You are here

Express determination of the pathogenic bacteria based on the immunosensor test-system

In recent years public concern about the safety of foods of animal origin has heightened due to problems that have arisen with outbreaks of foodborne bacterial infections, as well as growing concern about veterinary drug residues and microbial resistance to antibiotics. These problems have drawn attention to feeding practices within the livestock industry and have prompted health professionals and the feed industry to closely scrutinize food quality and safety problems that can arise in foods of animal origin as a result of animal feeding systems.

Identification and detection of pathogen bacteria is in general required for routine surveillance and monitoring, evaluation of the most common food sources responsible for specific foodborne, during regulatory actions or from investigation of a foodborne outbreak. A wide range of methods are available for pathogen bacteria identification and detection, in connection with these programs, for the prevention and identification of problems related to health and safety. The choice of the right method is a key factor for the detection and identification of pathogen microorganism and the intended use of the method, for instance whether for a qualitative or semi-quantitative screening, quantitative and/or confirmatory analysis, must be clearly defined. To reduce the likelihood of bacterial illnesses, the early detection of pathogens is the key. Conventional practice commonly involves cultivating sample cells in laboratory settings, making this procedure time-consuming and costly. Additionally, the cells become “stressed” during transportation from the farm or factory to the laboratory, and this can lead to false positives. The gathering, identification of pathogens and bacteriological defense measures in advance in order to be effective, is very difficult. The environment (water, air, soil) reside in different amounts pathogenic microbes and various organic compounds, so when identifying bacteriological pathogens difficulties arise.

Depending on pathogen and method, tests typically require 2–3 days to obtain a result. Rapid methods, however, are based on immunochemical or nucleic acid technologies. Commercially available rapid tests can provide results in 8–48 h but results from these screening tests are presumptive and require further isolation of organism as proof of contamination. Thus, the combination of speed and sensitivity is the key for any rapid detection method.

The article presents the results on the development of the immune biosensor test systems for the express-diagnostics of pathogenic bacteria, their detection in biological material and in the environment. Assessment of bacteria was carried out using an analytical device – immunosensor, with immobilized specific antibodies on the transducer surface.

Immunosensor development and application are exciting fields in applied microbiology. The basic idea is simple, but the actual operation is quite complex and involves much instrumentation. Basically, an immunosensor is a molecule or a group of molecules of biological origin attached to a signal recognition material. When an analyte comes in contact with the biosensor, the interaction will initiate a recognition signal that can be reported in an instrument. Many types of biosensors have been developed, including a large variety of enzymes, polyclonal and monoclonal antibodies, nucleic acids, and cellular materials.

Optical immuno-sensing technologies can be split into two categories, namely luminescence (fluorescence) sensors and label-free sensors. In the first case sensitive elements, such as proteins, antibodies, enzymes, nano-particles are conjugated with the fluorescent labels; binding analyte molecules to such receptors causes luminescence (fluorescence) or it’s quenching. As result, the response can be easily visualized either by naked eye or with a suitable photodetector. The example could be

the method of ELISA, which was established as a standard bio-sensing method in analytical laboratories, and other bio-sensing methods are commonly compared with it. Label-free optical methods based on the phenomenon of evanescent field or wave which appear as electromagnetic wave propagating along the interface between two materials with the different refractive indices when the light enter the material with lower refractive index at total internal reflection condition are the main focus of this article.

Phenomenon of the SPR in different modifications is widely used for biosensors creating. The most common SPR sensor platforms are based on the prism scheme and angular modulation, but recently, a lot of attention have been paid to the study of waveguides with optical phase detection technique since it demonstrates high sensitivity for the detection of bio-reactions. As a biological sensing elements the proteins (e.g., antibodies) and peptides are the most frequently used. In addition, it was shown that immobilization of biomolecules to the bare transducer surfaces has negative impact to their reactivity therefore different methods of previous surface preparation are used. The main aim of surface modification is to provide maximum interaction between selective biomolecule (ligand) and analyte.

The antibodies have interact with cell antigens, and the resulting shift value resonance angle recorded. Changing the angle depends on the amount of the immune complexes formed on the transducer surface. The antibody solution was applied on the prepared transducer surface, and after flushing saline - suspension cells with the appropriate concentration (10 cells in 1 ml and more orders). The interaction on the surface of immune complexes observed. This method can detect 10 cells in 1 ml at least. In our research we used surface plasmon resonance method, as transducer uses a thin film of gold (20 nm), on which was applied to a glass plate by evaporation in a vacuum. This surface allows to detect substances in the registration immune interactions with great sensitivity. Statistical significance of the analysis grows sharply with the increasing of concentrations. Sensitivity of the immunosensor is between 101–106 cells in 1 ml. Sensitivity analysis of the immune analyse can be significantly increased using highly specific affinity monoclonal antibodies.

