DEVELOPMENT OF AN ELECTROCHEMICAL SENSOR FOR MORE RAPID HIF-1Α DETECTION AS AN ALTERNATIVE TO WESTERN BLOT ANALYSIS

Abstract

Oxygen levels in the body/and organs significantly influence the regulation of metabolic reactions. Hypoxia, a decrease in oxygen levels, can potentially trigger various signals leading to serious health issues. This study aimed to develop an electrochemical immunosensor platform for rapidly and accurately detecting the hypoxia biomarker, HIF-1α protein. In this regard, screen-printed gold electrodes were modified using 11-mercaptoundecanoic acid and 3-mercapto-1-propanol (as a spacer) to generate functional carboxyl groups. Employing EDC-NHS chemistry facilitated the immobilization of HIF-1α antibodies, which were then utilized for the selective and specific recognition of their target. Electrochemical voltametric measurements were conducted using a potassium ferri/ferrocyanide redox couple in both hypoxia-cultured cell lysates and phosphate-buffered saline spiked with HIF-1α protein. In addition to electrochemical measurements, Western blotting (WB) was performed to compare findings with electrochemical results and to confirm the presence of HIF-1α in hypoxic cell lysates. While WB results only exhibited the qualitative presence of the respective antigen in lysates, significant signal decreases were observed in both Cyclic Voltammetry (CV) and Differential Pulse Voltammetry (DPV) measurements due to specific antibody-target binding, emphasizing the electrochemical sensor’s performance for more rapid and quantitative protein detection. The low limit of detection (1.46 nM) suggests the potential of our proposed immunosensor platform for detecting HIF-1α protein within a clinically significant range, which is highly desired for point-of-care applications. This study is one of its kind in the literature to develop an electrochemical immunosensor platform for the rapid detection of HIF-1α. The sensor's ability to provide inexpensive, rapid, and quantitative measurements is a significant advancement for HIF-1 α detection.

Keywords

• Hypoxia
• HIF-1α
• Electrochemical biosensing
• Voltammetry
• Western blotting

References

1. [1] Luo Z, Tian M, Yang G, et al Hypoxia signaling in human health and diseases: implications and prospects for therapeutics. Signal Transduct Target Ther, 2022;7:218
2. [2] Taylor CT, Scholz CC The effect of HIF on metabolism and immunity. Nat Rev Nephrol 2022;18:573–587
3. [3] Lee JW, Ko J, Ju C, Eltzschig HK. Hypoxia signaling in human diseases and therapeutic targets. Experimental & Molecular Medicine 2019;-(6):1-13. doi:10.1038/s12276-019-0235-1
4. [4] Rankin EB, Nam JM, Giaccia AJ. Hypoxia: signaling the metastatic cascade. Trends in Cancer 2016;2(6):295-304. doi:10.1016/j.trecan.2016.05.006
5. [5] Ruas JL, Poellinger L. Hypoxia-dependent activation of HIF into a transcriptional regulator. Seminars in Cell and Developmental Biology 2005;16(4-5):514-522. doi:10.1016/j.semcdb.2005.04.001
6. [6] Kim LC, Simon MC. Hypoxia-Inducible factors in cancer. Cancer Research 2022;82(2):195-196. doi:10.1158/0008-5472.can-21-3780
7. [7] Wilson WR, Hay MP. Targeting hypoxia in cancer therapy. Nature Reviews Cancer 2011;11(6):393-410. doi:10.1038/nrc3064
8. [8] Alique M, Sánchez-López E, Bodega G, Giannarelli C, Carracedo J, Ramírez R. Hypoxia-Inducible factor-1Α: the master regulator of endothelial cell senescence in vascular aging. Cells 2020;9(1):195. doi:10.3390/cells9010195

