Miniaturized Circulatory Measuring Chamber With Integrated Chemo- And/or Biosensor Elements

This application is a 371 of PCT/DE95/00339 filed on Mar. 10, 1995.

1. Field of the Invention

The invention relates to a miniaturized circulatory measuring chamber with integrated chemo- and biosensor elements, to a method for its production and to its utilization.

2. Discussion of Background

It is known that chemo- and biosensor elements are integrated in circulatory systems. The best known example is flow injection analysis (FIA) (e.g.: G. Schwedt: Taschenatlas der Analytik, Georg Thieme Verlag Stuttgart, 1992).

Similar devices are used as microdialysis systems for determining the glucose concentration in human tissue (e.g.: F. J. Schmidt et al.: Calibration of a wearable glucose sensor, The International Journal of Artificial Organs, Vol. 15, No. 1, 1992, Pages 055-061).

It is likewise known that chemo- and biosensor elements which operate with polymeric membranes and gel layers for substance recognition, can be produced in silicon technology according to the containment principle (DE 41 15 414 A1). In this respect the polymeric membranes and gel layers are not produced on the surface of silicon chips but in the interior of the chip. Compared to planar structures, this has considerable advantages as regards simplicity of manufacture and long-term stability.

These sensors can also be incorporated in silicon technology in prefabricated circulatory measuring cells.

A disadvantage of the described prior art is that until now no sensors have been described which operate on the containment principle and are incorporated in common with a circulatory measurement chamber on a chip.

Therefore the object underlying the invention is to integrate chemo- and biosensor elements according to the containment principle in common with a circulatory measurement chamber on a chip. The measuring chamber with the integrated sensors is to be produceable preferably in silicon technology, but also in other technology. In addition the sensors are to be capable of integration with the circulatory measuring chamber, e.g. in common with pumps, reaction paths, valves, etc. in microsystems.

Thus it is proposed according to the invention to dispose a duct-shaped cavity in a chip in such a way that it is contact with the smaller opening of the containment. Then the substance-recognition material inserted in the containment forms in the area of the small containment opening the active sensor surface, to which the liquid measuring medium is brought to the duct, and thus can come into contact with the sensor surface.

Preferably, in the circulatory measuring chamber, a Si substrate is used as a carrier. The invention however includes all other appropriate materials. The substance-recognition membrane materials usable for the sensor arrangement and the design of the containment and of the preferred Si carrier itself are known from DE 41 15 414. Therefore reference is made expressly to the disclosed content of this document.

There are preferably used as substance-recognition membrane materials those which, due to their flow behaviour through one of the openings, preferably the large opening, can be brought in on the front-side surface into the containment. Filling may for example be effected by means of an automatic dispenser device according to the ink jet principle, or also according to the method described in DE 41 15 414.

All the immobilizing materials previously known from prior art can be used for potentiometric and in particular amperometric biosensors. Examples of these are gelatin, collagen, alginate, agar, cellulose. triacetate, silicon rubber, polyvinyl alcohol, polyurethane, HEMA and all other known materials. Photolinkable materials can be cross-linked by UV radiation after filling through the upper or lower containment opening.

The active substance-recognition components such as enzymes or antibodies are immobilised in these materials. This may be effected according to known methods, such for example as F. Scheller, F. Schubert: Biosensors, Birkhauser Verlag, Berlin, 1989. The electrical contact layers which in fact lead into the containment filled with the substance-recognition membrane material, preferably are composed of films of noble metals such as platinum, gold or silver.

Likewise all other films can be used which are generated by the known thin-layer technology such as evaporating or sputtering and then photolithographic structuring. It is also possible to evaporate on or sputter on these metal film structures through shadow masks or to spray them on through a solution according to the electrospray method. Instead of the noble metals, however electrically conductive materials such as graphite or others can also be used. It is advantageous for the circulatory measuring chamber, as also described in DE 41 15 414, to cover the substrate with an insulating layer. Particularly suitable for this purpose are SiO.sub.2 and/or other dielectrics such for example as Si.sub.3 N.sub.4 It is further preferred to provide at least the upper side containment opening with an encapsulating layer.

According to the invention a carrier described as above, preferably an Si carrier, is connected with the inserted containments by the second surface by means of at least one plate, the inserted cavity being so formed that it is in contact with the smaller opening of the containment. Preferably, the duct-shaped cavity is inserted on the side of the Si carrier upon which the smaller opening of the containment is located. In this way only one duct structure can be formed which is in contact with the smaller opening of the containment. Alternatively, it is however also possible to incorporate the duct-shaped cavity, not behind the Si carrier, but in the plate connected to the Si carrier. The essential factor is that in each case a contact is produced between the duct-shaped cavity and the smaller opening of the containment. Therefore the duct-shaped cavity can be so formed that it is made by recesses both in the Si carrier and in the plate. The plate itself can consist either of glass, photostructurable glass, ceramic, green tape, plastic or other appropriate material. In a further development of the invention, metal films are incorporated in the duct-shaped recesses as electrodes by means of the known thin-layer technology.

The invention further relates to a method of manufacturing the miniaturized circulatory measuring chamber described above. The procedure in this method can be such that either the duct-shaped cavity is incorporated in the second surface of the Si wafer, and then this duct-shaped cavity is closed by means of a plate, or an appropriate structure is inserted in the plate and this latter is connected to the second surface of the carrier.

The insertion of the containment into the wafer is basically known from DE 41 15 414, with the example of the Si wafer. In this respect the invention also includes this previously known method for manufacturing the containment structure.