Key words: biosensor, microorganisms, bacteria, Pseudomonas aeruginosa, Salmonella spp., express-diagnostic, transducer, antibodies, antigen.

1. Antybiotykorezystentnist' klinichnyh shtamiv Pseudomonas Aeruginosa u hirurgichnyh stacionarah Ukrai'ny v 2010 roci / V.V. Lazoryshynec', A.G. Salmanov, V.F. Marijevs'kyj, M.K. Hobzej // Zdorov’ja nacii'. – 2011. – № 2. – C. 162–169.

2. The rapid alert system for food and feed 2013, Annual report. – Luxembourg: Publications Office of the European Union, 2014. –

3. Psevdomonoz ptyci: metodychni rekomendacii' / [Verbyc'kyj P.I., Kosenko M.V., Avdos'eva I.K. ta in.]. – K.: DDVM, 2000. – 16 s.

4. Smyrnov V.V. Bakterii' rodu Pseudomona / V.V. Smyrnov, Je.A. Kiprijanova. – K.: Nauk. dumka, 1990. – 263 s.

5. Rehm B.H.A. Pseudomonas: Model Organism, Pathogen, Cell Factory / B.H.A. Rehm. – Weinheim: Wiley-VCH, 2008. – 403 p.

6. Pyrogova L.V. Imobilizacija antygenu retrovirusu lejkozu velykoi' rogatoi' hudoby na poverhni imunnogo biosensora / L.V. Pyrogova, M.F. Starodub // Biotechnologia Acta. – 2008. – T. 1, № 2. – S. 52–58.

7. Detection of foodborne pathogens using surface plasmon resonance biosensors / V. Koubová, E. Brynda, L. Karasová [et al.] // Sensors and Actuators B: Chemical.2001.Vol. 74, № 1–3. – P. 100–105.

8. Magneto Actuated Biosensors for Foodborne Pathogens and Infection Diseases Affecting Global Health / I.M. Pividori, A.B. Aissa, D. Brandao [et al.] // Biosensors for Security and Bioterrorism Applications: Part of the series Advanced Sciences and Technologies for Security Applications, 13 March. – 2016. – P. 83–114.

9. Optical immune biosensor based on the surface plasmon resonance for the control of Salmonella tiphimurium level in solutions / N.F. Starodub, Ju.A. Ogorodnijchuk, V.O. Romanov [et al.] // Scientific Bulletin NUBiP Ukraine, Series "Veterinary medicine, quality and safety of food". – 2010. – Vol. 151, № 2. – P. 183–189.

10. Optical immune biosensor «Plasmon Test» for the determination of Salmonella typhimurium / N.F. Starodub, I. Ogorodniichuk, T. Lebedeva, P. Shpylovyy // Sensor Electronics and Microsystems Technology. – 2013. – Vol. 10, № 1. – P. 106–113.

11. Wang Y. Gold nanoparticle-labeled biosensor for rapid and sensitive detection of bacterial pathogens / Y. Wang, E. Alocilja // Journal of Biological Engineering. – 2015. – Vol. 9. – P. 16.

12. Starodub N. Efficiency of Instrumental Analytical Approaches at the Control of Bacterial Infections in Water, Foods and Feed, in Biosensors for Security and Bioterrorism Applications / N. Starodub, J. Ogorodniichuk, O. Novgorodova; edited by Dimitrios P. – Nikolelis, 2016. – 199 p.

13. Oh B.K. Surface plasmon resonance immunosensor using self-assembled protein G for the detection of Salmonella paratyphi / B.K. Oh, W. Lee, Y.K. Kim // J Biotechnol. – 2004. – Vol. 1, № 111 (1). – P. 1–8.

14. Abdelhamida H. Multifunctional graphene magnetic nanosheet decorated with chitosan for highly sensitive detection of pathogenic bacteria / H. Abdelhamida, H.-F. Wu // J. Mater. Chem. B. – 2013. – Vol. 1. – P. 3950–3961.

15. Qi С. Label-free Biosensors for Health Applications / С. Qi, G. Gao, G. Jin. // Biosensors for Health, Environment and Biosecurity; edited by Pier Andrea Serra. – InTech, 2011. – 550 p.

16. Starodub N.F. Efficiency of Biosensors in Environmental Monitoring / N.F. Starodub // Book of SERIES IN SENSORS: Portable Biosensing of Food Toxicants and Environmental Pollutants. – CRC Press, Taylor&Francis Croup Boca Raton London, NewYork, 2013. – P. 515–560.

PDF icon 2016_1_novgorodova_ua.pdf358.42 KB