9. [9] Sato T, Takeda N. The roles of HIF-1α signaling in cardiovascular diseases. Journal of Cardiology 2022;81(2):202-208. doi:10.1016/j.jjcc.2022.09.002
10. [10] Ullah MM, Basile DP. Role of renal hypoxia in the progression from acute kidney injury to chronic kidney disease. Seminars in Nephrology 2019;39(6):567-580. doi:10.1016/j.semnephrol.2019.10.006
11. [11] Wang B, Li ZL, Zhang YL, Wen Y, Gao YM, Liu BC. Hypoxia and chronic kidney disease. EBioMedicine 2022;77:103942. doi:10.1016/j.ebiom.2022.103942
12. [12] Ward JP, McMurtry IF. Mechanisms of hypoxic pulmonary vasoconstriction and their roles in pulmonary hypertension: new findings for an old problem. Current Opinion in Pharmacology 2009;9(3):287-296. doi:10.1016/j.coph.2009.02.006
13. [13] Nathan SD, Barbera JA, Gaine SP, et al. Pulmonary hypertension in chronic lung disease and hypoxia. European Respiratory Journal 2018;53(1):1801914. doi:10.1183/13993003.01914-2018
14. [14] Anderson GJ, Cipolla CM, Kennedy RT. Western blotting using capillary electrophoresis. Analytical Chemistry 2011;83(4):1350-1355. doi:10.1021/ac102671n
15. [15] Kurien B, Scofield R. Western blotting. Methods 2006;38(4):283-293. doi:10.1016/j.ymeth.2005.11.007
16. [16] Manotham K, Tanaka T, Ohse T, et al. A biologic role of HIF-1 in the renal medulla. Kidney International 2005;67(4):1428-1439. doi:10.1111/j.1523-1755.2005.00220.x
17. [17] Minet E, Arnould T, Michel G, et al. ERK activation upon hypoxia: involvement in HIF‐1 activation. FEBS Letters 2000;468(1):53-58. doi:10.1016/s0014-5793(00)01181-9
18. [18] Formento JL, Berra E, Ferrua B, et al. Enzyme-Linked immunosorbent assay for pharmacological studies targeting Hypoxia-Inducible Factor 1Α. Clinical and Vaccine Immunology 2005;12(5):660-664. doi:10.1128/cdli.12.5.660-664.2005
19. [19] Cimmino F, Avitabile M, Lasorsa VA, et al. HIF-1 transcription activity: HIF1A driven response in normoxia and in hypoxia. BMC Medical Genetics 2019;20(1):37. doi:10.1186/s12881-019-0767-1
20. [20] De Wilde L, Roels K, Deventer K, Van Eenoo P. Automated sample preparation for the detection and confirmation of hypoxia‐inducible factor stabilizers in urine. Biomedical Chromatography 2020;35(2):e4970. doi:10.1002/bmc.4970
21. [21] Mistry IN, Tavassoli A. Reprogramming the Transcriptional Response to Hypoxia with a Chromosomally Encoded Cyclic Peptide HIF-1 Inhibitor. ACS Synthetic Biology 2016;6(3):518-527. doi:10.1021/acssynbio.6b00219
22. [22] Hosseini S, Vázquez-Villegas P, Rito-Palomares M, Martinez-Chapa SO. Advantages, Disadvantages and modifications of conventional ELISA. In: SpringerBriefs in Applied Sciences and Technology 2017:67-115. doi:10.1007/978-981-10-6766-2_5
23. [23] Klein D. Quantification using real-time PCR technology: applications and limitations. Trends in Molecular Medicine 2002;8(6):257-260. doi:10.1016/s1471-4914(02)02355-9
24. [24] Bronsema KJ, Bischoff R, Van De Merbel NC. Internal standards in the quantitative determination of protein biopharmaceuticals using liquid chromatography coupled to mass spectrometry. Journal of Chromatography B., 2012;893-894:1-14. doi:10.1016/j.jchromb.2012.02.021
25. [25] Shaharuddin S, Rahman NMANA, Masarudin MJ, Alamassi MN, Saad FFA. HIF-1 sensor in detecting hypoxia tolerance at high altitude. Aerospace Medicine and Human Performance 2023;94(6):485-487. doi:10.3357/amhp.6166.2023
26. [26] Martín CMS, Gamella M, Pedrero M, et al. Magnetic beads-based electrochemical immunosensing of HIF-1α, a biomarker of tumoral hypoxia. Sensors and Actuators B Chemical 2019;307:127623. doi:10.1016/j.snb.2019.127623
27. [27] Shen G. Recent advances of flexible sensors for biomedical applications. Progress in Natural Science Materials International 2021;31(6):872-882. doi:10.1016/j.pnsc.2021.10.005
