SENSITIVE BLOCKS TO MASSES.
The present invention relates to a p-sensor, zoelectric for use in diagnostic / analytical procedures, especially for the immunochemical identification of specific binding partners, of relevance in diagnosis. Piezoelectric sensors as such are known, for example, from Dickert et al. (Che ie in unserer Zeit 28 (3), pages 147-152 (1994)). Such biosensors are also disclosed, for example, in U.S. Pat. 4,236,893 and 4,735,906. Such biosensors, which can be used for the identification of molecules of relevance in diagnosis (analysts), are usually composed of a transducer with a surface coated with a specific binding partner for the molecule to be identified, thereby it is able to selectively bind these molecules. Most often quartz piezoelectric crystals are used as a transducer. If the quartz crystal is mounted on a suitable electronic switching circuit, then it vibrates at a certain resonance frequency. If then the molecules to be identified accumulate, then the resonance frequency shifts. Such electronic switching circuits are known to the person skilled in the art, for example from DE-A 39 20 052. Even though these known biosensors already partially satisfy the functions assigned to them, it is nevertheless demonstrated that, in the methods employed in which they are attached to the quartz surface, for example, anti-bodies through silane derivatives, on the one hand that the procedure for the binding of the antibodies to the quartz surface is very complex and difficult to repress. on the other hand, that the procedure of the surface of the sensor by treatment with a reactive agent that must break the union between the antigen to be identified and the antibodies and separate the bound antigen is, on the other hand, hardly reproducible and leads to an uncontrolled degradation of the antibodies on the surface of the sensor. In a DAVI publication? et al. (Anal. Che. (1989), 61 (11), pages 1227-1230 it is described that the transducer can be coated with gold and bound to this protein A layer. Protein A, on the other hand, can reversibly bind antibodies for identification of molecules of relevance in diagnosis As the authors themselves conclude, by means of the coating with protein A the signal is strongly displaced and the measurement is made difficult, therefore, the present invention has as its mission to propose a biosensor for use in analytical procedures or diagnostic, which can be simply coated with a specific binding partner at the same time, must be regenerated in a simple and safe manner after the specific reaction has elapsed.This problem is solved according to the present invention by covering the sensor with a noble metal, preferably gold and being able to bind to this coating a participant in the specific union. This way the participant in the specific union can be separated, for example by the reagents known from DE 44 36 910. It is not essential for the present invention in which form of execution the transducer is or in what form and manner the change caused by the accumulation of analytes it becomes a measurement signal. The present invention can be used both in manual procedures in which, for example, the sensor is immersed in the r-ertri, as well as in automatic analysis devices. Basically, the procedure according to 1? The invention can be carried out in the following manner: a commercially available gold-coated pi-ezoelectric crystal -which is integrated in an integrated electronic switching circuit- (for example from the company Sepsotec), is coated with a participant in the specific binding for the analyte to be determined. A participant in the specific union of this type can be, for example, an antibody or monoclonal or polyclonal antibody fragment, a lectin or an antigen. The coating duration can range between 5 min and several hours, preferably between 10 and 120 min. After the coating the sensor surface is rinsed with washing buffer. The washing buffer preferably has a detergent. Optionally, after the specific coating, an inespecific coating can also be carried out, for example with inactivated BSA or POD, to prevent nonspecific binding. Such methods are known per se to the specialist. After a subsequent in-specific coating of this type, a washing step can also be carried out. The coated sensor is incubated with the sample, this being possible, for example, by immersion in the sample