Abstract:
A microfluidic diagnosis kit used in biochemical analysis obtains a premeasured amount of reagents in an applicator for an analysis system. The required reagents are proportioned as non-volatile agents and a microfluidic system is provided, whereby the reagents are automatically dissolved in a solvent and fed to a sensor module to carry out measurement.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application is based on and hereby claims priority to German Application No. 101 11 457.5 filed on Mar. 9, 2001, the contents of which are hereby incorporated by reference. This application is related to MODULE FOR AN ANALYSIS DEVICE, APPLICATOR AS AN EXCHANGE PART OF THE ANALYSIS DEVICE AND ANALYSIS DEVICE ASSOCIATED THEREWITH, filed concurrently by Walter Gumbrecht, Manfred Stanzel, Manfred Wossler and Jörg Zapf and incorporated by reference herein. 
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The invention relates to an analysis device for use in biochemical analytics, with an applicator for decentralized use, containing a first housing, a fluidic system and a sensor module, which together with a second housing forms a measuring and analysis system. 
     2. Description of the Related Art 
     One of the requirements for the decentralization of chemical-biological analyses in medical technology is that reagents are flexibly available. In the present context, decentralized means that the analyses are carried out, often not with a high throughput, as in large-scale clinical laboratories. Reagents for chemical-biological analysis are often very costly and greatly restricted in their service life/usability, at least after the container has been opened, for example outgassing of O 2  and CO 2  from blood-gas calibrating solutions or decomposition of biochemical components, so that efficient, low-cost use is made more difficult or impossible. 
     Decentralized analyses are therefore carried out particularly advantageously with so-called disposable kits, in which the reagents are provided in a pre-apportioned, individually packed amount required for the specific instance. Known for example is a system (i-STAT Corporation, 303A College Road East, Princeton, N.J. 08540; U.S. Pat. No. 5,096,669) in which a calibrating solution required for the calibration of blood-gas/electrolyte sensors is stored in a gastight aluminum/plastic bag with a content of &lt;1 ml for a disposable sensor and is opened during operation of the disposable sensor by “piercing” the bag wall. 
     Such a concept of providing calibrating solutions is not suitable for the use of reagents which in dissolved form are subjected to a decomposing process, such as for example enzymes, sensitive organic substances, such as in particular p-aminophenyl-phosphate, p-aminophenyl-β-galactoside. This procedure is also complex and expensive, and there is also the risk of the bags leaking and consequently the entire diagnosis of the blood gas analysis being falsified, for example by escaping gases. Furthermore, in the case of the prior art, only a single calibrating solution is realized and consequently only a one-point calibration is made possible, which casts doubt on the reliability of the results and consequently reduces the acceptance among customers. Although the theoretical possibility of providing more than one calibrating solution is mentioned in U.S. Pat. No. 5,096,669 A, this would increase the complexity, and consequently the production costs, of the disposable article. 
     Furthermore, the possibility of admixing dry reagents with the sample, i.e. for example the blood sample, is mentioned in U.S. Pat. No. 5,096,669 A. However, this does not solve the problems involved in providing reagents when, for complex diagnostic operations, a number of reagent solutions have to be passed over a sensor device, for example a sensor chip or sensor module, in series before and/or after entry of the sample fluid, for example in the case of analyses with the aid of so-called enzymatic amplification: this involves sequentially feeding in 1. buffer solution, 2. sample, 3. buffer solution, 4. enzyme label reagent, 5. buffer solution, 6. enzyme substrate. 
     Furthermore, in Dirks, G. et al. “Development of a disposable biosensor chipcard system”, Sens. Technol. Neth., Proc. Dutch Sens. Conf, 3rd (1988), pages 207 to 212, there is a description of a measuring system for biomedical applications in which a so-called chip card is made from a flat container with a number of cavities and a system of fluid channels, with an ISFET which serves as a sensor being introduced into the channel system. In the case of this system, it is in particular a matter of separately feeding a measuring fluid on the one hand and a calibrating or reagent fluid on the other hand to the sensor from separate containers. 
     Furthermore, in the monograph by Langereis, G.R. “An integrated sensor system for monitoring washing process”, ISBN 90, there is a description of systems with sensors concerned with integrating in fluidic devices sensors which have their signals electrically tapped. 
     SUMMARY OF THE INVENTION 
     The problems of feeding in reagents are not satisfactorily solved in the prior art. On the basis of the prior art, it is therefore an object of the invention to improve an analysis device of the type stated at the beginning for decentralized use. 
     In the case of the invention, the reagents are kept as solid substances in a pre-portioned form in a microfluidic system in the applicator and, in combination with a suitable operating mode, are automatically dissolved and fed to the analysis system, in particular from a single solvent reservoir for at least one complete analyzing operation, in a number of partial steps. The reagent solutions are consequently produced ‘in situ’ in the fed-in solvent and are provided only immediately before they are to be used. 
