RU2425382C1 - Method of blood plasma proximate analysis for cardiomyoglobin by means of electrochemical immunosensor - Google Patents

Abstract

FIELD: medicine. ^ SUBSTANCE: method of blood plasma proximate analysis for cardiomyoglobin by means of an electrochemical immunosensor consists in the fact that a blood plasma sample is applied on a surface of a main electrode pre-modified by colloidal gold and monoclonal cardiomyoglobin antibodies; the prepared sample immunosensor is kept, preincubated in a buffer solution; cardiomyoglobin is electrochemically recorded in the sample by the prepared calibration diagram under certain conditions. ^ EFFECT: more sensitive analysis procedure, and reduced analysis time. ^ 2 dwg, 2 tbl, 4 ex

Description

The invention relates to the field of analytical chemistry, electrochemistry and medical diagnostics and relates to a method for the rapid determination of cardiomyoglobin in blood plasma using an electrochemical immunosensor.

The invention can be used in medical practice for the diagnosis of myocardial infarction (MI). According to statistics, cardiovascular diseases take the first place among the causes of death in Russia (www.gks.ru) and cause almost half of the total number of deaths in Europe (www.ehnheart.org). Inefficiency or inaccessibility of early diagnosis systems also directly affects the situation. Over the past decade, a large number of studies have focused on the identification of cardiomarkers that can be used to diagnose heart pain. A cardiomarker is a biological substance whose increase in blood level is observed during a cardiovascular disease or immediately after damage to the heart muscle [1]. To date, 177 compounds markers of cardiovascular disease are known and research in this area is ongoing [2].

The determination of biomarkers in the blood of patients for the diagnosis of MI has become a standard procedure in medical practice abroad and is used in leading clinics of the Russian Federation. Nevertheless, a continuous search for new cardiomarkers is being conducted, as well as various statistical studies are carried out confirming the effectiveness of test systems for the diagnosis of MI. Currently, troponins I and T, creatine kinase-MV, and cardiomyoglobin are widely used to diagnose myocardial infarction. Serum troponins and creatine kinase-MV indicate death of myocardial cells. Serum cardiomyoglobin is one of the earliest biomarkers of MI. Cardiomyoglobin is a hemoprotein with a molecular weight of 17800 and due to its small size, it is rapidly released from damaged tissue into the blood. Its half-life in plasma is only 8.9 ± 1.5 min. Among patients with acute myocardial infarction, the general picture of changes in plasma cardiomyoglobin concentration is similar to the dependences for creatine kinase-MV and troponins. However, the release of troponins and creatine kinase-MB is slower, which allows them to be detected in the blood only 4-6 hours after the onset of symptoms; in some patients, an increase in the level of these markers does not appear within 12 hours. Serum cardiomyoglobin appears in the blood within 1 to 4 hours. Serum concentrations of cardiomyoglobin increase after damage to various tissues (especially skeletal muscle) or cocaine, or in patients with renal failure due to a decrease in clearance (clearance rate). Nevertheless, an early jump in the level of cardiomyoglobin and its ability to determine acute myocardial infarction before other biomarkers compensates for these shortcomings, especially in combination with troponins and creatine kinase-MV, as well as a series of tests [3, 4].

In various sources, the threshold concentrations of cardiomyoglobin in the blood, the excess of which indicates MI, vary within a fairly wide range (from 70-90 μg / L [5-8] to 200 μg / L [9]) and, apparently, depend on individual characteristics of the body. However, most researchers adhere to an average threshold value of 100 μg / L [10-14].

To date, the following tests and bench-top immunoassay systems for cardiomarker proteins produced by foreign manufacturers are presented on the market: Stratus® CS STAT (Dade Behring Inc), i-STAT® (Abbott), Triage® Cardiac Panel (Biosite), RAMP® (Response Biomedical Corp), Cardiac Reader ™ (Roche), PATHFAST® (Mitsubishi Chemical Europe GmbH), MGL-CHECK-1 (Vedalab), HEXAGON TROPONIN (HUMAN GmbH).

The published work on the determination of cardiomyoglobin is a “sandwich” immunoassay. The method is based on the selective binding of marker proteins with appropriate antibodies attached to the surface of the sensor. Signal detection is carried out due to the second coating layer of antibodies with different labels (Table 1).

However, all of the above methods are quite complex, time consuming. None of them are currently approved for clinical use. As can be seen from the data presented, only one immunoassay technique is adapted for working with blood. None of the authors cites data on working with real samples of patients with MI. The accuracy and other analytical characteristics of the determination in this case will depend on many factors: the activity of the antibodies themselves, the enzyme label, the concentration of substrates, etc.

