RU2408012C1 - Method of detecting amino acid phenylalanine in aqueous solutions using piezoelectric sensor modified with molecular imprinted polymer - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: method involves taking samples, modification of the surface of a piezoelectric sensor by using a chromotography microsyringe to deposit molecular imprinted polymers colloxylin+phenylalanine, or polyamide acid+phenylalanine onto the piezoelectric sensor in form of an AT-section resonator with nominal resonance frequency of 2.5 MHz in an amount which is such that, after removing the solvent, the mass of the film is 1.0-2.0 mcg, holding the piezoelectric sensor in distilled water for 24 hours so as to remove template molecules of phenylalanine and then holding said sensor in a detection cell for 3-7 minutes to stabilise the zero signal, putting the sample of aqueous solution of amino acid phenylalanine into the detection cell and then detection by picking up the analytical signal with a frequency metre in form of resonance frequency of the piezoelectric sensor and estimating differential frequency offset of the piezoelectric sensor.

EFFECT: easy implementation, cutting on number of chemical reagents used, high selectivity, high acid- and heat-resistance.

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Description

The invention relates to chemistry and can be used in clinical and laboratory diagnostics of physiologically active substances, materials, quality control of products of the chemical and food industries.

Closest to the claimed device is a piezosensor with silver electrodes based on molecular imprints, used as an immunosensor for flow-injection determination of high and low molecular weight organic compounds [Kalmykova EN, Ermolaeva TN, Eremin S.A. Development of flow-injection analysis of high and low molecular weight compounds. Bulletin of Moscow State University Chemistry. - 2006 No. 20]. The installation for flow-injection analysis consists of a Gilson peristaltic pump, a dispenser, a 15-20 μl flow cell for detection, including commercially available domestic AT-cut piezoelectric quartz sensors with silver electrodes with a diameter of 8 mm and a natural oscillation frequency of 8-10 MHz, at surfaces of which immobilize immunoreagents. Sensors contact on one side with the analyzed liquid phase. Silicone tubes with a diameter of 0.16 mm connect the microcell with a peristaltic pump and a dispenser. The fluid flow rate is 30 μl / min. As a carrier solution, 5-50 mM phosphate buffer (pH 6.8; 7.0; 7.2) is used. The change in the oscillation frequency of the sensor with an interval of 1 minute is recorded with a frequency meter.

The disadvantages are the complexity of the hardware design, the presence of many chemicals, low selectivity, low acid and heat resistance.

The aim of the invention is to simplify the hardware design, reducing the number of chemicals used, increasing selectivity, increasing acid and heat resistance.

This goal is achieved by the fact that in the device the surface of the piezosensor is modified with a polymer with a molecular imprint:

colloxylin + phenylalanine, or polyamido acid + phenylalanine.

The method is as follows.

Preparing the piezosensor for work. When performing the experiment, AT-cut resonators with a nominal resonant frequency of 2.5 MHz were used.

Modification of sensors. As surface modifiers of the electrodes of piezoelectric quartz sensors, a colloxylin-based polymer with a molecular imprint of the phenylalanine amino acid, or polyamide acid with a molecular imprint of the phenylalanine amino acid, is used.

Example 1

Preparation of colloxylin with a molecular imprint of phenylalanine (a molecularly imprinted colloxylin-based polymer).

Receive colloxylin according to the method described in [E.V. Kuznetsov, I.P. Prokhorov, D.A. Fayzullina. An album of technological schemes for the production of polymers and plastics based on them. Second edition, revised and supplemented. - M: Chemistry, 1976].

Molecules of the phenylalanine template are introduced. To obtain 0.5 g of a polymer with molecular imprints, 0.045 g (accurately weighed) of phenylalanine is dissolved in 3-5 ml of distilled water, solution A is prepared. A mixture of ethanol and diethyl ether in the ratio 1: 3 is prepared - this is solution B. Add to solution B. 0.3 ml of solution A, solution C is obtained, while solution C remains transparent. Colloxylin is added to solution C, stirred with a glass rod. A viscous solution is formed - collodion. The polymer is applied to the piezosensor. The electrodes of the piezosensors are modified by applying a polymer with molecular imprints of phenylalanine by a chromatographic microsyringe so that, after removal of the solvents, the film mass is 1.0-2.0 μg. The sensor is then placed for 24 hours in distilled water to remove the phenylalanine template molecule from the polymer. Then the sensor is placed in the detection cell for 3-7 minutes to stabilize the zero signal.