     By contrast with the prior art—the invention advantageously achieves a technical realization of a number of reagent solutions from just one solvent reservoir for at least one analyzing operation. In the case of the prior art, and specifically in U.S. Pat. No. 5,096,669 A, it is not stated whether, and in particular how, a number of different reagent solutions could be sequentially provided from dry reagents. 
     In the case of the invention, the reagents are preferably kept in solid form or dissolved in a solid adjuvant, for example water-soluble polymer. An example is the provision of means for prescribing a defined pCO 2  value for medical diagnostics: for this purpose, apart from the salts required, such as, inter alia, NaCl and KCl, a solid base substance, for example NaHCO3, and a solid acid substance, for example citric acid, are also introduced. During the dissolving of the reagents, the solid base substance and solid acid substance react, as known in the prior art for example from effervescent tablets, and produce a defined amount of CO 2 . Since significantly smaller concentrations than in the case of effervescent tablets are required, no formation of bubbles occurs. 
     Furthermore, the provision of a number of reagent solutions for complex analyzing operations is possible. An advantageous example is an immunoassay with enzymatic amplification. In this case, a washing step with a buffer solution may have to be performed after the sample fluid has been applied to the sensor or sensor module. This may take place either directly from the reservoir or advantageously by dissolving solid buffer substance, for example dissolved in water-soluble polymer and placed in a micro-throughflow channel from a water reservoir, which may be placed in the applicator or in the second housing. This is followed by enzyme label being fed in, to be precise advantageously likewise placed as a solid substance, if appropriate dissolved in the water-soluble polymer, in the micro-throughflow channel, which for its part is then dissolved from the buffer reservoir or advantageously from the same water reservoir. Finally, by analogy with the previous steps, the preparation and feeding in of enzyme substrate solution takes place. 
     Chemical equilibriums and the rate of reaction of chemical or biochemical enzymatic reactions are subject to a strong temperature influence. For example, the partial pressures of the dissolved blood gases O 2  and CO 2  are dependent on temperature and, in the case of laboratory equipment, are therefore always measured at 37° C. With sensors based on silicon technology and microelectronic circuitry, it is now possible to measure and control the temperature of the sensor chip, and consequently also the temperature of the sample. A restriction in this respect was until now constituted by the fact that, although a silicon chip can be electrically heated up, for example by resistance heating, it cannot be electrically cooled. This is achieved by an advantageous development of the invention. 
     A further advantageous application possibility of the invention is the amplification of DNA/RNA (deoxyribonucleic acid/ribonucleic acid) samples by the exponential replication method with the so-called PCR (Polymer Chain Reaction), i.e. the polymerase chain reaction method. For this purpose, the sample fluid must be cycled 20 to 40 times between two temperatures, typically between 40° C. and 95° C. In the case of the prior art, the cooling process is speed-determining for this thermal cycling. 
     The latter problems can also be solved in a practical way by the invention: for a specific application, a particularly advantageous embodiment similar to the chip module of a chip card comes into consideration as the applicator. 
     In the case of the chip card module, the silicon chip is advantageously mounted on a gold-coated copper layer only approximately 50 μm thick. This is the middle metal zone of known chip card modules, which is not used for electrical contacting points in the card reader. This free zone can consequently be used in the card reader, which acts here equally as an evaluation device, for directly contacting a cooling element, for example a Peltier cooler, to the corresponding location of the chip card module. On account of the placement (50 μm thick metallic contact with respect to the chip), an efficient heat transfer is consequently possible, so that a defined temperature can be set very quickly, in particular also by cooling. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       These and other objects and advantages of the present invention will become more apparent and more readily appreciated from the following description of exemplary embodiments, taken in conjunction with the accompanying drawings of which: 
         FIGS. 1 to 3  are cross-sectional views of three different embodiments of a so-called diagnosis kit an applicator and a reader, 
         FIG. 4  is a sectional view of a reader with an integrated cooling element for direct thermal coupling to a chip-card contacting zone, 
         FIG. 5  is a plan view of the contacting zone of the module according to  FIG. 4 , 
         FIG. 6  is a plan view of a sample and multichannel reagent feed with a distribution system in the reader and 
         FIGS. 7 and 8  are plan views of a multichannel reagent feed, modified with respect  FIG. 6 , by displacing the chip card into two positions. 
     
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
     Reference will now be made in detail to the preferred embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like reference numerals refer to like elements throughout. 
     In  FIGS. 1 to 4 , an applicator with a sensor module is designated throughout by  10 , while in  FIGS. 6 and 7  a modified applicator is designated by  60  or  70 . For measuring, such an applicator  10 ,  60  or  70  is pushed into a reader  20  or  80 . 
