Method of streptomycin detection by means of piezoelectric immunosensor

FIELD: medicine.

SUBSTANCE: invention describes a method of immunochemical streptomycin detection wherein on a surface of a piezoelectric immunosensor, a receptor coating with a streptomycin-protein (bovine serum albumin) conjugate is immobilised, a settled number of streptomycin antibodies is added to a sample and kept for 2-3 min to produce an immune complex which is introduced in a cell in a phosphate buffer solution of pH = 7.1-7.5 and to record the sensor oscillation frequency variations when reacting with the streptomycin-protein conjugate with streptomycin antibodies, an analytical signal is inversely proportional to the concentration of streptomycin in the analysed sample, while the concentration is derived from the calibration chart, the receptor coating is regenerated by applying 0.03 mM potassium thiocyanate solution on the surface.

EFFECT: higher sensitivity of streptomycin detection in composite mixtures, ensured multiple use of the immunosensor after regeneration of the bioreceptor coating that reduces analysis price.

13 dwg, 1 tbl

 

The invention relates to the field of analytical chemistry and can be recommended for the selective determination of streptomycin in food.

At the present time to determine streptomycin used methods: microbiological [Haasnoot W. Immunochemical detection of aminoglycosides in milk and kidney/ W.Haasnoot, P.Stouten, G.Cazemier, A.Lommen, Jacques F.M. Nouws, Henk J. Keukens // State Institute for Quality Control of Agricultural Products (RIKILT-DLO), Borrsesteeg 45. - 1999; Jin y], the drawback of such methods is the low sensitivity (detection limit of 0.01 mg/l); chromatographic [Bruijnsvoort M. Determination of streptomycin and dihydrostreptomycin in milk and honey by liquid chromatography with tandem mass spectrometry/ M. Bruijnsvoort, S.J.M. Ottink, C.M. Jonker, E.Boer // J.Chromatogr. - 2004. - V.1-2. - P.137-142; Kaufmamn A. Determination of 11 Aminoglycosides in Meat and liver by liquid chromatography with tandem mass spectrometry / A. Kaufmamn, K. Maden // Journal of AOAC International. - 2005 - V.88. - P.1118-1125], with a fairly complicated procedure sample preparation requiring to increase the selectivity to apply sophisticated separation of the sample. The closest technique is the method of fluorescence polarization immunoassay (PFIA) [Kathryn S. Schwenzer Automated Fluorescence Polarisation Immunoassay for Monitoring Streptomycin / Kathryn S. Schwenzer, John P. Anhalt // Antimicrobial Agents and Chemotherapy. - 1983. - V.23 supported, No. 5. - P.683-687], the method is rapid, selective, however, the sensitivity of this method is low, the detection limit 2,41 mg/L.

The present invention is to reduce the detection limit to what the susceptibility and increase detection sensitivity, automation of analysis, measurements in a closed loop, which includes the regeneration of the sensor.

The problem is solved in that the analyzed samples are prepared, and then conduct a competitive analysis by flow-injection determination.

For the determination of trace concentrations of streptomycin on the electrode surface of the sensor was immobilized conjugate of bovine serum albumin with streptomycin, while the sample containing the analyte, has introduced a pre-set amount of antibodies, corresponding to 50%of specific binding. The analytical signal of the sensor is inversely proportional to the content of streptomycin in the analyzed sample. After each measuring cycle perform the regeneration of the receptor coating, causing the surface of 0.03 mm solution of potassium thiocyanate.

Distinctive features of the proposed method are:

- high sensitivity of the method allows the determination of streptomycin in food products within the concentration range from 1.0 to 50.0 ng/ml, with a detection limit equal to 0.6 ng/ml;

the high selectivity of the determination of streptomycin in complex mixtures, including in the presence of related compounds (PR% <6,00%);

- multiple (more than 20 times) using immunosensor as a result of regeneration bioreceptors cover on the Les of each measurement cycle;

- relatively low duration of analysis (15-20 min).

This allows the determination of streptomycin in the concentration range from 1.0 to 50.0 ng/ml in food. High selectivity is provided by the use of specific immunoreagents - polyclonal antibodies to streptomycin. Multiple (more than 20 times) using immunosensor after regeneration bioreceptors coating reduces the implementation cost of analysis.

The method is as follows.

