according to the invention, a card according to the invention being about to be introduced into the housing of the biosensor; FIG. 2 represents a vertical transverse schematic sectional view of a card according to the invention in a service position. The card is shown inserted in a receiving block of a housing of a biosensor according to the invention, in the spatial configuration which it occupies preferably in the service position. The V axis represents a vertical direction.

  In the present description, the adjectives upper, lower, right, left, vertical, horizontal, etc. are used with reference to FIG.

  Disassembly means any operation to move enough support out of the service position to allow its replacement by a second support. The support is not necessarily detached from the housing during disassembly. For example, a carousel type device for successively inserting different media is possible.

  The biosensor 10 shown in FIG. 1 comprises a housing 12, a removable card 14 incorporating an immobilization support, in the form of a window 16, for immobilizing bioelements 18, for example bioluminescent bacteria, and means optoelectronic transduction system 20.

  The housing 12 is preferably transportable and has for example a length of about 60 cm, a width of about 40 cm and a height of about 35 cm. It comprises a reception block 22 (FIG. 2) presenting, on the front face 23 of the housing 12, a slot 24 in which the removable card 14 can be inserted to a service position where the transducing means 20 can detect a signal emitted by the bacteria by reaction with a target agent present in their environment. This radiation is for example a visible light.

  The transduction means 20 have the function of sensing the signal emitted by the bioelements, in this case the bioluminescence of the bacteria immobilized on the support 16, and converting it into information on its nature and / or its intensity. This information may for example take the form of an electrical signal, a visible light signal, an image, etc. The nature and the intensity of the signal can for example be deduced from the amplitude and the frequency of the electrical signal or the color and the luminous intensity of the light signal or of a luminous trace on the image.

  The transducing means 20 are known per se. They may for example comprise a photomultiplier, a photodiode, a CMOS circuit, a CCD camera, a photographic film, etc. Preferably they allow a continuous measurement of the bioluminescence.

  The card 14 has for example a parallelepiped shape of 35 * 35 mm side and 17 mm thick.

  The card 14 comprises a frame 30 and the optical pane 16, fixed on the frame 30. The pane 16 is preferably made of a borosilicate glass, but any transparent material making it possible to immobilize the bioelements and to transmit the signal they emit could suit. By way of example, mention may be made of a plastic based on PVC, for example PEEK.

  Preferably, the frame 30 is made of ERTACETAL plastic.

  Advantageously, this material is chemically and biologically inert and can be autoclaved at 120 ° C. while ensuring a good heat capacity.

  The frame 30 is pierced with a central through bore 32 of horizontal axis A opening through openings 34 and 36 on the right left and right faces 38 of the frame 30. Preferably, the bore 32 has a diameter of about 13 mm. The transparent optical pane 16 is inserted transversely inside the bore 32 so as to seal the latter.

  The pane 16 is preferably bonded to the frame 30, preferably by bearing on a shoulder 42 of the bore 32. Such bonding is delicate insofar as it is a matter of hermetically joining two materials with different physical properties, namely a borosilicate glass and a very inert plastic, the bonding having to be resistant to organic solvents such as isopropanol or ethanol while maintaining full biological compatibility. Adhesives operating by vulcanization have no effect on ERTACETAL, and polyvinyl glues commonly used for adhesion between different materials do not allow the maintenance of the optical glass 16 beyond one or two uses when the cards are exposed. to organic solvents.

  After extensive research, the inventors have managed to obtain an effective and prolonged adhesion by using a thermal glue. This adhesive remains fluid and inefficient beyond about 70 to 80 C, but it has the required adhesion properties once stiffened at room temperature. By way of example, mention may be made of Rocafix thermal glue. Silicone adhesives are also suitable.

  A right face 46 of the window 16, on which the bacteria are attached, defines a bottom of a measuring chamber 50.

  The bacteria are immobilized on the right face 46 of the optical pane 16.

  All known immobilization techniques are applicable: For example, the bacteria can be attached to heat directly on the right face 46, then covered with a fine mesh grid. They can also be immobilized by molecular functionalization, interacting for example with antibodies fixed on the right side 46.

  They can still be immobilized in a polymer (or immobilizer), of the PVA type, alginate, agarose or agar. A suspension of bacteria is then mixed with the polymer at a temperature where the latter is liquid. This mixture is then cast on the right face 46, preferably via the opening 36 of the bore 32, the card being arranged substantially horizontally. Then, it is treated so that it takes in mass, for example by letting it cool.