     In  FIGS. 1 to 3 , a sensor module  15 , for example a silicon chip  1  with a sensitive area  2 , has been introduced into the applicator  10 , encapsulated and electrically contacted on a carrier. Such a sensor module is the subject, inter alia, of a corresponding patent application with the same priority. On the sensor module  15  there is a microfluidic channel  11 , to which a channel  110 , in which reagents or adjuvants  16 ,  16 ′, . . . ,  16 ′″ are arranged, leads from an inlet  12  with a valve arrangement/seal. Behind the sensor module  15 , i.e. after the measurement, the substance is taken up by an outlet channel  18 . 
     The reader  20  has in the housing fluid channels  21 , with water, for example, being brought into the applicator  10  in the first channel  21 , from a solvent store outside or inside the device, via a seal  22 . The used measuring fluid is pumped via the seal of the outlet  23  by a pump  25  to a waste container, not represented in  FIG. 1 , inside or outside the reader. 
     The arrangement according to  FIG. 2  corresponds substantially to  FIG. 1 , with the modifications that a solvent reservoir  29  has been placed in the second housing of the reader and, after the sensor module  15 , the microfluid channel  11  has a widening or lengthening as a collecting container  28  for the purpose of taking up used solution or analyzed sample. If appropriate, such a widening is adequate as a reservoir for waste. In this case, only air is passed into the reader by the pump  25  via the outlet  13  with valves or seals  13  or  23 . 
     In a corresponding development, the applicator  10  according to  FIG. 3  includes a separate container  39  for a solvent store, i.e. for water. The water feed from the external device  20  is not necessary here. It is merely the case that the valve  12  from  FIG. 1  is specifically formed as an air-admitting valve  38 . 
     In  FIG. 4 , the applicator with the sensor module is formed substantially in a way corresponding to  FIG. 1 . Specifically in the reader, a heating and/or cooling element, for example a Peltier element  30 , is arranged at the position of the sensor module  15  with the applicator pushed in. The Peltier element  30  has a cooling plate  31 . With the Peltier element  30 , effective and rapid cooling of the sensor module  15  to a defined temperature is possible. 
     This arrangement can preferably also be used for the amplification of DNA/RNA (deoxyribonucleic acid/ribonucleic acid) by the exponential replication method, the so-called PCR (Polymer Chain Reaction). For this purpose, the DNA/RNA sample and required reagents, such as for example nucleotide triphosphates, primer DNA/RNA and polymerase in buffer solution are fed to the sensitive area of the sensor chip via the microfluidic channels. The reaction space (space over the sensitive area of the chip with a height of up to several hundred μm), is then cycled approximately 20 to 40 times between two temperatures, typically between 40° C. and 95° C. In the case of this arrangement, the entire DNA/RNA replication process can be carried out in a few minutes. 
     The operating principle of the chip module  15 , and in particular of the actual sensor chip, is illustrated in  FIG. 5 . On the electrical contact side  3 , i.e. the rear side, of the module  15  with the sensor chip  1 , contacting zones  3   I , . . . ,  3   VIII  can be seen as individual terminals, which correspond to the customary contacting points for chips which can be integrated into a card. On the sensitive side  2  of the chip  1 , bonding pads run from the corners of the chip to the contacts of the contacting zones  3   I , . . .  3   VIII . 
     The latter arrangement is the subject of a parallel application with the same priority date (German patent application number 101 11 458.5-52 of 09.03.2001), to the disclosure of which reference is expressly made. 
     It is evident from  FIG. 5 , in the plan view, that for the case of chip card technology with a silicon chip and rear area contacts  3   I  to  3   VIII , as known from customary chip cards, the Peltier element  30  directly touches the effective area of the sensor on the rear side, and consequently brings about effective heat transfer. 
     Represented in  FIG. 6 , in the plan view, is a chip card  60  which has a sensor module  15  with a rear Peltier element  30  and electrical chip contacts  3 ′ to  3   VIII . There is a sample port  68  as a sample feed opening and also a sample channel  69  for feeding the sample to the sensor module  15 . Also present are reagent channels with non-volatile reagents in a pre-measured amount. There is a first reagent channel  61 , which is connected to a water inlet  62 . Furthermore, there is a second reagent channel  61 ′, which runs parallel to the first reagent channel  61  and, by contrast with the reagent channel  61 , is not yet filled with solvent in the representation of  FIG. 6 , and consequently does not yet contain any reagent solution. The second reagent channel  61 ′ can be connected to a second water inlet  62 ′. Further parallel-connected reagent channels  61 ″ may be provided, with water inlets  62 ″, which are respectively parallel-connected, so that altogether n reagent channels and n water inlets are present. After flowing past the sensor module  15 , there is an outlet  63 . In the reader  20  there is a water distribution system with valves. 
