Method of blood plasma proximate analysis for cardiomyoglobin by means of electrochemical immunosensor

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

 

The invention relates to the field of analytical chemistry, Elektrokhimiya and medical diagnostics and relates to a method of rapid determination of cardiomegaly in plasma using electrochemical immunosensor.

The invention can be used in medical practice for the diagnosis of myocardial infarction (mi). According to statistics, cardiovascular diseases occupy the first place among the causes of death of the population in Russia (www.gks.ru and cause almost half of the total number of deaths in Europe (www.ehnheart.org). Inefficiency or lack of early diagnosis also directly affects the situation. Over the last decade, a large number of studies aimed at the identification of cardiometer, which can be used for diagnosis for heart pain. Cardiometer is a biological substance, raising the level in the blood is observed for cardiovascular diseases or immediately after damage to the heart muscle [1]. Today we know 177 compounds markers of cardiovascular diseases and research in this area continues [2].

Determination of biomarkers in the blood of patients to diagnose THEM has become a standard procedure in medical practice abroad and is used in leading clinics of the Russian Federation. Nevertheless continuously conducted by the SC of new cardiometer, and a variety of statistical studies confirming the effectiveness of test-systems for diagnostics of THEM. At the present time to diagnose THEM widespread troponin I and T, creatine kinase-MB and cardiomegaly. Serum troponin and creatine kinase-MB point to the death of myocardial cells. Whey cardiomegaly is one of the earliest biomarkers THEM. Cardiomegaly - hemoprotein with a molecular weight of 17,800 and due to its small size, it is rapidly released from damaged tissue in the blood. Its half-life in plasma is only 8.9±1.5 minutes Among patients with acute period of THEM the overall picture of changes in the concentration of cardiomegaly plasma with similar dependencies for creatine kinase-MB and troponin. However, the release of troponin and creatine kinase-MB slower, allowing you to detect them in the blood only after 4-6 hours after onset of symptoms, some patients have increased levels of these markers does not manifest within 12 hours. Whey cardiomegaly appears in the blood within 1 to 4 hours. Serum concentrations of cardiomegaly higher after the damage of various tissues (especially skeletal muscle) or taking cocaine, or in patients with renal failure by reducing clearance (coefficient of purification). However, m is her early racing level cardiomegaly and its ability to determine the acute THEM before other biomarkers compensates for these disadvantages, especially in combination with troponin and creatine kinase-MB, and conducting a series of tests [3, 4].

In various sources, the threshold concentration of cardiomegaly in the blood, the excess of which proves THEY vary in a fairly wide range (from 70-90 µg/l [5-8] to 200 µg/l [9]) and seems to depend on the individual characteristics of the organism. However, the average threshold of 100 µg/l adhere to most researchers [10-14].

In today's market presents the following tests and system table immunoassay proteins cardiometer produced by foreign manufacturers: 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).

Published work on the definition of cardiomegaly is "sandwichy" immunoassay. The method is based on the selective binding of proteins markers corresponding antibodies attached to the surface of the sensor. The detection signal is due to the second top layer of antibodies with different labels (PL. 1).

However, all these methods are quite complex, requiring large expenditures of time. None of them are allowed today to clinical application. As can be seen, and the presented data, only one method of immunoassay adapted for use with blood. None of the authors gives no information about working with real samples of patients with THEM. Accuracy and other analytical characteristics definition in this case will depend on many factors: the activity of antibodies, enzyme-labels, substrate concentration, etc.

The redox activity of iron heme serves as a kind of token hemoproteins and allows for their direct detection, and outside influence on the active center of the enzyme. Myoglobin belongs to the family of hemoproteins - electroactive proteins, the molecules of which contain iron ion. The effect of direct electron transfer from the electrode to the heme iron is widely used for manufacturing electrochemical sensors for hydrogen peroxide and nitrite ion. For registration and modification of the electrode surface are used surfactants [19-21], polymer hydrogels [21], membranophone complexes of polyelectrolytes and other

The aim of the present invention is to develop a method for determining cardiomyogenesis in plasma samples using electrochemical immunosensor. The method of obtaining electrochemical immunosensor for the direct determination of cardiomegaly described in the patent [22]. Previously it was shown that the selective determination of cardiomyo is of Aubin in buffer solutions. The Express-analysis of cardiomegaly in plasma samples required significant revision of the methodology for determining and making immunosensors. The main objectives were: 1) increasing the detection sensitivity and 2) a significant reduction of analysis time.

Unlike most foreign researchers, implementing "sandwich" the analysis scheme, the proposed method is based on own electroactivity of cardiomegaly and direct detection of specific interaction between molecules of hemoprotein and corresponding antibodies. The proposed scheme direct registration of myoglobin due to direct electron transfer from the electrode to the heme iron ion catalyzed oxygen:

The contribution to the signal amplification contribute:

1) gold nanoparticles;

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

3) method of reception signal is a square-wave voltammetry.

The need for the use of antibodies based on the task of selective determination of cardiotomy enzyme released into the blood when the heart muscle is damaged. The analytical signal is the magnitude of the detected current of the electron transfer from the heme iron to the surface of the graphite is of the first electrode, modified by gold nanoparticles. Deposited on the surface of the electrode colloidal gold particles work as an ensemble of microelectrodes and allow not only to increase the area of the active surface of the sensor, but also to improve the ratio signal/noise. The transition from the flat electrode to the system of microelectrodes with radii comparable with the thickness of the diffusion layer, leads to edge effect when the value of the limiting current is directly proportional to the radius of microelectrode.