The number of analyzes without repeated modification of the piezosensor electrode 10-15 measurements; analysis time, including the stage of sensor regeneration, not more than 15 minutes The analysis data are presented in table 1.

Example 2

Obtaining a polymer with a molecular imprint of phenylalanine based on polyamic acid (a molecularly imprinted polymer based on polyamido acid).

Phenylalanine in the amount of 0.045 g (accurately weighed) is placed in the bottle and dissolved in an aqueous-ethanol-butanol solution (12: 5: 4). Then polyamic acid is added to the bottle. The resulting mixture is applied to a piezosensor using a chromatographic microsyringe. To remove the template molecule from the polymer, the piezosensor is placed for 24 hours in distilled water. Then the sensor is placed in the detection cell for 3-7 minutes to stabilize the zero signal.

The number of analyzes without repeated modification of the piezosensor electrode is 20-30 measurements; analysis time, including the stage of sensor regeneration, not more than 15 minutes The analysis data are presented in table 1.

The decrease in the operating frequency of the oscillations of piezoelectric quartz sensors on body-acoustic waves is calculated according to the Sauerbrey G.G. Messung von plattenschwingungen sehr kleiner amplitude durch lichtstrom-modulation // Z. Phys. - 1964. - Bd. 178. - S. 457-471]:

where m is the mass of the modifier, g; f ₀ is the resonant frequency of the piezosensor, MHz;

Δf is the change in the frequency of the resonator, Hz; A is the surface area of the modifier, cm ² .

For piezoelectric quartz resonators with a nominal frequency of 2.5 MHz, the response after modification was f _c ≈ 2-6 kHz.

Detection is carried out after sampling by an automatic micropipette of an aqueous solution of the phenylalanine amino acid of concentrations 1 · 10 ^-2 M, 1 · 10 ^-3 M, 1 · 10 ^-4 M, 1 · 10 ^-5 M, 1 · 10 ^-6 M in the detection cell and register the analytical signal with a frequency meter in the form of a resonant frequency of the sensor. The relative frequency shift Δf _{a is} calculated by the equation:

Δf _a = f ₀ -f ₁ ,

where f ₀ and f ₁ are the oscillation frequencies of the sensor before and after analysis, Hz.

Table 1 Examples of the method using a piezosensor modified with a molecular imprint polymer or colloxylin + phenylalanine, or polyamido acid + phenylalanine Sensor Electrode Modifiers The ratio of solvent, vol. hours Concentration, M Δf, MHz one 2 3 four Example 1 A colloxylin-based polymer with molecular imprints of an amino acid, e.g. phenylalanine 1: 3 1 · 10 ^-2 235897 1 · 10 ^-3 196800 1 · 10 ^-4 131049 1 · 10 ^-5 65616 1 · 10 ^-6 35763 Example 2 Polyamide acid polymer with molecular imprints of an amino acid, e.g. phenylalanine 12: 5: 4 1 · 10 ^-2 235760 1 · 10 ^-3 196640 1 · 10 ^-4 130957 1 · 10 ^-5 65425 1 · 10 ^-6 35528

Using the proposed method allows us to simplify the design, reduce the number of chemicals used, increase selectivity, increase acid and heat resistance for polyamic acid.

Claims (1)

A method for determining the amino acid phenylalanine in aqueous solutions with a piezosensor modified with a molecular imprint polymer, including sampling, modifying the surface of the piezosensor by applying a molecular imprinted polymer of colloxylin + phenylalanine, or polyamide acid + phenylalanine to the piezosensor, in the form of a resonance resonant with a T resonance of 2 5 MHz in such a quantity that, after removal of the solvent, the mass of the film was 1.0-2.0 μg, placing the piezosensor at 24 h into distilled water to remove the phenylalanine template molecule, then placing it in the detection cell for 3-7 minutes to stabilize the zero signal, introducing a sample of the phenylalanine amino acid aqueous solution into the detection cell, followed by detection by recording the analytical signal with a frequency meter in the form of a resonant frequency of the piezosensor and estimates of the relative frequency shift of the piezosensor.