     The operating principle of an arrangement modified with respect to the arrangement of  FIG. 6  is illustrated on the basis of two subfigures  7  and  8 . On the applicator  70  there are in turn a sample feed opening  78 , as the sample port, and also a sample channel  79  for feeding the sample to the sensor module  15 . Also present are reagent channels  71  to  71   n ′ and an outlet  73 . In the case of this arrangement, in the reader  80  there is a single inflow channel, which has a single inflow opening  81  and a single outflow opening  83 . In the position illustrated in  FIG. 7 , the first reagent channel  71  is congruent with the inflow opening  81 , while in the position illustrated in  FIG. 8 , the second reagent channel  71 ′ is congruent with the inflow opening  81 . The outlet opening  83  is in this case formed as a slit opening, so that in both positions of the applicator and also in further positions it is always possible for the outlet  73  to be toward the outlet  83  of the reader  80 . 
     In the case of the arrangements described, it is important for the microfluidic analysis/diagnosis system that it is possible to store each time a defined amount of at least one reagent, to store the reagent in a stable form, to store the reagent as a pure and solid substance or to store the reagent in a dissolved or mixed form in a further substance (adjuvant). Such an adjuvant may be solid or liquid. A solid adjuvant may be, for example, a water-soluble polymer such as polyvinyl alcohol. The adjuvant may serve the purpose of diluting reagent (for example when using enzymes which are to be used in very small amounts) and/or placing them in a container in such a way that they are geometrically defined and have good adhesion. 
     Irrespective of the representation in the drawings, the applicator has a defined geometry as a plastic housing. In the plastic housing are micro-channels with a cross section of for example 1 mm×0.1 mm and a length of several mm, which form a fluid system. Reagent dissolved in the adjuvant may be placed in a defined quantitative gradient along a micro-channel. The plastic housing may contain a defined store of solvent. Furthermore, the plastic housing may contain a defined empty volume for the disposal of waste. 
     In the case of all the examples, the plastic housing as the applicator in combination with the reader and the suitable operating mode allow reagent and solvent to be brought together. The plastic housing is connected by at least one micro-channel to a reader. The reader contains a storage container in which there is, in the simplest case, water, adequate for a number of analyses. The reader may contain a container for the disposal of the waste from a number of analyses and also contains means for conveying the solvent through the micro-channels to the sensor module and further to the waste container in the plastic housing or in the reader. The solvent, no matter from which store, is passed over the geometrically placed reagent-adjuvant mixture in such a way that a defined solution can be produced, under some circumstances by the solvent remaining for a time over the solid substance, pumping forward and back, heating or the like. 
     In the way described, even uncritical reagent solutions, such as buffer solutions or the like, can be generated in the analysis kit. Although stable buffer solutions could also be fed in from a storage container in the reader, with the applicator removed the interfaces between the reader and the applicator are susceptible to evaporation of the solvent and consequently precipitation of solid substance (for example salt) and soiling/encrusting of the fluidic interfaces. This is not to be feared in the case in which pure solvent is stored in the reader. What is more, this method allows a number of reagent solutions to be realized in a simple way by arranging the reagent channels from just one solvent reservoir in parallel. 
     A special case exists when providing reagent for sensors of dissolved gases, for example in the case of sensors for determining the blood gases oxygen and carbon dioxide. Here, the sensors must be calibrated with media, for example solutions, which have a defined concentration of the respective gases. 
     In the case of blood-gas sensors, which for example for so-called “point of care diagnostics” have to be calibrated once before they are used a single time, the sensors for pO 2  and pCO 2  have to be brought into contact with buffer solutions of known pO 2  and pCO 2  values. While previously a single solution with known pO 2  and pCO 2  values, already prepared during the production of the module, was filled into a small gastight bag and fitted into the diagnosis module, now the calibration can be performed as desired, in particular as a two-point calibration. 
     This consequently provides an analysis device which can be used in a variety of ways in biochemical analytics, for example for use in medical diagnostics, forensics, for food monitoring and for environmental measuring technology. The decentralized use of the applicator and reader allows time-saving low-cost examination on the spot, in particular in clinics and doctors&#39; own practices, of for example blood, liquor, saliva and smears, for example for viruses of infectious diseases. This may include, if necessary, not only simple typing of the germs, but also the determination of any resistances to antibiotics, which significantly improves the quality of the therapy and consequently can reduce the duration and cost of the illness. Apart from the diagnosis of infectious diseases, the diagnosis system is for example also suitable in medicine for blood gas/blood electrolyte analysis, for therapy control, for early detection of cancer and for the determination of genetic predispositions. 
     The invention has been described in detail with particular reference to preferred embodiments thereof and examples, but it will be understood that variations and modifications can be effected within the spirit and scope of the invention.