To create immunosensor used piezoquartz mass-sensitive resonator at-cut with gold electrodes with a diameter of 5-8 mm and natural frequency (5-10 MHz)±1 Hz. After thoroughly cleaning and degreasing the surface of the silver electrode piezoquartz resonator is treated with 0.6 μl of a 2.5%aqueous solution of γ-aminopropyltriethoxysilane in acetone, dried in air and incubated for 30-40 minutes at 90°C. After the application of 15 μl of 2.5% freshly prepared solution of glutaraldehyde and 10 μl of a 0.05% solution of streptomycin-protein conjugate is strong pinning bioloy. The sensor can withstand 10-12 hours at 4°C in a humid chamber

Before starting the first measurement piezoquartz immunosensor incubated in phosphate buffer solution (pH of 7.2) for 30 min to stabilize the si is Nala sensor. In the sample to determine the antibiotic was administered a fixed number of antibody (100 ml) and kept for 2-3 minutes to complete the formation of homogeneous immune complex-defined connections with the appropriate antibodies. Then, the sample was injected into a stream of solution vehicle and after entering the cell detection was measured analytical signal of the sensor as a result of interaction unbound antibodies with drug-protein conjugate on the surface of the electrodes of the sensor.

After measuring the analytical signal of the sensor was carried out by the destruction of the formed immunocomplex and regeneration bioloy. The oscillation frequency of the sensor when it is returned to the original value. After preliminary preparation, described above, was determined by the concentration of streptomycin in the sample of pre-constructed calibration curve.

To construct the calibration curve to 50 ál of the test solution with a concentration of streptomycin 0,05, 0,1, 5,0, 10,0, 30,0, 50,0, 70,0 ng/ml was added a solution of antibodies, corresponding to 50%of specific binding, the mixture was adjusted with phosphate buffer solution to 1 ml and incubated until completion of the reaction.

The value of the analytical signal is inversely proportional to the content of the analyte in the sample.

The calibration graph for the determination of streptomycin linear in u is not of concentrations from 1.0 to 50.0 ng/ml: Δf=-1,2+ 112,0, where Δf is the analytical signal; C is the concentration of streptomycin in the sample.

Example 1. The streptomycin solution with a concentration of 0.05 ng/ml volume of 200 µl was injected into the cell in phosphate buffer solution (pH of 7.1 and 7.5) and registered the change of the analytical signal due to the formation of the complex. Pre-K solution was added to a fixed number of antibody to streptomycin and maintained for 3 minutes until the formation of the immunocomplex. The analytical response of the sensor is inversely proportional to the content of streptomycin in solution.

Regeneration bioacoustical cover piezoquartz sensor was carried out by coating the surface of the KCNS solution 0.03 mm. The concentration of streptomycin was carried out on the calibration curve, constructed using the standard samples.

Analytical signal Δf=111,8 Hz; sensitivity definition 11184,0 Hz/ng, the limit of detection of streptomycin - 0.5 ng/ml.

Example 2. The streptomycin solution with a concentration of 0.1 ng/ml volume of 200 µl was injected into the cell in phosphate buffer solution (pH of 7.1 and 7.5) and registered the change of the analytical signal due to the formation of the complex. Pre-K solution was added to a fixed number of antibody to streptomycin and maintained for 3 minutes until the formation of the immunocomplex. Analytical response Sens the RA is inversely proportional to the content of streptomycin in solution.

Regeneration bioacoustical cover piezoquartz sensor was carried out by coating the surface of the KCNS solution 0.03 mm. The concentration of streptomycin was carried out on the calibration curve, constructed using the standard samples.

Analytical signal Δf=111,7 Hz; sensitivity definition 5585,0 Hz/ng, the limit of detection of streptomycin - 0.5 ng/ml.

Example 3. The solution of streptomycin concentration of streptomycin 5.0 ng/ml volume of 200 µl was injected into the cell in phosphate buffer solution (pH of 7.1 and 7.5) and registered the change of the analytical signal due to the formation of the complex. Pre-K solution was added to a fixed number of antibody to streptomycin and maintained for 3 minutes until the formation of the immunocomplex. The analytical response of the sensor is inversely proportional to the content of streptomycin in solution.

Regeneration bioacoustical cover piezoquartz sensor was carried out by coating the surface of the KCNS solution 0.03 mm. The concentration of streptomycin was carried out on the calibration curve, constructed using the standard samples.

