Development of an immunochromatographic test system with nanozyme amplification for detecting the phytopathogen Erwinia amylovora

S. Razo^1,2, V. G. Panferov¹, I. V. Safenkova¹, N. V. Drenova³, Yu. A. Varitsev⁴, A. V. Zherdev¹, E. N. Pakina², B. B. Dzantiev^1
1Federal Research Centre “Fundamentals of Biotechnology” of the Russian Academy of Sciences, Leninskii prosp., 33, str. 2, Moskva, 119071, Russian Federation
²RUDN University (Peoples’ Friendship University of Russia), ul. Miklukho-Maklaya, 6, Moskva, 117198, Russian Federation
³All-Russian Center of Plant Quarantine, ul. Pogranichnaya, 32, pos. Bykovo, Ramenskii r-n, Moskovskaya obl., 140150, Russian Federation
⁴Lorkh Federal Research Center on Potato, ul. Lorkha, 23, pos. Kraskovo, Lyuberetskii r-n, Moskovskaya obl., 140051, Russian Federation

Abstract. The study aimed to develop a highly sensitive immunochromatographic test system for detecting the causative agent of bacterial blight Erwinia amylovora using Pt-containing nanozyme as an optical signal amplifier. The test system included antibodies specific to E. amylovora immobilized in the test area of ​​the nitrocellulose membrane and on the surface of the nanoscale label. Nanozymes consisting of an Au-containing core and a Pt-containing shell were synthesized as a label. Au-Pt nanoparticles and their conjugates with antibodies were catalytically (peroxidase-like) active. The activity developed during 1–2 min amplifying signal on the test strip. During the analysis, the test strip was immersed in the sample. Then a chromogenic substrate based on 3,3’-diaminobenzidine, oxidized by nanozyme, was applied to the nitrocellulose membrane. As a result, in the presence of E. amylovora in the sample, the test and control zones were painted black. In the absence of E. amylovora in the sample (buffer or plant extract of a healthy plant served as negative control), only the control zone was painted. The limit of detection for E. amylovora was 5×103 CFU mL–1, which was 10 times lower compared to testing without nanozyme amplification. The analysis lasted 15 minutes. Apple leaf extracts infected with bacterial blight according to PCR results were tested using the developed test system. According to the results of the immunochromatographic analysis with signal amplification, E. amylovora was also detected in all samples. The developed immunochromatographic test system based on the catalytic properties of Au-Pt nanoparticles provided highly sensitive detection of E. amylovora and, due to simplicity of use, can be used in the field.

Keywords: detection; phytopathogens; Erwinia amylovora; bacterial blight of fruit crops; immunochromatographic analysis; nanozymes; Au-Pt nanoparticles.

Author Details: S. Razo, post graduate student (e-mail: Этот адрес электронной почты защищён от спам-ботов. У вас должен быть включен JavaScript для просмотра.); V. G. Panferov, Cand. Sc. (Chem.), research fellow (e-mail: Этот адрес электронной почты защищён от спам-ботов. У вас должен быть включен JavaScript для просмотра.); I. V. Safenkova, Cand. Sc. (Biol.), senior research fellow (e-mail: Этот адрес электронной почты защищён от спам-ботов. У вас должен быть включен JavaScript для просмотра.); N. V. Drenova, research fellow (e-mail: Этот адрес электронной почты защищён от спам-ботов. У вас должен быть включен JavaScript для просмотра.); Yu. A. Varitsev, Cand. Sc. (Biol.), senior research fellow (e-mail: Этот адрес электронной почты защищён от спам-ботов. У вас должен быть включен JavaScript для просмотра.); A. V. Zherdev, Cand. Sc. (Biol.), leading research fellow (e-mail: Этот адрес электронной почты защищён от спам-ботов. У вас должен быть включен JavaScript для просмотра.); E. N. Pakina, D. Sc. (Agr.), assoc. prof. (e-mail: Этот адрес электронной почты защищён от спам-ботов. У вас должен быть включен JavaScript для просмотра.); B. B. Dzantiev, D. Sc. (Chem.),head of laboratory (e-mail: Этот адрес электронной почты защищён от спам-ботов. У вас должен быть включен JavaScript для просмотра.).

For citation: Razo S, Panferov VG, Safenkova IV, et al. [Development of an immunochromatographic test system with nanozyme amplification for detecting the phytopathogen Erwinia amylovora]. Dostizheniya nauki i tekhniki APK. 2022;36(1):34-9. Russian. doi: 10.53859/02352451_2022_36_1_34.