I.I. Bobrinetskii - Dr.Sc.(Eng.), Senior Research Scientist, Scientific Educational Center «Probe Microscopy and Nanotechnology» National Research University of Electronic Technology (MIET) (Zelenograd, Moscow)
K.K. Lavrentyev - Student, National Research University of Electronic Technology (MIET) (Zelenograd, Moscow)
M.V. Mezentseva - Dr.Sc.(Biol.), Head of the Laboratory of Cell Cultures, Federal State Institution D. I. Ivanovskiy Institute of Virology Russian Ministry of Health
V. K. Nevolin - Dr.Sc.(Phys.-Math.), Professor, Chief Researcher, National Research University of Electronic Technology (MIET) (Zelenograd, Moscow)
L.I. Russu - Research Scientist, Cell Cultures of Federal State Institution D. I. Ivanovskiy Institute of Virology Russian Ministry of Health
I.A. Suetina - Ph.D.(Biol.), Senior Research Scientist, Federal State Institution D. I. Ivanovskiy Institute of Virology Russian Ministry of Health
K. A. Tsarik - Ph.D.(Eng.), Lead Process Engineer, Scientific Educational Center «Probe Microscopy and Nanotechnology» National Research University of Electronic Technology (MIET) (Zelenograd, Moscow)

For the development of bio-nitride nano-electronic sensor systems requires the development of technological procedures lowdefect layers forming AlN, identifying patterns of functional characteristics of SAW sensors, as well as research of interaction with biological objects and environments. On sapphire substrates were grown thick AlN films by molecular beam epitaxy with a surface roughness of less than 3-5 nm. On the final substrate created groups pairs of interdigital transducers (IDT). For the investigated AlN films measured value of the SAW velocity on AlN, which is equal to 5730 m/s for the signal delay between input and output IDT 1,05 microseconds. Analysis of the AFC and PFC in the operating parameters was performed on a vector network analyzer PNA Network Analyzer E8362B (Aglient Technologies). The maximum amplitude of the signal is at a frequency of 95,5 MHz with 24 dB attenuation. At the operating frequency the phase shift of a reference signal whose phase is taken as 0 °, was -2154 ± 1°. To evaluate the sensory properties experiment was conducted with the application of various aqueous salt solutions (NaCl) or sucrose (C12H22O11) of 1 l to the area between two IDTs. For comparison, taking the complementary sensor. Sensitivity was defined as the difference between the frequency shift Δφ, measured on a structure with a solution and a complementary clean structure. To investigate the biocompatibility formed structures was carried out cultivation of normal human embryonic fibroblast cells (HEB-T) of Collection of Cell Cultures D.I. Ivanovsky Institute of Virology Russian Ministry of Health. Incubation was performed in a CO2 incubator for 72 hours at 5% CO2 and 37 º C. Viability and proliferation of cells on the AlN was evaluated using a standard MTT test to detect changes in functional activity of mitochondrial enzymes (degidrogenez). By comparing the optical density of reflecting the concentration of enzyme reaction product (formazan) in the test and control samples, the MTT test was performed. Cell enzyme activity was evaluated by the color intensity of the solution by measuring the optical density at a wavelength of 492 nm and 545 nm on a reader. The resulting values of the coefficient of proliferation (CP) for different wavelengths are close to the limits of error and are: СP492 nm = 1,1 ± 0,4, СP545 nm = 1,3 ± 0,5. High error associated with the small size of the crystals. Coefficient of proliferation values indicate that the metallization and sensor nitride epitaxial film are non-toxic. For the researching the morphology and location of the cells on the structures surface by the methodes AFM and scanning electron microscopy (SEM NANOFAB NTC-100, JSC "Nanotechnology-MDT") fixation of cells on the surface of AlN structures is conducted. For this purpose, after removing the substrate from the culture medium they were fixed for 30 minutes in 2,5% glutaraldehyde, and then washed in phosphate buffer 2-3 times for 2 minutes, and the dehydrogenated sequentially in solutions of 50%, 70% and 96% ethanol for 2 min each. The organization of cells on a substrate with lithography was marked. Also analyzed the gene expression of cytokine by the presence of their mRNAs in an HEB cells grown on glass and the sensor substrate. It can be concluded that the presence of the nitride substrate initiates the immune system cells, in particular, induces expression of interferon gamma (IFN-γ). Nevertheless, the suppression of expression of interleukin IL-12, IL-18 and IFN-α can indicate the dynamic character of the changes and the need for more detailed investigations of an immune cell response in the operation of the sensor device. These results demonstrate the ability to create nanobiosensors based on AlN SAW structures.

