Early diagnosis of renal cell carcinoma: use of a nanowire biosensor for detection of small nucleolar ribonucleic acid SNORA77 in patient’s blood

Renal cell carcinoma is the most common form of kidney cancer (more than 90 % of all oncological pathologies of the kidney). At an early stage of development, renal cell carcinoma can be asymptomatic, and this significantly complicates its diagnosis. Commonly used methods for diagnosing renal cell carcinoma do not allow for timely detection of this disease at early stages, thus it is necessary to develop effective and non-invasive methods for its diagnosis using biological macromolecules detectable in blood – biomarkers of this type of cancer. Small nucleolar RNAs are of great interest as such biological macromolecules. In this study, a SiNW biosensor was designed and manufactured for the direct detection of small nucleolar RNA SNORA77 in the blood, associated with renal cell carcinoma. The key element of the SiNW biosensor developed is a nanowire chip based on “siliconon- insulator” structures. The chip is manufactured using a technology similar to Smart Cut, and contains an array of silicon nanowires with n-type conductivity, on whose surface DNA oligonucleotide probes are covalently immobilized. To ensure the specificity of the analysis, the nucleotide sequence of the immobilized DNA probes is complementary to the target sequence of the small nucleolar RNA SNORA77. Purified buffer solutions containing various concentrations of synthetic DNA oligonucleotides, whose sequence is similar to the target detectable sequence of SNORA77, have been analyzed. Using the SiNW biosensor developed, the detection limit of SNORA77 was determined to be approximately 10–17 M. The SiNW biosensor has allowed us to detect an elevated level of SNORA77 in a sample isolated from the blood plasma of a patient with confirmed diagnosis of renal cell carcinoma in comparison with that in a control sample isolated from the plasma of a patient with a non-oncologic disease. The results of the study will be useful for further development of early diagnostic systems for renal cell carcinoma.

1. Tran J., Ornstein M. C. Clinical review on the management of metastatic renal cell carcinoma. JCO Oncology Practice, 18(3), 187–196 (2022). https://doi.org/10.1200/OP.21.00419

2. Hsieh J. J. et al. Renal cell carcinoma. Nature Reviews Disease Primers, 3(1), 1–19 (2017). https://doi.org/10.1038/nrdp.2017.9

3. Ljungberg B., Purdue M. P., Signoretti S. et al. European Association of Urology guidelines on renal cell carcinoma: the 2022 update. European Urology, 82(4), 399–410 (2022). https://doi.org/10.1016/j.eururo.2022.03.006

4. Sung H., Ferlay J., Siegel R. L. et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: a Cancer Journal for Clinicians, 71(3), 209–249 (2021). https://doi.org/10.3322/caac.21660

5. Bosma N. A., Warkentin M. T., Gan C. L et al. Effi cacy and safety of fi rst-line systemic therapy for metastatic renal cell carcinoma: a systematic review and network meta-analysis. European Urology Open Science, 37, 14–26 (2022). https://doi.org/10.1016/j.euros.2021.12.007

6. Tenold M., Ravi P., Kumar M. et al. Current approaches to the treatment of advanced or metastatic renal cell carcinoma. American Society of Clinical Oncology Educational Book, 40, 187–196 (2020). https://doi.org/10.1200/EDBK_279881

7. Motzer R. J., Bander N. H., Nanus D. M. Renal-cell carcinoma. New England Journal of Medicine, 335(12), 865–875 (1996). https://doi.org/10.1056/NEJM199609193351207

8. Gibbons R. P., Montie J. E., Correa Jr. R. J. et al. Manifestations of renal cell carcinoma. Urology, 8(3), 201–206 (1976). https://doi.org/10.1016/0090-4295(76)90366-6

9. McLaughlin J. K., Lipworth L., Tarone R. E. Epidemiologic aspects of renal cell carcinoma. Seminars in oncology. WB Saunders, 33(5), 527–533 (2006). https://doi.org/10.1053/j.seminoncol.2006.06.010