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

Preparation of the solution zolotoporfirovoe acid.

Weigh (3.9) mg trihydrate zolotoporfirovoe acid (HAuCl4×3H2(O)transfer sample in Eppendorf and dissolved in 1 ml of distilled water. The solution is stored in Eppendorf at 4°C not longer than 1 month.

Preparation of the solution dimethyldioctadecylammonium bromide (DDAB) (0.1 M).

Weigh (46.3) mg dimethyldioctadecylammonium bromide, transferred to the sample in Eppendorf and dissolved in 1 ml of chloroform. The solution is stored in Eppendorf at 4°C not longer than 1 month.

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

Weigh (15.2) mg sodium borohydride, carry it in Eppendorf and dissolved in 1 ml of distilled water. The solution is not stored.

Preparation colloides the solution of gold (5 mm).

A round bottom flask is fixed on a tripod over a magnetic stirrer. Into the flask using a pipette, transfer 1 ml of 0.1 M solution DAB. With vigorous stirring to a solution of DAB add 0.5 ml of 10 mm solution zolotoporfirovoe acid, selected by the eyedropper. Then, with vigorous stirring, added dropwise 0.2 ml of freshly prepared solution of sodium borohydride. After 2 hours dyed organic layer is separated from the water.

EXAMPLE 2. Fabrication of electrochemical immunosensors on cardiomyogenic in plasma.

Preparation of 6 M NaOH

Weigh (24.0 g of sodium hydroxide, transfer the sample into a measuring flask of 100 ml and adjusted with distilled water to the mark. Solution store in plastic dishes under a thrust not exceeding 1 month.

Preparation of phosphate buffer solution

Weigh (1.33 g of potassium dihydrophosphate (KH2PO4and (0,29) g sodium chloride (NaCl). Sample salts transferred into a volumetric flask of 100 ml and adjusted with distilled water to the mark. The solution is thoroughly mixed on a magnetic stirrer until complete dissolution of the salts. On the pH meter using 6 M NaOH solution was adjusted pH to 6.5. The solution is stored in a plastic container with the lid tightly closed at 4°C not longer than 1 month.

Preparation of the solution of antibodies to cardiomegaly

The original solution of antibodies to cardiomyo is the Aubin (firm USBio, USA, cat. No. m-16A) were diluted 10 times with phosphate buffer solution pH 6.5±0,1. In a test tube "Eppendorf" transfer an aliquot of 10 µl of the antibody solution and add 90 ál of buffer solution. The solution is stored in a test tube "Eppendorf" at -20±2°C not more than 1 month.

Preparation of electrochemical biosensors for cardiomegaly

On the surface of the graphite working electrode (manufactured using carbon paste firm "Gwent", England; manufacturer firm "Rosens", Moscow) put 1 ál of freshly prepared colloidal solution of gold (5 mm). We allow the chloroform to evaporate for 10 minutes Then put 1 μl of the antibody solution, give the drop to dry for 10 min and incubated for biosensor in the refrigerator with a temperature of 4±2°C during the night. Biosensors stored at 4±2°C not more than 1 month.

EXAMPLE 3. Measuring the concentration of cardiomegaly using electrochemical immunosensor in plasma samples.

Preparation for measurement

On the working surface electrochemical biosensor with a pipette and put a 1 μl sample of blood plasma and placed in an oven with a temperature of 37±1°C in 15 minutes

The parameters of the measurement signal

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

Method: Quadrantanopia voltammetry;

Incubation time:900 s;

Frequency: 10 Hz;

The initial potential: 0.1;

The final potential: -0.6 V;

Step potential: 0.005;

Range: 0.020;

The signal measurement

In the cell make 1 ml of phosphate buffer solution. Fix biosensor coated with a break in the measuring cell, immersed in a buffer solution and begin the procedure of the measurement signal. The obtained voltammetric curve remembered as a separate file.

The calculation of the concentration of myoglobin

If the received voltamperometry peak is observed (Emax=-200...-300 mV), then using the device to produce a definition of the area received the maximum recovery of myoglobin. On the previously constructed calibration curve to determine the concentration of myoglobin, corresponding to the received value of the peak area.

EXAMPLE 4. The definition of cardiomegaly in plasma samples of patients with myocardial infarction and healthy donors.

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

Table 2
Characteristics of plasma samples
ID sampleThe concentration of cardiomegaly, ng/ml
0001120±12
000294±9
0004118±12
K-00157±7
K-00218±2

The following figure 1 and 2 shows a typical square-wave voltamperometry reduction of iron of heme cardiomegaly obtained according to the developed method of Express-analysis for different samples: serum without cardiomegaly (control sample), serum from 14 µM by kardiomiogeneza and plasma sample of the patient NAMED (ID 0001) (Fig 1)and also shows a linear relationship between the concentration of cardiomegaly determined using the analyzer RAMP® (Response Biomedical Corp, Canada) and signal immunosensor (peak area reduction of iron of heme cardiomegaly)obtained according to the developed methodology (figure 2).

The method of rapid determination of cardiomegaly in plasma using electrochemical immunosensor, namely, that on the surface of the working electrode, pre-modified colloidal gold and monoclonal antibodies to cardiomegaly, put a 1 μl sample of blood plasma, can withstand the resulting immunosensor with a break of 15 min at 37±1°C, perform incubation for 15 min in the working phosphate buffer solution at rn,5±0,1, conduct electrochemical check cardiomegaly by measuring the peak area recovery heme iron by the method of square-wave voltammetry and determine the content of cardiomegaly in the sample prior calibration.



 

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