The redox activity of heme iron serves as a kind of marker of hemoproteins and makes it possible both for their direct determination and for external influence on the active center of the enzyme. Myoglobin belongs to the family of hemoproteins - electroactive proteins whose molecules contain an iron ion. The effect of direct electron transfer from the electrode to the heme iron is widely used in the creation of electrochemical sensors for hydrogen peroxide and nitrite ion. Surfactants [19-21], polymer hydrogels [21], membrane-like complexes of polyelectrolytes, etc. are used for its registration and modification of the electrode surface.

The aim of the present invention is to develop a method for determination of cardiomyoglobin in blood plasma samples using an electrochemical immunosensor. A method for producing an electrochemical immunosensor for the direct determination of cardiomyoglobin is described in the patent [22]. The possibility of selective determination of cardiomyoglobin in buffer solutions was previously shown. The rapid analysis of cardiomyoglobin in blood plasma samples required significant improvements in the methodology for the determination and manufacture of immunosensors. The main tasks were: 1) increasing the sensitivity of the determination and 2) a significant reduction in analysis time.

Unlike most foreign researchers who implement the “sandwich” analysis scheme, the proposed method is based on the cardiomyoglobin’s own electroactivity and direct detection of the specific interaction between hemoprotein molecules and the corresponding antibodies. A scheme for direct registration of myoglobin due to direct electron transfer from the electrode to the heme iron ion catalyzed by air oxygen is proposed:

The following factors contribute to signal amplification:

1) gold nanoparticles;

2) the stabilizer of gold nanoparticles - DDAB, sorbing oxygen from the background electrolyte;

3) the method of signal registration - square wave voltammetry.

The need for the use of antibodies is due to the task of selective determination of the cardioisoform of the enzyme released into the blood when damage to the heart muscle. An analytical signal is the value of the recorded electron transfer current from heme iron to the surface of a graphite electrode modified with gold nanoparticles. Colloidal gold particles deposited on the surface of the electrode work as an ensemble of microelectrodes and not only increase the area of the active surface of the sensor, but also improve the signal-to-noise ratio. The transition from a flat electrode to a system of microelectrodes with radii commensurate with the thickness of the diffusion layer leads to the appearance of a boundary effect when the magnitude of the limiting current is directly proportional to the radius of the microelectrode.

EXAMPLE 1. The method of preparation of a colloidal solution of gold

Preparation of a solution of hydrochloric acid.

(3.9) mg of trichlorohydrochloric acid (HAuCl ₄ × 3H ₂ O) is weighed, transferred to Eppendorf and dissolved in 1 ml of distilled water. The solution is stored in eppendorf at 4 ° C for not more than 1 month.

Preparation of a solution of dimethyldidodecylammonium bromide (DDAB) (0.1 M).

(46.3) mg of dimethyldidodecylammonium bromide is weighed, weighed into eppendorf and dissolved in 1 ml of chloroform. The solution is stored in eppendorf at 4 ° C for not more than 1 month.

Preparation of a solution of sodium borohydride (0.4 M).

(15.2) mg of sodium borohydride is weighed, the weighed portion is transferred to eppendorf and dissolved in 1 ml of distilled water. The solution is not stored.

Preparation of a colloidal solution of gold (5 mm).

A round-bottom flask is mounted on a tripod above a magnetic stirrer. Using a pipette, transfer 1 ml of a 0.1 M DDAB solution to a flask. With vigorous stirring, 0.5 ml of a 10 mM solution of hydrochloric acid, selected by pipette, is added to the DDAB solution. Then, with vigorous stirring, 0.2 ml of a freshly prepared sodium borohydride solution is added dropwise. After 2 hours, the colored organic layer was separated from water.

EXAMPLE 2. Production of electrochemical immunosensors for cardiomyoglobin in blood plasma.

Preparation of 6 M NaOH

Weigh (24.0) g of sodium hydroxide, transfer the sample to a 100 ml volumetric flask, and make up with distilled water to the mark. The solution is stored in a plastic container under traction for no more than 1 month.

Preparation of phosphate buffered saline

(1.33) g of potassium dihydrogen phosphate (KH ₂ PO ₄ ) and (0.29) g of sodium chloride (NaCl) are weighed. Samples of salts are transferred to a 100 ml volumetric flask and adjusted to the mark with distilled water. The solution is thoroughly mixed on a magnetic stirrer until the salts are completely dissolved. On a pH meter, the pH was adjusted to 6.5 with a 6 M NaOH solution. The solution is stored in a plastic container with a tightly closed lid at 4 ° C for no more than 1 month.

