Cardiac troponin I determination by elisa immuno assay on magnetic particles with electrochemical detection

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Abstract

A high sensitive method for the quantitative rapid determination of cardiac Troponin I in human serum has been developed. The method is based on an enzyme-linked immunosorbent assay on magnetic particles in the volume of a blood serum sample, which can significantly reduce the diffusion limits typical for common ELISA. Alkaline phosphatase which is a high-performance enzyme was used as an enzyme label. The enzyme demonstrated a catalytic efficiency (kcat/Km) = 26500 1/(s∙mM) in combination with the substrate 1-naphtyl phosphate monosodium salt. The planar electrochemical sensors manufactured by industrial screen-printing technology were used for signal detection. The detection was carried out in differential pulse voltammetry mode. The calculated limit of detection by the enzymatic reaction product was 0.075 μM which significantly exceeded the sensitivity of colorimetric methods. The combination of the proposed methods and approaches makes it possible to obtain a quantitative analysis for cardiac TnI in human serum within 20 minutes with an estimated detection limit of 7 pg/mL and an upper reference limit of normal analyte concentration (99-th percentile) of 22 pg/mL.

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About the authors

O. N. Sorokina

Emanuel Institute of Biochemical Physics, Russian Academy of Sciences

Author for correspondence.
Email: alsiona@gmail.com
Russian Federation, Moscow

T. S. Konstantinova

Emanuel Institute of Biochemical Physics, Russian Academy of Sciences

Email: alsiona@gmail.com
Russian Federation, Moscow

A. K. Vorobyova

Emanuel Institute of Biochemical Physics, Russian Academy of Sciences

Email: alsiona@gmail.com
Russian Federation, Moscow

A. D. Vasilyeva

Emanuel Institute of Biochemical Physics, Russian Academy of Sciences

Email: alsiona@gmail.com
Russian Federation, Moscow

L. V. Yurina

Emanuel Institute of Biochemical Physics, Russian Academy of Sciences

Email: alsiona@gmail.com
Russian Federation, Moscow

A. V. Eremenko

Emanuel Institute of Biochemical Physics, Russian Academy of Sciences

Email: alsiona@gmail.com
Russian Federation, Moscow

A. V. Lyzhenkova

Bauman City Clinical Hospital No.29, Health Department of Moscow

Email: alsiona@gmail.com
Russian Federation, Moscow

L. O. Minushkina

“Central State Medical Academy” Department of the President of the Russian Federation

Email: alsiona@gmail.com
Russian Federation, Moscow

D. A. Zateyshchikov

Bauman City Clinical Hospital No.29, Health Department of Moscow; “Central State Medical Academy” Department of the President of the Russian Federation

Email: alsiona@gmail.com
Russian Federation, Moscow; Moscow

I. N. Kurochkin

Emanuel Institute of Biochemical Physics, Russian Academy of Sciences; Lomonosov Moscow State University

Email: alsiona@gmail.com
Russian Federation, Moscow; Moscow

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2. Fig. 1. Dependences of the electrochemical signal on the number of magnetic particles (a) and on the concentration of AT conjugated with the enzyme label (b) for different concentrations of cTn I in the sample ■ – 1 ng/ml, • – 10 ng/ml, ▲ – 25 ng/ml.

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3. Fig. 2. Dependence of the intensity of the electrochemical signal on the temperature conditions of the assembly of the immunological complex on magnetic particles and the production of the enzymatic reaction product; 1 – the assembly temperature of the immunological complex is 25 °C, the production temperature of the enzymatic reaction product is 25 °C; 2 – the assembly temperature of the immunological complex is 25 °C, the production temperature of the enzymatic reaction product is 37 °C; 3 – the assembly temperature of the immunological complex is 37 °C, the production temperature of the enzymatic reaction product is 37 °C.

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4. Fig. 3. Calibration dependence of the intensity of the electrochemical signal on the concentration of the troponin complex TnITC in the sample (•); inset – initial section of the calibration dependence; ▲ – intensity of TnI signals in patients’ blood serum samples depending on the concentration of Tn I according to City Clinical Hospital No. 29.

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