Analytical signal Δf=to 106.0 Hz; sensitivity determination to 106.0 Hz/ng, the limit of detection of streptomycin and 0.6 ng/ml.

Example 4. The streptomycin solution with a concentration of 10.0 ng/ml volume of 200 the CL was injected into the cell in phosphate buffer solution (pH of 7.1 and 7.5) and registered the change of the analytical signal due to the formation of the complex. Pre-K solution was added to a fixed number of antibody to streptomycin and maintained for 3 minutes until the formation of the immunocomplex. The analytical response of the sensor is inversely proportional to the content of streptomycin in solution.

Regeneration bioacoustical cover piezoquartz sensor was carried out by coating the surface of the KCNS solution 0.03 mm. The concentration of streptomycin was carried out on the calibration curve, constructed using the standard samples.

Analytical signal Δf=100.0 Hz; sensitivity definition 50.0 Hz/ng, the limit of detection of streptomycin and 0.6 ng/ml.

Example 5. The streptomycin solution with a concentration of 30.0 ng/ml volume of 200 µl was injected into the cell in phosphate buffer solution (pH of 7.1 and 7.5) and registered the change of the analytical signal due to the formation of the complex. Pre-K solution was added to a fixed number of antibody to streptomycin and maintained for 3 minutes until the formation of the immunocomplex. The analytical response of the sensor is inversely proportional to the content of streptomycin in solution.

Regeneration bioacoustical cover piezoquartz sensor was carried out by coating the surface of the KCNS solution 0.03 mm. The concentration of streptomycin was carried out on the calibration curve, built the mu using standard samples.

Analytical signal Δf=76,0 Hz; sensitivity determination of 12.7 Hz/ng, the limit of detection of streptomycin and 0.6 ng/ml.

Example 6. The streptomycin solution with a concentration of 50.0 ng/ml volume of 200 µl was injected into the cell in phosphate buffer solution (pH of 7.1 and 7.5) and registered the change of the analytical signal due to the formation of the complex. Pre-K solution was added to a fixed number of antibody to streptomycin and maintained for 3 minutes until the formation of the immunocomplex. The analytical response of the sensor is inversely proportional to the content of streptomycin in solution.

Regeneration bioacoustical cover piezoquartz sensor was carried out by coating the surface of the KCNS solution 0.03 mm. The concentration of streptomycin was carried out on the calibration curve, constructed using the standard samples.

Analytical signal Δf=52,0 Hz; sensitivity definition 10.4 Hz/ng, the limit of detection of streptomycin and 0.6 ng/ml.

Example 7. The solution of streptomycin concentration 70.0 ng/ml volume of 200 µl was injected into the cell in phosphate buffer solution (pH of 7.1 and 7.5) and registered the change of the analytical signal due to the formation of the complex. Pre-K solution was added to a fixed number of antibody to streptomycin and maintained for 3 minutes to education is complex. The analytical response of the sensor is inversely proportional to the content of streptomycin in solution.

Regeneration bioacoustical cover piezoquartz sensor was carried out by coating the surface of the KCNS solution 0.03 mm. The concentration of streptomycin was carried out on the calibration curve, constructed using the standard samples.

Analytical signal Δf=30.0 Hz; sensitivity determination of 4.3 Hz/ng, the limit of detection of streptomycin - 0.7 ng/ml

Example 8. The solution of streptomycin containing streptomycin and structural similar - gentamicin concentration ratio of 1:1, volume of 200 µl was injected into the cell in phosphate buffer solution (pH of 7.1 and 7.5) and registered the change of the analytical signal due to the formation of the complex. Next, similarly to example 1.

Introduced streptomycin:Found streptomycin:
of 10.0 ng/ml10,00±0,02

Δf=100,1 Hz; sensitivity definition 100 Hz/ng. The presence of a structural analogue of (gentamicin) does not affect the determination of streptomycin.

Example 9. The solution of streptomycin containing streptomycin and structural similar - tetracycline in the concentration ratio of 1:1, volume of 200 μl enter the whether the cell in phosphate buffer solution (pH of 7.1 and 7.5) and registered the change of the analytical signal due to the formation of the complex. Next, similarly to example 1.