  Preferably, the bore 32 has on the right side of the window 16, close to the right face 46, a circumferential groove 52, preferably about 1 mm, in which the mixture of the immobilizer and bacteria can flow is. After caking, the bacteria membrane 18 thus formed can therefore advantageously be firmly held in the frame 30.

  The bacteria are chosen according to the target agent or target agents to be detected or measured. For example the strain TBT3 can be chosen for the detection of tributyltin.

  The portion of the bore 32 located to the right of the window 16 defines a side wall 54 of the measuring chamber 50.

  An inlet duct 56 is formed in the frame 30 and opens on the one hand into the lower part of the lateral wall 54 of the measurement chamber 50 and on the other hand onto a lower face of the frame 30 through an opening of entrance 58.

  An outlet duct 60 is formed in the frame 30 and opens on the one hand into the upper part of the lateral wall 54 of the measurement chamber 50 and on the other hand onto an upper face of the frame 30 via an outlet opening 64 .

  Preferably, the inlet ducts 56 and 60, intended for the introduction and extraction of test fluids in the measuring chamber 50, are aligned along a substantially vertical axis B.

  The block 22 has upper 70 and lower 72 discharge and fluid supply channels, opening on upper faces 74 and lower 76 of the slot 24 through outlet openings 78 and inlet 80, respectively. Preferably the upper and lower channels 70 and 70 are coaxial with axis B. In the operating position, the inlet and outlet openings 58 and 64 of the card 14 are opposite the inlet and outlet openings 80 and 78 of the receiving block 22.

  Sealing means, for example in the form of O-rings 82 and 84, are provided to provide a tight connection between the inlet 58 and outlet 64 openings of the board 14 and the inlet and outlet openings 80 78 of the receiving block 22, respectively.

  In the service position, the bacterial membrane thus extends substantially vertically, the flow of fluid being effected from the bottom upwards. Advantageously, this type of circulation makes it possible to reduce the zones of hydraulic inertia in the measuring chamber 50.

  In the service position, the right opening 36 of the bore 32 is closed by the right face 86 of the slot 24 of the block 22. Preferably, a seal 88 prevents leakage of fluid from the measuring chamber 50 through the right opening 35 36 of the bore 32.

  In a variant of the invention, not shown, the bore 32 does not open on the face 40 of the frame 30, but forms a blind hole. Advantageously, the volumes of the measuring chamber 50 and the zones of inertia (regions in which the speed of the fluid to be analyzed is likely to decrease or even to cancel during the analysis) are reduced. In addition, no seal 88 is no longer needed. Finally, the card change no longer requires cleaning the right face 86 of the slot 24, the latter never again in contact with the fluids analyzed.

  This embodiment however makes it more difficult to place the bacteria on the window 16, no direct access to the right face 46 is then available. An improvement consists in making an opening bore, as shown in FIG. 2, so as to be able to easily access the right face 46 of the window 16 to place the bacteria therein, then to plug the right opening 36 of the bore means of a waterproof cap.

  In a variant, the frame 40 could also be formed in two half-shells shaped so as to allow access to the right face 46 of the window 16 in the disassembled position of the two half-shells while ensuring a sealing of the measuring chamber 50 relative to at the right face 86 of the slot 24 in the assembled position of the two half-shells.

  Preferably, the seals 82, 84 and 88 are attached to the surface of the frame 30, for example by a force fit in grooves arranged around the corresponding openings. The depth of the grooves is determined to allow the sliding of the card 14 in the slot 24 while ensuring a crushing of the joints sufficient to ensure the seal between the card 14 and the block 22.

  The total volume of the measuring chamber 50 is preferably about 1.32 cm 3.

  The block 22 also comprises a bore 90, preferably substantially horizontal axis A, serving as a housing for an optical fiber bundle 92. The optical fiber bundle 92 comprises a core 93 formed by a plurality of optical fibers, and, Preferably, a sheath 94 surrounding the core 93 to protect and optically isolate it.

  The bore 90 opens into the slot 24, so that the end faces of the optical fibers of the core 93 are opposite the left face 95 of the optical pane 16.

  The optical fiber bundle 92 is secured to the block 22 by means of a ferrule 96. The ferrule 96 advantageously allows precise positioning of the ends of the fibers of the optical fiber bundle 92 along a plane Po parallel to the plane P, of the left face 95 of the optical window 16.

  Fixing the fiber bundle 92 by means of a ferrule 96 also makes it possible to identically reproduce the position of the core of the bundle of fibers 96 after possible maintenance operations. The ferrule 96 allows a fast and simple positioning of the fiber bundle 92, avoiding the use of a more complex system in which the signal emitted by the bacteria is focused in the core of the fiber bundle by a biconvex lens, which requires an extremely high geometry. precise. The present invention, however, also relates to a biosensor comprising such a system.

