Method of restoring sensitive layer of biosensor

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology. The method involves treating the sensitive layer of a biosensor with solution of a common fraction of protease of hepatopancreas of king crab in a buffer comprising tris-HCl 50 mM, CaCl2 3 mM, NaCl 100 mM, pH 8.0 at solution temperature of 35-40°C. The process is carried out in multiple successive steps with a solution of a solution of a common fraction of protease of hepatopancreas of king crab in said solvent at temperature of 35-40°C followed by exposure. The sensitive layer of the biosensor is successively washed with dodecyl sulphate dissolved in the same buffer in concentration of 0.1% and then with water at each step. The treatment process is carried out three times.

EFFECT: invention reduces the duration of the process.

2 cl

 

The invention relates to the field of biosensor analysis and relates to a method of recovery of the sensitive layer of the biosensor.

The biosensor chips are a high-tech product, which leads to the fact that non-factory of their production is impossible. At the same time they are essentially disposable items, reuse, which is not envisaged, although the cost of the chip is high enough, and the cost of one analysis can be very significant. In many cases this is not an obstacle for the use of biosensors as when performing a vital analysis or the development of new drugs price analysis is not the determining factor. At the same time there are studies, for which you need to spend tens or even hundreds of chips. In many cases this is not possible for economic reasons.

In most cases, if immobilized component in the study of the interaction is a protein or polypeptide, its immobilization on the chip is made by the user. From the factory chips are produced without immobilizovannoi components, but only prepared for immobilization. This is because protein and polypeptides immobilized on the chip surface stored worse than separately chips and sewn components, for which the nutrient is possible to provide optimal storage conditions for each.

Immobilizovannyi component must be put firmly on the surface of the chip. Otherwise, if this component is washed away from the chip, there is a strong drift, interfering with the measurements. In addition, the loss of the immobilized component leads to a continuous decrease in the sensitivity of the biosensor. Therefore, usually used for immobilization irreversible presume, leading to the formation of covalent bonds between the substrate of the chip and immobilizovannoi molecule. Most often used immobilizatsiya for the amino group on carboxymethylamino dextranase substrate activated with a mixture of EDC/NHS (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide -hydroxysuccinimide). In addition, it is possible to commit macromolecules for carboxyl group, sulfhydryl group, etc. Using any of these methods leads to irreversible fixation of macromolecules, after which they remain on the surface of the chip and cannot be washed off with her, detergents, solvents and other cleaning agents. Therefore, over time, after degradation of the immobilized component used chip already unfit for any further use. At the same time, the substrate of such a chip is quite suitable for the immobilization of other macromolecules, but to use it to re-immobilization in most cases, it is not because some places (usually Carbo is strong groups) immobilization deactivated or occupied in the previous landing, and the remaining from the previous immobilization of molecules can lead to unpredictable artifacts and errors of measurement.

To get used chip in a state close to that in which it comes from the factory, it is necessary to more fully resolve with its surface remains of previously immobilized macromolecules and, if necessary, to restore to the original values of the surface density of carboxyl groups. At the moment there is no way aiming to break the covalent bond formed during immobilization, however, have the ability to delete all items sewn macromolecule in addition, which is directly sewn to the substrate.

This can lead to the surface of the chip in contact with a solution of enzyme able to hydrolyze communication between the components of macromolecules. For proteins, and proteins should be used protease, and nucleic acids - nuclease - Rncse, Tnkz[1, 2, 5].

There is a wide choice of proteolytic enzymes, but not all of them are suitable for carrying out regeneration [3], as most proteases cut the peptide bond only on certain amino acids, which, in addition, must be available for exposure of the enzyme, that is located outside of the protein globule. Such proteases are not able gidrolizovat is sewn proteins. Always remain areas that do not contain the right amino acids, and which will not be hydrolyzed and removed. In addition, even if the protein molecule will be cut in several places, the cut pieces will not be separated from the protein, because the protein chain in globule strongly intertwined with itself, and cut off pieces can't leave globule.

There are enzymes that can cut from the protein chain amino acid for amino acid with a C or N end (this is different enzymes, respectively-peptidases and N-peptidases), which in principle can hydrolyze the entire protein residue. However, such enzymes are still not solve the problem for several reasons. There are proteins in which the C-end, N-end, or both hidden inside the protein [3].

Such proteins are invulnerable to the appropriate type of end proteases or to both. Because in most cases, the immobilization of a protein globule is not at the end of the chain, and for any internal link for complete cleaning sensitive surfaces of the biosensor must be present in regenerating the solution of both types of end proteases. At the same time, these proteases are normally resistant to its effects, but vulnerable to proteases of another type. And finally, it is quite expensive proteins which are not always available for purchase. At the same time, given the other activities of such enzymes is generally low, and for effective removal of proteins within an acceptable time they need to apply in large numbers.

There is a method of regeneration of optical elements of the biosensor by hydrolysis of the immobilized protein with a solution of proteolytic enzyme Pronase that is, the Solution of the specified enzyme concentration 1 mg/ml were prepared in buffer solution composition is 20 mm phosphate, 150 mm sodium chloride, pH 7,2.

The chip was placed in the solution for about 15-17 hours at room temperature. Then it was rinsed in distilled water. There was obtained a satisfactory level of recovery properties of the chip, however, the main drawback of the method is the high consumption of proteolytic enzyme, its high cost (http://www.sigmaaldrich.com/life-science/biochemicals/biochemical-products.html?TablePage=16410626 and the lack of production of this drug. In this regard, the cost recovery of the optical chip by this method is too high [4].