 

1. Famulok M., Mayer G. Aptamer Modules as Sensors and Detectors // Accounts of Chemical Research. 2011. V. 44. № 12. P. 1349 - 1358.
2. Di Pietrantonio F., Cannatà D., Benetti M., Verona E., Varriale A., Staiano M., D'Auria S. Detection of odorant molecules via surface acoustic wave biosensor array based on odorant-binding proteins // Biosensors and Bioelectronics. 2013. V.41. P. 328 - 334.
3. Horiguchi Y., Miyachi S., Nagasaki Y. High-Performance Surface Acoustic Wave Immunosensing System on a PEG/Aptamer Hybridized Surface // Langmuir. 2013. V.29(24). P. 7369 - 7376.
4. Länge K., Rapp B.E., Rapp M. Surface acoustic wave biosensors: a review // Analytical and Bioanalytical Chemistry. 2008. V.391. P.1509 - 1519.
5. Flory C.A., Baer R.L. Surface transverse wave mode analysis and coupling to interdigital transducers // IEEE Ultrasonics Symposium. 1987. P.313 - 318.
6. Moriizumi T., Unno Y., Shiokawa S. New sensor in liquid using leaky SAW // IEEE Ultrasonics Symposium. 1987. P.579 - 582.
7. Andle J.C., Weaver J.T., Vetelino J.F., McAllister D.J. Selective acoustic plate mode DNA sensor // Journal of Sensor and Actuator Networks B. 1995. V.24. P.129 - 133.
8. Andle J.C., Vetelino J.F., Lade M.W., McAllister D.J. An acoustic plate mode device for biosensing applications // Proceedings of International Conference Solid-State Sensors and Actuators, San Francisco. USA. 1991. P. 483 - 485.
9. Rufer L., Torres A., Mir S., Alam M. O., Lalinsky T., Chan Y. C. SAW chemical sensors based on AlGaN/GaN piezoelectric material system: acoustic design and packaging considerations// International Symposium on Electronics Materials and Packaging. 2005. P.204 - 208
10. Kukushkin S.A., Osipov A.V., Bessolov V.N., Medvedev B.K., Nevolin V.K., Tsarik K.A. Substrates for epitaxy of gallium nitride: new materialsa and techniques // Reviews on Advanced Materials Science». 2008. V.17. P.1 - 32.
11. Malavé A., Schlecht U., Gronewold T.M.A., Perpeet M., and Tewes M. // IEEE Sensors. 2006. P.604.
12. Bergaoui Y., Zerrouki C., Fougnion J. M., Fourati N., Abdelghani A. Sensitivity Estimation and Biosensing Potential of Lithium Tantalate Shear Horizontal  Surface Acoustic Wave Sensor //  Sensor Letters. 2009. V.7. P.1001 - 1005.
13. Yotter R. A., Wilson D. M. Sensor technologies for monitoring metabolic activity in single cells-part II: nonoptical methods and applications // Sensors Journal. IEEE. 2004. V. 4. №. 4. P. 412 - 429.
14. Katalog «Vsesoyuznaya Kollekcziya kletochny'x kul'tur». Nauka: RAEN. 1991. S. 118.
15. Xarbiev R.U. Rukovodstvo po e'ksperimental'nomu (doklinicheskomu) izucheniyu novy'x farmakologicheskix veshhestv. M.: Mediczina. 2005. 832 s.
16. Bobrineczkij I.I., Morozov R.A., Seleznev A.S., Podchernyaeva R.Ja., Lopatina O.A. Issledovaniya proliferativnoj aktivnosti i zhiznesposobnosti kletok fibroblasta i glioblastomy' na razlichny'x tipax uglerodny'x nanotrubok // Byulleten' e'ksperimental'noj biologii i medicziny'. 2012. T. 153. № 2. S. 227 - 232.