  Unlike bioluminescent bacterial biosensors immobilized phase according to the prior art where the bacterial membrane is directly attached to the surface of a bundle of optical fibers, the use of a removable card 14 according to the invention imposes a separation of the left face 95 of the optical pane 16 and the fiber bundle 92. Preferably, the spacing α between the planes Po and P, however, is less than about 1 mm. Advantageously, the dispersion of the photons between the bacteria and the optical fibers is limited.

  In addition, preferably, the pane 16 isolates the measuring chamber 50 from the optical fiber bundle 92. No fluid therefore circulates between the bacteria 18 and the optical fiber bundle 92. Advantageously, it is thus possible to analyze samples disposed in the measuring chamber 50 which impede the transmission of the signal emitted by the bacteria, for example opaque samples or disorders.

  The slot 24 of the block 22 is shaped to ensure accurate positioning of the card 14 relative to the optical fiber bundle 92, while allowing the seals 82, 84 and 88 to be crushed. When the card 14 is inserted in the block 22, the optical pane 16 is located exactly in front of the ferrule 96.

  Preferably, the block 22 finally comprises a housing 98 situated a few millimeters from the optical pane 16 and receiving a heating element 100. The heat transfer from this heating element 100 to the card 14 is achieved by simple thermal conduction due to the thermal properties ERTACETAL plastic.

  The housing 12 then preferably incorporates temperature control means, not shown, for maintaining the bacteria at a set temperature by means of the heating element 100.

  The card 14 is advantageously completely devoid of any electrical or electronic component. It can therefore be replaced in a few minutes depending on the measurement desired by an operator without special qualifications.

  The card 14 is preferably equipped with a gripping means, for example a not shown ring, facilitating its introduction and its manual extraction.

  The housing 12 preferably further incorporates a hydraulic circuit 102 (see FIG. 1) making it possible to introduce and extract various fluids, in particular liquids containing one or more target agents, simultaneously or successively, into the measuring chamber 50. Preferably the hydraulic circuit 102 allows the separate passage, alternatively, of at least three fluid samples extracted from sources 104 in the measuring chamber 50, ensuring recovery of waste. This recovery advantageously allows biological decontamination and avoids any release, even accidental bioelements in the environment.

  More preferably, the housing 12 incorporates unrepresented ventilation means, preferably continuous operation, in order to limit both the heating of electrical appliances and possible phenomena of condensation.

  A source of electrical energy or electrical connection means, not shown, are also provided for supplying the various electrical consumers of the biosensor.

  The operation of the biosensor described above is as follows.

  To detect or measure a target agent, the operator inserts a card 14 in the slot 24 so that the window 16 separates the optical fiber bundle 92 from the bacteria membrane 18. Keying means can be provided to prevent any error during of this step.

  The card 14 is inserted to the service position shown in FIG. 2. In the operating position, the inlet and outlet openings 58 of the card 14 are opposite the entry and exit openings 80. 78 of the receiving block 22 respectively, and the window 16 faces the optical fiber bundle 92. Preferably, stops, not shown, are provided in the housing 12 to allow the depression of the card 14 in the slot 24 only to the service position. The operator thus encounters no difficulty in positioning the card 14 in this position.

  If necessary, the operator sets a set temperature, assisted for example by a controller displaying in real time the temperature of the block 22 and controlling the heating element 100. This temperature can for example be measured in real time by means of a controller. type J thermocouple, not shown, placed in block 22.

  As the plastic ERTACETAL has excellent thermal conductivity, the temperature of the block 22 measured by the thermocouple can advantageously be considered equal to that of the card 14.

  The operator then controls the hydraulic circuit 102, in particular by setting the flow rate of the fluid injected into the measuring chamber 50, the sequence of the different fluids to be passed through the measuring chamber 50, and the times during which these fluids must be in contact. contact with bacteria 18 in the measuring chamber 50.

  The arrows in thick lines in Figure 2 represent the circulation of the fluid.

  The fluids diffuse into the immobilization matrix and react the immobilized bacteria that emit light. This light passes through the window 16, then the air space between the window 16 and the fiber bundle 92. It then enters the optical fiber bundle 92 which transmits it to a converter which ensures their conversion into an electrical signal. This signal can be recorded, interpreted or viewed, preferably in real time.

  The housing 12 is preferably sufficiently hermetic to light to allow continuous measurement of the bacterial bioluminescence.