10. Padala S. A., Barsouk A., Thandra K. C. et al. Epidemiology of renal cell carcinoma. World Journal of Oncology, 11(3), 79 (2020). https://doi.org/10.14740/wjon1279

11. Wein A. J., Barsouk A., etc. Campbell-Walsh Urology, 11th ed. Elsevier Health Sciences, Philadelphia (2016).

12. Vasudev N. S., Wilson M., Stewart G. D. et al. Challenges of early renal cancer detection: symptom patterns and incidental diagnosis rate in a multicentre prospective UK cohort of patients presenting with suspected renal cancer. BMJ Open, 10(5), e035938 (2020). https://doi.org/10.1136/bmjopen-2019-035938

13. Wajahat M., Bracken C. P., Orang A. Emerging functions for snoRNAs and snoRNA-derived fragments. International Journal of Molecular Sciences, 22(19), 10193 (2021). https://doi.org/10.3390/ijms221910193

14. Huang Z., Du Y., Wen J, et al. SnoRNAs: functions and mechanisms in biological processes, and roles in tumor pathophysiology. Cell Death Discovery, 8(1), 259 (2022). https://doi.org/10.1038/s41420-022-01056-8

15. Lu B., Chen X., Liu X. et al. C/D box small nucleolar RNA SNORD104 promotes endometrial cancer by regulating the 2ʹ-O-methylation of PARP1. Journal of Translational Medicine, 20(1), 618 (2022). https://doi.org/10.1186/s12967-022-03802-z

16. Chow R. D., Chen S. Sno-derived RNAs are prevalent molecular markers of cancer immunity. Oncogene, 37(50), 6442–6462 (2018). https://doi.org/10.1038/s41388-018-0420-z

17. Mannoor K., Liao J., Jiang F. Small nucleolar RNAs in cancer. Biochimica et Biophysica Acta (BBA) – Reviews on Cancer, 1826(1), 121–128 (2012). https://doi.org/10.1016/j.bbcan.2012.03.005

18. Yang S., Rothman R. E. PCR-based diagnostics for infectious diseases: uses, limitations, and future applications in acute-care settings. The Lancet Infectious Diseases, 4(6), 337–348 (2004). https://doi.org/10.1016/S1473-3099(04)01044-8

19. Ro S., Park C., Jin J. et al. A PCR-based method for detection and quantifi cation of small RNAs. Biochemical and Biophysical Research Communications, 351(3), 756–763 (2006). https://doi.org/10.1016/j.bbrc.2006.10.105

20. Yang T., Zhang M., Zhang N. Modifi ed Northern blot protocol for easy detection of mRNAs in total RNA using radiolabeled probes. BMC Genomics, 23(1), 66 (2022). https://doi.org/10.1186/s12864-021-08275-w

21. Ivanov Y. D., Romanova T.S., Malsagova K.A. et al. Use of silicon nanowire sensors for early cancer diagnosis. Molecules, 26(12), 3734 (2021). https://doi.org/10.3390/molecules26123734

22. Ambhorkar P., Wang Z., Ko H. et al. Nanowire-based biosensors: from growth to applications. Micromachines, 9(12), 679 (2018). https://doi.org/10.3390/mi9120679

23. Kim K., Park C., Kwon D. et al. Silicon nanowire biosensors for detection of cardiac troponin I (cTnI) with high sensitivity. Biosensors and Bioelectronics, 77, 695–701 (2016). https://doi.org/10.1016/j.bios.2015.10.008

24. Ivanov Y. D., Malsagova K. A., Pleshakova T. O., et al. Aptamer-Sensitized nanoribbon biosensor for ovarian cancer marker detection in plasma. Chemosensors, 9(8), 222 (2021). https://doi.org/10.3390/chemosensors9080222