or by applying a sample on the surface of the sensor. Also advantageous is an embodiment in which the surface of the sensor is shaped so that it can be measured in the throughput. It is easily understandable to the specialist what form the respective sensor should have depending on its use. By means of the reaction of the analyte with the participant in the specific connection, the magnitude of the measurement determined by the electronic switching circuit, for example the resonance frequency, is displaced. A part.r: r >; the variation of the magnitude of the measurement, can ciea'-c; The amount of bound analyte is measured, for example by comparison with a reference curve. The arrangement is also suitable for calibrating directly in units of mass. To determine the variation of the magnitude of the measurement, for example, a reference electrode that is not coated with the participant in the specific joint can be used. Advantageously, after completion of the incubation with the sample, another washing step is also carried out, the incubation with the sample can be carried out in a manner between 1 and 100 min, particularly advantageously between 5 and 60 min. very particularly advantageous form between 13 and 30 min. The regeneration can be carried out as described in DE 44 36 910. In this case, in 1: use of noble metals, preferably gold, certain reducing or oxidizing agents, such as, for example, sodium borohydride, are used as regeneration phase for the regeneration. , tetrabutylammonium hydroxide, with or without the addition of detergents. The following exemplary embodiment serves for clarification, not for the limitation of the invention. Example Quantitative determination of human IgE Support: Piezoelectric crystal of the firm Sensotec, coated with gold, set in a Teflon ring (external diameter, 36 mm). Diameter of the gold surface: 9 mm. Gold surface: 14 mm2. Coating: 50 μl of a polyclonal antibody (rabbit) against human IgE is applied to the sensor. The concentration of the antibody is 5 μg / ml. The solution also contains 75 mM Na phosphate, 75 mM NaCl, 100 g / 1 Na2? 04. The pH value is 6, 0. The antibody solution is left for 1 hour at 37 ° C or overnight at room temperature. Washing step: The supernatant is removed and the sensor is rinsed 5 times in each case with 250 μl of washing buffer. The washing buffer is composed of a solution of 50 mM tris (hydroxymethyl) aminoethane (TRIS) and 50 M citric acid, pH 7.4. Posterior lining: 50 μl of inactivated peroxidase (POD) is applied to the sensor. The POD concentration amounts to] g / 1. The solution also contains 75 mM Na phosphate, 75 mM NaCl, 10] g / 1 Na2SO4. The pH value is 6.0. The solution is left for 1 hour at 37 ° C or overnight at room temperature. Washing step: The supernatant is removed and the sensor is rinsed 5 times in each case with 250 μl of washing buffer. The washing buffer is composed of a solution of 50 mM tris (hydroxymethyl) amino-ethane (TRIS) and 50 mM citric acid, pH 4. 4. Incubation of the sample: 50 μl of human serum containing 50 μM is added to the sensor. defined amounts of IgE and incubated at 37 c for 30 min Wash step: The supernatant is removed and the sensor is rinsed 5 times in each case with 250 μl of wash buffer (5 mM Na phosphate, 85 mM NaCl, 1 g / 1 Tween 20, 0.5 g / 1 phenol, pH 6.5).
Regeneration of the solid phase: 250 μl of a 20% (wt%) solution of tetrabutylammonium hydroxide are applied to the sensor and incubated at 37 aC for 1 hour. Washing stage: After removal of the supernatant, the sensor is rinsedtimes with deionized water. 2nd regeneration: 250 μl of a 1% solution (% by weight) e * is incubated on the sensor for 15 minutes in the third environment. ? BH4. The solution also contains 2-cyclohexaic acid. ir.ce: anosulphonic (CHES) 50 mM. The pH is 10.0. Washing step: After removal of the supernatant rinse the sensor 3 times with deionized water and 3 times with a phosphate buffered sodium chloride solution (pH 7.2). Results: * 1. Determination with 100 IU / ml of human IgE = 329 Ua "2. Determination with 100 IU / ml of human IgE = 576 Ua 3. Determination with 100 IU / ml of human IgE = -23 t ^ Determination with 0 IU / ml of human IgE = 19 Ua determination with 100 IU / ml of human IgE = "or" 'Ja Determination with 100 IU / ml of human IgE (control = coating antibody inespcJ i:: -o) = 14 Ua Mean values of n = 2. Arbitrary units.