The formation of the duct structure in the second surface of the wafer is preferably effected by anisotropic or isotropic etching processes. The subsequent closure of the duct is effected according to the method of anodic bonding with a plate, e.g. a glass lid, so that an entirely covered duct results, through which the measuring medium can then be brought on to the active surface of the sensor. The duct structured in the wafer can also be covered by a polymer film of photostructurable dry resist by lamination, or with a plastic film which is connected to the plate-shaped carrier by means of an adhesive.

When the duct-shaped recess is to be located in the plate itself, this latter may be effected for example by etching, and the plate thus structured connected to the wafer by means of anodic bonding. As already stated above, it is also possible to combine the two variant methods described above, i.e. on the one hand to produce a recess both in the second surface of the wafer and on the other hand to produce a recess in the plate and thus make a cavity.

The measurements to be carried out with this apparatus may be carried out in a known way, depending on the analyte and substance-recognition membrane material, according to the potentiometric or amperometric principle. For this purpose the measuring chamber with the chemo- or biosensor is preferably inserted in a FIA arrangement. It is however just as possible to use the measuring chamber and sensor in other circulatory apparatus. In addition, the sensors with the circulatory measuring chamber can be integrated with the aid of the known microstructure technologies in common with pumps, reaction paths, valves and other system components in Microsystems.

The advantages achieved by the invention reside in particular in the fact that sensor and circulatory chamber are produced on a carrier (chip) and thus integration of chemo- and biosensors is possible in Microsystems. Thus the known advantages of microstructure technology as regards feasibility of mass production, reliability and capacity for miniaturizing can be utilised.

Further features, details and advantages of the invention will become apparent from the following description of preferred embodiments of the invention, and with reference to the drawings, which show:

FIG. 1: a sensor according to the containment principle with a duct structure in the Si carrier, in a perspective view;

FIG. 2: a cross-section of the apparatus in FIG. 1;

FIG. 3: a sensor according to the containment principle analogous to FIG. 1 in a perspective view, yet with two containments;

FIG. 4: a perspective view of a sensor according to the containment principle, the duct structure in this case being produced in the plate itself;

FIG. 5: a simplified view of the possible methods of introduction and removal of the liquid medium.

FIG. 1 shows a sensor according to the containment principle comprising a silicon wafer 1 with containment structure 4, metal film 7 and a substance-recognition membrane material 8. The manufacturing process is known from DE 41 15 414. In this the silicon is covered with an insulating coating 15 of SiO.sub.2 and if necessary additionally with another dielectric (Si.sub.3 N.sub.4). The containment 4 is in a tapering structure from the frontal surface 2 to the second surface 3.

Before or after production of this sensor structure, the duct-shaped recess 6 is produced in the silicon by known anisotropic or isotropic etching processes. After the method of anodic bonding, this duct 6 is closed by a glass lid 5, so that a covered duct results, through which the measuring medium can be passed over the active surface of the sensor (small opening 9). The dimensions of the duct lie in the range of 10 to 1,000 .mu.m, depending on requirements, the overall height of the chip comes to 100 to 1,000 .mu.m.

In FIG. 2, the arrangement according to FIG. 1 is shown in cross-section. The cross-section extends along the duct-shaped recess. In addition to FIG. 1, it is shown in FIG. 2 how the membrane-filled containment structure is covered with an encapsulating layer 10.

FIG. 3 shows an arrangement with two containment sensors according to FIG. 1. The two active sensor surfaces (openings 9 and 9') in this case have contact with the measuring medium, which is passed through the duct (6").

Alternatively to the example in FIG. 1, in this case the cover for the duct (6"), is produced by a polymer film (5"). This film can for example be a photostructurable dry resist, which is laminated on according to the known method. The film may also however comprise a plastics film, which is connected to the plate-shaped carrier by adhesive.

For use as a glucose sensor, the substance-recognition membrane materials in the first containment (4) consist for example of gelatin with the enzyme GOD, and in the second containment (4') of gelatin without the enzyme GOD. Measurement is in this case effected according to the amperometric principle by application of an electrical voltage of approximately 0.6 volts respectively between the platinum films (7) and (7') and a reference electrode arranged externally or in the duct 6", and by measurement of the current.

If more than one containment structure is used in the circulatory arrangement, there results a multi-sensor system in a circulatory measuring chamber.

FIG. 4 shows an embodiment in which the duct-shaped recess (6') is produced for example by etching in the cover plate (5'). This cover plate, provided with the duct-shaped recess, is connected to the silicon wafer by anodic bonding. The Si wafer in this case has a thickness of 300 .mu.m.

Metallic films may also be inserted in the duct-shaped recesses according to FIGS. 1 to 4 as electrodes (not shown). If for example a silver film is applied to the inner wall of the duct and electrolytically chloridated, there is obtained an Ag/AgCl--reference electrode, by means of which measurements may be carried out potentiometrically or amperometrically against one or more containment sensors.

Alternatively to the embodiments in FIGS. 1 to 4, the plate-shaped carrier may also comprise glass, ceramics, green tape, plastics or other materials.

A possible means of introduction and removal of the liquid measuring medium is shown in FIG. 5.

For reasons of simplicity, the sensor is shown with the containment (4), the substance-recognition membrane material (8) and the encapsulating layer (10), but without insulating layers (15) and metal films (7).

In this case the duct-shaped recess (6) of the circulatory arrangement is connected with at least two openings (11) and (11'), which connect the duct-shaped recess with the first surface (2) of the silicon wafer.

This circulatory arrangement is connected to a plastic block 12, which has at least two openings (13) and (13'), through which the liquid measuring medium is introduced and removed. Seal rings (14) and (14') are provided for sealing between the silicon chip and the plastic block.