In accordance with the invention describes a method of restoring the sensitive layer of the biosensor by multistage processing solution the total fraction of proteases hepatopancreas crab in a buffer solution composition of Tris-HCl 50 mm, CaCl23 mm, NaCl 100 mm, PH 8.0 at a solution temperature of 35-40° C, followed by exposure and sequence is through washing sensitive layer first dodecyl sulfate and then with water after each stage.

The total fraction of proteases from hepatopancreas crab is a very effective set of enzymes designed and are highly adapted for destruction and transformation into oligopeptides and single amino acids in any protein molecules. This mixture is able to hydrolyze any proteins regardless of their primary and secondary structure, with a very high speed and at a temperature from +2°C and above. These properties are determined by habitat conditions and physiology of the red king crab and optimized over millions of years of evolution. The total fraction of proteases is a by-product in the processing of red king crab in the food products, so the cost is not high [7].

According to literature data [6], the optimal conditions for storing and actions enzyme preparation collagenase crab are the following: for storage - potassium phosphate 100 mm, 50 mm potassium citrate (pH of 4.8); and to work Tris-HCl 50 mm, CaCl23 mm, NaCl 100 mm, (pH 8.0). The best activity of the drug is achieved at a temperature of 35-40°C.

The objective of the invention is to develop a method to restore sensitive layer of the biosensor.

Directly the process of regeneration of the sensitive layer of the biosensor is as follows.

Used the biosensor chip is released from the protective shell, if it exists. Sachemship placed in a hermetically closed container minimum volume, enough to chip fit in it entirely. Prepare a solution of the total fraction of proteases of hepatopancreas crab in a buffer solution composition of Tris-HCl 50 mm, CaCl23 mm, NaCl 100 mm (pH 8.0). The temperature of the solution is maintained within the range of 35-40°C. For this purpose a portion of the dry lyophilized drug is dissolved to a concentration of 0.1-0.01 mg/ml Smaller values result in longer procedures, and large waste of the drug. The resulting solution is poured into the container placed in a chip so that the sensitive surface of the chip was coated with the resulting solution, and the lid of the container tightly closed. The container is placed in a thermostat at a temperature of 35-40°C. After 24 hours exposure, the chip is removed from the container with a solution of protease and washed in a solution of sodium dodecyl sulfate for one hour, then 5 minutes in running distilled water and again placed in a solution of protease. This cycle is repeated three times, after which the chip was washed for one hour in flowing distilled water. Thus, the total duration of the recovery of the chip is about 76 hours. The solution proteases hepatopancreas crab saves suitability to work within one month from the date of dissolution in case of storage at a temperature not exceeding 8°C [6, 7].

After okonchatelnaya optical surface of the chip blow clean compressed air to remove dust, return the chip in a protective shell and placed in a plastic bag for storage. To assess the quality of recovery chip is placed into the sensor and make sure that the provisions of the lines sensogram correspond to the position of the lines characteristic of intact dextran or approaching such. To estimate the number of carboxyl groups they control preconcentration any reference lipid. If the speed of preconcentration lipid considerably reduced, it is possible to restore the number of carboxyl groups the following procedure:

The whole procedure of regeneration sensitive surfaces can be produced directly in the biosensor, as the applied solutions (solution proteases, a solution of dodecyl sulfate and water) do not cause corrosion and do not destroy plastics, or held separately in the supporting tubes or specially adapted containers. When conducting regeneration outside of the device removed restrictions on the duration of operations and requirements decrease of the specific activity of the protease complex. The duration of the procedures you can bring up to several days without compromising performance.

Experimental verification showed that the total fraction of proteases hepatopancreas crab quickly and effectively removes sensitive surface of the biosensor chip more than the 99% of the previously immobilized protein. Thus liberated from the results of the previous planting chip it is possible to plant a new batch of protein not less than 90%, which sits on a new chipmoondang increase the number of carboxyl groups on the dextran chip is not needed.

Used in the described method, the total fraction of proteases hepatopancreas crab has high proteolytic activity and low cost, because it is produced in large quantities as a by-product of the food industry.

References

1. Patent RU No. 2027181.

2. maxXbond: the First regeneration system for twisted DNA-columns https://www.applichem.com/ru/o-produktakh/maxxbond-pervaja-sistema-regeneracii-dnk-kolonok/.

3. Handbook of Proteolytic Enzymes / Eds Barrett AJ, Rawlings N.D., Woessner J.F. London: Acad. Press, 1998.

4. Ronald C. Chatelier, Thomas R. Gengenbach, Hans J. Griesser, Michael Brigham-Burke, t and Daniel J. O Shannessyt'(1995). A General Method to Recondition and Reuse BIAcore Sensor Chips Fouled with Covalently Immobilized Protein/Peptide. ANALYTICAL BIOCHEMISTRY 229, 112-118.

5. Patent RU No. 2239827.

6. THE 2639-001-45554109-98. The drug, the enzyme collagenase from hydrobionts.

7. Patent RU No. 2280076.

1. The method of restoring the sensitive layer of the biosensor by treating it with a solution of enzyme, characterized in that the quality of an enzyme solution used solution the total fraction of proteases hepatopancreas red king crab in the buffer composition of Tris-HCl 50 mm , CaCl23 mm, NaCl 100 mm, pH 8.0 at a temperature of the solution is in the range of 35-40 °With, and the process is carried out in several successive stages by processing the sensitive layer of the biosensor solution the total fraction of proteases hepatopancreas red king crab in the specified solvent at a temperature in the range of 35-40°With subsequent exposure and sequential leaching sensitive layer dodecyl sulfate dissolved in the same buffer at a concentration of 0.1 %, then the water at each stage.

2. The method according to claim 1, characterized in that the loop processing is carried out three times.



 

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