28. [28] Güzel FD, Ghorbanpoor H, Dizaji AN, et al. Label‐free molecular detection of antibiotic susceptibility for Mycobacterium smegmatis using a low cost electrode format. Biotechnology and Applied Biochemistry 2020;68(6):1159-1166. doi:10.1002/bab.2037
29. [29] Haleem A, Javaid M, Singh RP, Suman R, Rab S. Biosensors applications in medical field: A brief review. Sensors International 2021;2:100100. doi:10.1016/j.sintl.2021.100100
30. [30] Mohankumar P, Ajayan J, Mohanraj T, Yasodharan R. Recent developments in biosensors for healthcare and biomedical applications: A review. Measurement 2020;167:108293. doi:10.1016/j.measurement.2020.108293
31. [31] Chadha U, Bhardwaj P, Agarwal R, et al. Recent progress and growth in biosensors technology: A critical review. Journal of Industrial and Engineering Chemistry 2022;109:21-51. doi:10.1016/j.jiec.2022.02.010
32. [32] Shi X, Sung SHP, Lee MMS, et al. A lipophilic AIEgen for lipid droplet imaging and evaluation of the efficacy of HIF-1 targeting drugs. Journal of Materials Chemistry B., 2020;8(7):1516-1523. doi:10.1039/c9tb02848j
33. [33] Wotzlaw C, Otto T, Berchner‐Pfannschmidt U, Metzen E, Acker H, Fandrey J. Optical analysis of the HIF‐1 complex in living cells by FRET and FRAP. The FASEB Journal 2006;21(3):700-707. doi:10.1096/fj.06-6280com
34. [34] Kaur J, Ghorbanpoor H, Öztürk Y, et al. On‐chip label‐free impedance‐based detection of antibiotic permeation. IET Nanobiotechnology 2021;15(1):100-106. doi:10.1049/nbt2.12019
35. [35] Ghorbanpoor H, Dizaji AN, Akcakoca I, et al. A fully integrated rapid on-chip antibiotic susceptibility test – A case study for Mycobacterium smegmatis. Sensors and Actuators a Physical 2022;339:113515. doi:10.1016/j.sna.2022.113515
36. [36] Ucar A, González-Fernández E, Staderini M, et al. Miniaturisation of a peptide-based electrochemical protease activity sensor using platinum microelectrodes. The Analyst 2019;145(3):975-982. doi:10.1039/c9an02321f
37. [37] Da Silva Santos F, Da Silva LV, Campos PVS, De Medeiros Strunkis C, Ribeiro CMG, Salles MO. Review—Recent Advances of Electrochemical Techniques in food, energy, environment, and Forensic Applications. ECS Sensors Plus 2022;1(1):013603. doi:10.1149/2754-2726/ac5cdf
38. [38] Pu X, Zhao D, Fu C, et al. Understanding and calibration of charge storage mechanism in cyclic voltammetry curves. Angewandte Chemie International Edition 2021;60(39):21310-21318. doi:10.1002/anie.202104167
39. [39] Baluta S, Meloni F, Halicka K, et al. Differential pulse voltammetry and chronoamperometry as analytical tools for epinephrine detection using a tyrosinase-based electrochemical biosensor. RSC Advances 2022;12(39):25342-25353. doi:10.1039/d2ra04045j
40. [40] Mradula, Raj R, Mishra S. Voltammetric immunosensor for selective thyroxine detection using Cu‐MOF@PANI composite. Electrochemical Science Advances 2021;2(5). doi:10.1002/elsa.202100051
41. [41] Liu J, Xu Y, Liu S, Yu S, Yu Z, Low SS. Application and progress of chemometrics in voltammetric biosensing. Biosensors 2022;12(7):494. doi:10.3390/bios12070494
42. [42] Yang PC, Mahmood T. Western blot: Technique, theory, and trouble shooting. North American Journal of Medical Sciences 2012;4(9):429. doi:10.4103/1947-2714.100998
43. [43] Ghosh R, Gilda JE, Gomes AV. The necessity of and strategies for improving confidence in the accuracy of western blots. Expert Review of Proteomics 2014;11(5):549-560. doi:10.1586/14789450.2014.939635
44. [44] Shepherd JL, Kell A, Chung E, Sinclar CW, Workentin MS, Bizzotto D. Selective reductive desorption of a SAM-Coated gold electrode revealed using fluorescence microscopy. Journal of the American Chemical Society 2004;126(26):8329-8335. doi:10.1021/ja0494095
45. [45] Bhadra P, Shajahan MS, Bhattacharya E, Chadha A. Studies on varying n-alkanethiol chain lengths on a gold coated surface and their effect on antibody–antigen binding efficiency. RSC Advances 2015;5(98):80480-80487. doi:10.1039/c5ra11725a