  The collection of photons by the fiber bundle 92 is improved by increasing the diameter of the core 93 of the fiber bundle 92. Preferably, this diameter reaches that of the optical pane 16 (preferably about 6.35 mm). However, a fiber core diameter of 1 mm is already satisfactory.

  At the end of the measurement, the operator stops the flow of fluid in the measuring chamber 50. He can then extract the card 14 and replace it with another, for example to measure new target agents or because the first card 14 is now defective. The card 14 may be designed to be disposable after use without the cost of operating the biosensor is impaired.

  As is clear now, the manipulation of the biosensor according to the invention is particularly simple, the intervention of the operator being minimal.

  In addition, the biosensor can be automated. The saving of time is then very significant.

  The possibility of easily replacing one card with another also advantageously reduces the maintenance of the biosensor. Ready-to-use cards can be easily manufactured and marketed, regardless of the means of transduction.

  Of course, the present invention is not limited to the embodiment described and shown provided by way of illustrative and non-limiting example.

  In particular, the biological part of the biosensor is not limited to a membrane of bioluminescent bacteria attached to a support. Other bioelements than bacteria or other microorganisms are possible. For example, particular proteins, enzymes or antibodies may be used.

  The response of the bioelements is not necessarily in the form of a light signal, other electromagnetic radiation being conceivable. The response of bioelements can also result, for example, in a change in the pH of the fluid present in the measuring chamber or its oxygen content or carbon dioxide. In the case of an oxygen measurement, the support is preferably a Teflon membrane permeable to oxygen but impermeable to bioelements. The transducing means are then adapted accordingly.

  Additional organs may also be provided depending on the bioelements. For example, a light source may be needed to activate certain bioelements. Preferably, this source illuminates the bioelements via the optical fiber bundle and the glass. In general, the biosensor may comprise means for transmitting a signal arranged so as to irradiate the support in the service position and means for receiving this radiation after interaction with the support, the bioelements being able to modify said interaction under the effect of the target agent to be detected.

  A substrate may also be introduced into the measurement chamber to enable the generation of a sensitive reaction of the bioelements to the presence of the target agent.

  The transduction means are not limited to those described either. For example, the biosensor could comprise a photodiode arranged to receive the light emitted by the bioelements and capable of directly transforming this light into information on its nature and / or its intensity, in particular in an electrical signal.

  The card according to the invention can also be "multichannel", that is to say be able to detect or measure several target agents, simultaneously or not.

  The shapes and dimensions of the card, block or housing are not limited. It is the same for the orientation of the biological part and the hydraulic circuit lines. The bores can also be of any shape.

  The measurement chamber and the fluid supply and discharge means opening into this chamber could also be located to the left of the window, provided that the fluids to be analyzed are sufficiently transparent to the radiation emitted by the bioelements to reach the beam. of optical fibers. The fluid would then circulate between the immobilized bacteria on the left side of the glass and the optical fiber bundle with which it would be in contact.

  Finally, the biosensor and the card according to the invention can be used to detect and / or measure a target agent, in the laboratory or on site, to analyze any fluid, liquid or gaseous.

Claims (9)

  1. Biosensor comprising a housing (12), an immobilization support (16) of bioelements (18) and transducing means (20), the transducing means (20) being suitable, when the support (16) is mounted on the housing (12) in a service position, receiving a signal emitted by said bioelements (18) and converting it into information on its nature and / or intensity, characterized in that the support (16) is removable of the housing (12) independently of the transducing means (20).   2. Biosensor according to claim 1, wherein the support (16) is manually removable.   A biosensor according to any one of claims 1 and 2, wherein the support (16) is arranged to physically isolate, in the service position, the bioelements (18) and the transducing means (20).   4. Biosensor according to any one of the preceding claims, wherein the support (16) is transparent to said signal.   A biosensor according to any one of the preceding claims, wherein the carrier (16) is secured to a frame (30) of a card (14) plugable into a slot (24) of the housing (12).   6. Biosensor according to claim 5, wherein the support (16) is bonded to the frame (30) by means of a thermal glue.   7. Biosensor according to any one of the preceding claims, comprising communication means (102) able to put in fluid relation, in the operating position, the bioelements (18) and an external fluid source (104) to the biosensor.   8. Biosensor according to any one of the preceding claims wherein, in the operating position, the support (16) extends substantially vertically.   9. Biosensor according to any one of the preceding claims comprising means for circulating a fluid from bottom to top along the support (16) in the service position.   10. A plug-in card (24) of a housing (12) of a biosensor according to any one of the preceding claims, comprising an immobilization support (16) of bioelements (18) fixed to a frame ( 30).