Introduced streptomycin:Found streptomycin:
of 10.0 ng/ml10,00±0,03

Δf=100,5 Hz; sensitivity definition 101 Hz/ng. The presence of a structural analogue of (tetracycline) does not affect the determination of streptomycin.

Example 10. The solution of streptomycin containing streptomycin and structural similar - bacitracin in the concentration ratio of 1:1, volume of 200 µl was injected into the cell in phosphate buffer solution (pH of 7.1 -7,5) and registered the change of the analytical signal due to the formation of the complex. Next, similarly to example 1.

Introduced streptomycin:Found streptomycin:
of 10.0 ng/ml10,00±0,04

Δf=100,7 Hz; sensitivity definition 101 Hz/ng. The presence of a structural analogue of (bacitracin) exerts no impact on the determination of streptomycin.

Example 11. The solution of streptomycin containing streptomycin and structural similar - gentamicin concentration ratio of 1:4, volume of 200 µl was injected into the cell in phosphate buffer solution (pH of 7.1 and 7.5) and recorded is ovali change the analytical signal due to the formation of the complex. Next, similarly to example 1.

Introduced streptomycin:Found streptomycin:
of 10.0 ng/ml10,00±0,05

Δf=100.0 Hz; sensitivity definition 100 Hz/ng. The presence of a structural analogue of (gentamicin) does not affect the determination of streptomycin.

Example 12. The solution of streptomycin containing streptomycin and structural similar - tetracycline in the concentration ratio of 1:4, volume of 200 µl was injected into the cell in phosphate buffer solution (pH of 7.1 and 7.5) and registered the change of the analytical signal due to the formation of the complex. Next, similarly to example 1.

Introduced streptomycin:Found streptomycin:
of 10.0 ng/ml10,00±0,04

Δf=100 Hz; sensitivity definition 100 Hz/ng. The presence of a structural analogue of (tetracycline) does not affect the determination of streptomycin.

Example 13. The solution of streptomycin containing streptomycin and structural similar - bacitracin in the concentration ratio of 1:4, volume of 200 µl was injected into the cell in phosphate buffer solution (pH of 7.1 and 7.5) and recorded is ovali change the analytical signal due to the formation of the complex. Next, similarly to example 1.

Introduced streptomycin:Found streptomycin:
of 10.0 ng/ml10,00±0,06

Δf=99,9 Hz; sensitivity definition 100 Hz/ng. The presence of a structural analogue of (bacitracin) exerts no impact on the determination of streptomycin.

This method can significantly increase the sensitivity of the determination of streptomycin in food products, and also provides multiple use immunosensor after regeneration bioreceptors coating that reduces the cost of implementation of the analysis. The limit of detection of streptomycin equal to 0.6 ng/ml

Comparative characteristics of known and proposed method of determination of streptomycin in the table.

Table
Comparative characteristics of known and proposed method of detection of streptomycin in liquid media
IndicatorsThe known method (fluorescence polarization immunoassay)The proposed method
The range of the designated contents of streptomycin300,0-5000,0 ng/mlfrom 1.0 to 50.0 ng/ml
The limit of detection of streptomycin116,0 ng/ml0,6 ng/ml
The use of additional labels streptomycinNot requiredNot required
The volume of the analyzed sample1000 ál200 ál
Analysis streptomycinDiscrete/directDiscrete/direct
Regeneration immunoreagentsNoAfter each measuring cycle
The duration of the measurement of the analytical signal, min streptomycin7-10 min15-20 min
The multiplicity of using immunoreagents120
Determination in honey (MPC-20 ng/ml)impossiblemay

<> The method of determination of streptomycin immunochemical method, characterized in that the surface of piezoquartz immunosensor immobilized receptor coating on the basis of streptomycin-protein (bovine serum albumin) conjugate to the sample add a fixed number of antibody to streptomycin and incubated for 2-3 min before the formation of the immunocomplex, is introduced into the cell in phosphate buffer solution with a pH of 7.1-7.5 and register the change of the frequency of oscillation of the sensor in the interaction of streptomycin-protein conjugate with antibodies to streptomycin, the analytical signal is inversely proportional to the concentration of streptomycin in the analyzed sample, the concentration is determined by the calibration curve, the regeneration of the receptor coating is performed by coating the surface of 0.03 mm solution of potassium thiocyanate.



 

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