46. [46] Koç Y, Moralı U, Erol S, Avci H. Electrochemical investigation of gold based screen printed electrodes: an application for a seafood toxin detection. Electroanalysis 2020;33(4):1033-1048. doi:10.1002/elan.202060433
47. [47] Blackler RJ, Müller-Loennies S, Pokorny-Lehrer B, et al. Antigen binding by conformational selection in near-germline antibodies. Journal of Biological Chemistry 2022;298(5):101901. doi:10.1016/j.jbc.2022.101901
48. [48] De Carvalho HJC, Seth A, Speidel R, et al. A 2D microfluidic Paper-Based analytical device for diagnosis of canine visceral leishmaniasis via mass Spectrometry-Based immunoassay. Analytical Chemistry 2025;97(13):7089-7097. doi:10.1021/acs.analchem.4c05962
49. [49] Yildirim-Tirgil N, Ayni E, Kaya K. Enhanced electrochemical detection of SARS-CoV-2 IgG using magnetic nanocomplexes: evaluation of preparation processes and sensor stability. Journal of Nanoparticle Research 2025;27(2). doi:10.1007/s11051-025-06240-2
50. [50] Ghorbanpoor H, Akcakoca I, Dizaji AN, et al. Simple and low‐cost antibiotic susceptibility testing for Mycobacterium tuberculosis using screen‐printed electrodes. Biotechnology and Applied Biochemistry 2023;70(3):1397-1406. doi:10.1002/bab.2448
51. [51] Martín CMS, Gamella M, Pedrero M, et al. Anticipating metastasis through electrochemical immunosensing of tumor hypoxia biomarkers. Analytical and Bioanalytical Chemistry 2021;414(1):399-412. doi:10.1007/s00216-021-03240-8
52. [52] Al-Qaoud KM, Obeidat YM, Al-Omari T, et al. The development of an electrochemical immunosensor utilizing chicken IgY anti-spike antibody for the detection of SARS-CoV-2. Scientific Reports 2024;14(1):748. doi:10.1038/s41598-023-50501-w
53. [53] Ranjan P, Sadique MA, Yadav S, Khan R. An Electrochemical Immunosensor Based on Gold-Graphene Oxide Nanocomposites with Ionic Liquid for Detecting the Breast Cancer CD44 Biomarker. ACS Applied Materials & Interfaces 2022;14(18):20802-20812. doi:10.1021/acsami.2c03905
54. [54] Rebelo TSCR, Ribeiro JA, Sales MGF, Pereira CM. Electrochemical immunosensor for detection of CA 15-3 biomarker in point-of-care. Sensing and Bio-Sensing Research 2021;33:100445. doi:10.1016/j.sbsr.2021.100445
55. [55] Barhoum A, Forster RJ. Label-free electrochemical immunosensor for picomolar detection of the cervical cancer biomarker MCM5. Analytica Chimica Acta 2022;1225:340226. doi:10.1016/j.aca.2022.340226
56. [56] Li J, Yang H, Cai R, Tan W. Ultrahighly sensitive Sandwich-Type electrochemical immunosensor for selective detection of tumor biomarkers. ACS Applied Materials & Interfaces 2022;14(39):44222-44227. doi:10.1021/acsami.2c13891
57. [57] Carneiro P, Loureiro JA, Delerue-Matos C, Morais S, Pereira MDC. Nanostructured label–free electrochemical immunosensor for detection of a Parkinson’s disease biomarker. Talanta 2022;252:123838. doi:10.1016/j.talanta.2022.123838
58. [58] Uçar A, Hajool ZA, Ghorbanpoor H, Didarian R, Güzel FD. Effect of microfluidic channel integration onto gold microelectrode on its redox electrochemistry. Turkısh Journal of Chemıstry 2023;47(1):232-241. doi:10.55730/1300-0527.3532
59. [59] Ghorbanpoor H, Corri̇Gan D, Guzel FD. Effect of microchannel dimensions in electrochemical impedance spectroscopy using gold microelectrode. Sakarya University Journal of Science 2022;26(1):120-127. doi:10.16984/saufenbilder.982707
60. [60] Yang N, Li H, Yin T, Huang Y, Sun L, Li G. A new method to assay hypoxia-inducible factor-1 based on small molecule binding DNA. Analytica Chimica Acta 2014;838:31-36. doi:10.1016/j.aca.2014.05.045
61. [61] Wang X, Zhang Z, Wu G, et al. Applications of electrochemical biosensors based on functional antibody-modified screen-printed electrodes: a review. Analytical Methods 2021;14(1):7-16. doi:10.1039/d1ay01570b
62. [62] Guzel FD, Avci H. Fabrication of nanopores in an Ultra-Thin polyimide membrane for biomolecule sensing. IEEE Sensors Journal 2018;18(7):2641-2646. doi:10.1109/jsen.2018.2794781