ANALYTICAL CHARACTERISTICS OF FLUORESCENT SUSPENSION NANOCRYSTAL-ENCODED MICROARRAY ADAPTED FOR SIMULTANEOUS QUANTITATIVE DETECTION OF FREE AND TOTAL PSA IN SERUM SAMPLES

Multiplexed suspension systems of anew generation are capable to provide precise quantitative profiling of mul- tipledisease-specific markers in human body fluids. We have developed suspension microarrays based on microbeads encoded with fluorescent nanociystals, that show undeniable advantages overavailable analogues in terms of improved multiplexing capabilities, physical and optical properties, low cost and simplicity of analysisperformance. We have adapted QD-encoded suspension microarrays for the simultaneous detection of two forms of prostate-specific antigen (PSA) in human serum by means of classical flow cytometiy. In the present study, we describe in detail the designed system properties including evaluation the microarray for quantitative analysis of serum markers in comparison to standard clinical approach ELISA, as well asestimation of the most important analytical characteristics, such as analytic sensitivity (limit of detection), reproducibility, reliability and accuracy of the analysis, the linear rangesfor detectable- marker concentrations. Experimental data suggested that the developed diagnostic system quantifies two forms of PSA in blood serum samples of patients with high accuracy, precision and reliability.

квантовые точки, простатический специфический антиген, суспензионные микрочипы, многопараметрический анализ, проточная цитометрия, fluorescent semiconductor nanocrystals, quantum dots, cancer markers, prostate-specific antigen, microbeads, suspension microarrays, multiplexed analysis, flow cytometry

1. Бражник К.И. Много параметрический анализ сывороточных онко маркеров с помощью суспензионных систем на основе микросфер, кодированных флуоресцентными нанокристаллами. - Дис.. канд. биол. наук. - Москва, 2015.

2. Бражник К.И., Барышникова МА., Соколова З.А. и др. Новые направления в исследовании и ранней диагностике рака с применением детекционных систем на основе флуоресцентных нанокристаллов // Российский биотерапевтический журнал. - 2013. - Т. 12, № 3. - С. 11-24.

3. Бражник К.И., Соколова ЗА., Барышникова МА. и др. Разработка суспензионных микрочипов на основе микросфер, кодированных флуоресцентными нанокристаллами, и принципы их использования для многопараметрической диагностики онкологических заболеваний // Российский биотерапевтический журнал. - 2014. - Т. 13, № 4. - С. 3-10.

4. Blohm D.H., Guiseppi-Elie A. New developments in microarray technology // CurrOpinBiotechnol. - 2001. -12(1).-P.41-7.

5. Brazhnik K., Grinevich R., Efimov A. et al. Development and potential applications of microarrays based on fluorescent nanocrystal-encoded beads for multiplexed cancer diagnostics // Proceedings of SPIE. Biophotonics: Photonic Solutions for Better Health Care IV. - 2014. - 9129. - Article 91292C.

6. Brazhnik K., Sokolova Z., Baryshnikova M. et a/.Quantum dot-based lab-on-a-bead system for multiplexed detection of free and total prostate-specific antigens in clinical human serum samples // Nanomedicine: NBM- 2015. -doi: 10.1016/j.nano.2015.03.003.

7. Dupont N.C., Wang K., Wadhwa P.D. et a/.Validation and comparison of luminex multiplex cytokine analysis kits with ELISA: determinations of a panel of nine cytokines in clinical sample culture supernatants // J Reprodlmmunol. - 2005. - 66(2). - P. 175-91.

8. Ellington A.A., Kullo I.J., Bailey K.R. et al. Antibody-based protein multiplex platforms: technical and operational challenges // Clin Chem. - 2010. - 56(2). - P. 186-93.

9. Elshal M.F., McCoy J.P. Multiplex bead array assays: performance evaluation and comparison of sensitivity to ELISA // Methods. - 2006. - 38(4). - P. 317-23.

10. Fuzery А.К., Levin J., Chan M.M. et al. Translation of proteomic biomarkers into FDA approved cancer diagnostics: issues and challenges // Clin Proteomics. - 2013. - 10(1). - P. 13-27.

11. Gershon D. Microarray technology: an array of opportunities // Nature. - 2002. - 416(6883). - P. 885-91.

12. Gonzalez-GonzalezM., Jara-Acevedo R., Matarraz S. et al. Nanotechniques in proteomics: protein microarrays and novel detection platforms // Eur J Pharm Sci. - 2012. - 45(4). - P. 499-506.

13. Kellar K.L., Douglass J.P. Multiplexed microsphere-based flow cytometric immunoassays for human cytokines // J Immunol Methods. - 2003. - 279(1-2). - P. 277-85.

14. Kodadek T. Protein microarrays: prospects and problems // Chem Biol. - 2001. - 8(2). - P. 105-15.

15. Liotta L.A., Espina V, Mehta A.I. et a/Protein microarrays: meeting analytical challenges for clinical applications // Cancer Cell. - 2003. - 3(4). - P. 317-25.

16. Nabiev 1, Sukhanova A., Artemyev M., Oleinikov V. Fluorescent colloidal particles as a detection tools in biotechnology systems: In Colloidal nanoparticles in Biotechnology / Eds. by A. Elissari. - London- Singapore-NY: Wiley, 2008 -P. 133-68.

17. Nolan J.P., Sklar L.A. Suspension array technology: evolution of the flat-array paradigm // Trends Biotechnol.-2002.-20(1).-P. 9-12.

18. Sturgeon C.M., Viljoen A. Analytical error and interference in immunoassay: minimizing risk // Ann Clin- Biochem. - 2011. - 48(Pt 5). - P. 418-32.

19. Sukhanova A., Nabiev I. Fluorescent nanocrystal-encoded microbeads for multiplexed cancer imaging and diagnosis // Crit Rev OncolHematol. - 2008. - 68(1). - P. 39-59.

20. Sukhanova A., Susha A.S., Bek A. et a/Nanocrystal-encoded fluorescent microbeads for proteomics: antibody profiling and diagnostics of autoimmune diseases // Nano Lett. - 2007. - 7(8). - P. 2322-7.

21. Sukhanova A., Devy J., Venteo L. et al. Biocompatible fluorescent nanocrystals for inununolabeling of membrane proteins and cells // Anal Biochem. - 2004. - 324. - P. 60-7.

22. Sukhanova A., Even-Desrumeaux K., Kisserli A. et al. Oriented conjugates of single-domain antibodies and quantum dots: toward a new generation of ultrasmall diagnostic nanoprobes // Nanomedicine. - 2012. - 8. - P. 516-25.