Preparation of cardiomyoglobin antibody solution

The initial solution of antibodies to cardiomyoglobin (USBio, USA, Cat. No. M9800-16A) is diluted 10 times with a phosphate buffer solution pH 6.5 ± 0.1. An aliquot of 10 μl of antibody solution was transferred to an eppendorf tube and 90 μl of buffer solution was added. The solution is stored in an eppendorf tube at -20 ± 2 ° C for not more than 1 month.

Preparation of electrochemical biosensors for cardiomyoglobin

On the surface of a working graphite electrode (made using carbon paste from Gwent, England; manufacturer is Rusens, Moscow), 1 μl of a freshly prepared colloidal gold solution (5 mM) is applied. Allow chloroform to evaporate for 10 minutes. Then, 1 μl of antibody solution is applied, the drop is allowed to dry for 10 minutes, and the biosensor is kept in the refrigerator with a temperature of 4 ± 2 ° С overnight. Biosensors are stored at 4 ± 2 ° C for not more than 1 month.

EXAMPLE 3. Measurement of the concentration of cardiomyoglobin using an electrochemical immunosensor in blood plasma samples.

Preparation for measurement

On the working surface of the electrochemical biosensor, 1 μl of the test blood plasma sample is applied with a pipette and placed in an oven with a temperature of 37 ± 1 ° C for 15 minutes.

Signal Measurement Parameters

In the program to the device set the following parameters of the signal measurement (figure 1):

Method: Square wave voltammetry;

Incubation time: 900 s;

Frequency: 10 Hz;

Initial Potential: 0.1 V;

Final potential: -0.6 V;

Potential step: 0.005 V;

Amplitude: 0.020 V;

Signal measurement

1 ml of phosphate buffer solution is added to the cell. The biosensor with the applied sample is fixed in the measuring cell, lowered into the buffer solution and the signal measurement procedure is started. The resulting voltammetric curve is stored as an independent file.

Calculation of myoglobin concentration

If a peak is observed on the obtained voltammogram (E _max = -200 ... -300 mV), then using the program to determine the area of the obtained peak recovery of myoglobin using the program. According to the calibration curve constructed earlier, the concentration of myoglobin corresponding to the obtained peak area value is determined.

EXAMPLE 4. Determination of cardiomyoglobin in blood plasma samples of patients with myocardial infarction and healthy donors.

The developed biosensor and the method for express determination of cardiomyoglobin were tested on 5 plasma samples of patients and healthy people. Table 2 shows the values of cardiomyoglobin concentrations obtained immediately after blood sampling using the RAMP system (Response Biomedical Corp), for each of the samples according to the instructions for the device. The resulting samples were stored at -70 ± 2 ° C and thawed immediately before measurements.

table 2 Characteristics of plasma samples Sample id Cardiomyoglobin concentration, ng / ml 0001 120 ± 12 0002 94 ± 9 0004 118 ± 12 K-001 57 ± 7 K-002 18 ± 2

Figures 1 and 2 below show typical square-wave voltammograms of the reduction of iron of heme cardiomyoglobin obtained according to the developed rapid analysis method for different samples: blood serum without cardiomyoglobin (control sample), blood serum with 14 μM cardiomyoglobin, as well as a plasma sample MI (ID 0001) (FIG. 1), and a linear relationship is shown between the concentration of cardiomyoglobin determined using a RAMP® analyzer (Response Biomedical Corp, Canada) and an immunosensor signal (pi area ka recovery of iron heme cardiomyoglobin) obtained according to the developed methodology (figure 2).

Claims (1)

The method of rapid determination of cardiomyoglobin in blood plasma using an electrochemical immunosensor, which consists in the fact that 1 μl of a blood plasma sample is applied to the surface of a working electrode pre-modified with colloidal gold and monoclonal antibodies to cardiomyoglobin, and the obtained immunosensor is maintained with a sample of 15 min at 37 ± 1 ° C, preincubation is carried out for 15 min in a working phosphate buffer solution at pH 6.5 ± 0.1, electrochemical registration of cardiomyoglobin is carried out by measuring the area peak recovery of heme iron by the method of square-wave voltammetry and determine the content of cardiomyoglobin in the sample according to a previously obtained calibration graph.

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