25. Ivanov Y., Pleshakova T., Malsagova K. et al. Detection of marker miRNAs, associated with prostate cancer, in plasma using SOI-NW biosensor in direct and inversion modes. Sensors, 19(23), 5248 (2019). https://doi.org/10.3390/s19235248

26. Ivanov Y. D., Malsagova K. A., Popov V. P. et al. Nanoribbon-based electronic detection of a glioma-associated circular miRNA. Biosensors, 11(7), 237 (2021). https://doi.org/10.3390/bios11070237

27. Ivanov Y. D., Nevedrova E. D., Vinogradova A. V. et al. Detection of colorectal cancer associated circular RNAs hsa_circ_0031263, hsa_circ_0072715, and hsa_circ_0136666 in plasma with nanowire chips. Almanac of Clinical Medicine, 52(3), 120–131 (2024). (In Russ.)]. https://doi.org/10.18786/2072-0505-2024-52-016

28. Patolsky F., Zheng G., Hayden O. et al. Electrical detection of single viruses. Proceedings of the National Academy of Sciences of the United States of America, 101(39), 14017–14022 (2004). https://doi.org/10.1073/pnas.0406159101

29. Malsagova K. A., Pleshakova T. O., Kozlov A. F. et al. Detection of infl uenza virus using a SOI-nanoribbon chip, based on an N-type fi eld-effect transistor. Biosensors, 11(4), 119 (2021). https://doi.org/10.3390/bios11040119

30. Popov V. P., Antonova A. I., Frantsuzov P. A. et al. Properties of silicon-on-insulator structures and devices. Semiconductors, 35, 1030–1037 (2001). https://doi.org/10.1134/1.1403567

31. Ivanov Y., Pleshakova T., Malsagova K. et al. Detection of marker miRNAs, associated with prostate cancer, in plasma using SOI-NW biosensor in direct and inversion modes. Sensors, 19(23), 5248 (2019). https://doi.org/10.3390/s19235248

32. Mattson G., Conklin E., Desai S. et al. A practical approach to crosslinking. Molecular Biology Reports, 17, 167–183 (1993). https://doi.org/10.1007/BF00986726

33. Stern E., Wagner R., Sigworth F. J. et al. Importance of the Debye screening length on nanowire fi eld effect transistor sensors. Nano Letters, 7(11), pp. 3405–3409 (2007). https://doi.org/10.1021/nl071792z

34. Laborde C., Pittino F., Verhoeven H. A. et al. Real-time imaging of microparticles and living cells with CMOS nanocapacitor arrays. Nature Nanotechnology, 10(9), pp. 791–795 (2015). https://doi.org/10.1038/nnano.2015.163

35. Namdari P., Daraee H., Eatemadi A. Recent advances in silicon nanowire biosensors: synthesis methods, properties, and applications. Nanoscale Research Letters, 11, 1–16 (2016). https://doi.org/10.1186/s11671-016-1618-z

36. Zhang H., Kikuchi N., Ohshima N. et al. Design and fabrication of silicon nanowire-based biosensors with integration of critical factors: toward ultrasensitive specifi c detection of biomolecules. American Chemical Society Applied Materials and Interfaces, 12(46), 51808–51819 (2020). https://doi.org/10.1021/acsami.0c13984

37. Rissin D. M., Kan C. W., Campbell T. G. et al. Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations. Nature Biotechnology, 28(6), 595–599 (2010). https://doi.org/10.1038/nbt.1641

38. Banerjee D., Tateishi-Karimata H., Ohyama T. et al. Improved nearest-neighbor parameters for the stability of RNA/DNA hybrids under a physiological condition. Nucleic Acids Research, 48(21), 12042–12054 (2020). https://doi.org/10.1093/nar/gkaa572

39. SantaLucia J., Hicks D. The thermodynamics of DNA structural motifs. Annual Review of Biophysics, 33, 415–440 (2004). https://doi.org/10.1146/annurev.biophys.32.110601.141800