QCM-based biosensor for the Detection of Homocysteine

Year 2020, Issue: 20, 835 - 843, 31.12.2020

Fatma Ayhan

https://doi.org/10.31590/ejosat.777852

Abstract

High plasma homocysteine levels can cause an increased risk of cardiovascular, cerebrovascular, and peripheral arterial diseases. Besides this, Alzheimer’s disease and other dementias, osteoporosis, diabetes and renal disease due to folate and B-vitamin deficiency, various drugs or pre-existing atherosclerotic diseases may be the result of high homocysteine levels. The presented research work aimedto perform the detection of Homocysteine (Hcy) by using Quartz Crystal Microbalance (QCM) biosensor. The temperature controlled QCM system was a home-made designed and constructed equipment which can use silver electrod quartz crystal. The modification of silver electrod quartz crystals surfaces was achieved by the surface cleaning process with sodium hydroxide, acetone and methanol in a consecutive manner. Then self-assembled monolayer of cysteamine and chemical coupling of glutaraldehyde (GA) to free end of monolayer was achieved to create the new functional surface in order to complete the formation of spacer arm/ligand. Homocysteine specific recognizing ligand, anti-Homocysteine antibody was immobilized to glutaraldehyde coupled surfaces. The change in resonance frequency values were measured for each modification step. The optimization of dilution ratio of the antibody solution was performed to modified surfaces. The least dilution ratio of antibody, 1/10000 v/v, was determined as optimum antibody ratio. The detection of homocystein was analysed at a detection limit of 0.1 µM and the linear ranges of calibration curves were estimated as 0.1-2.0 µM and 10-50 µM. Homocysteine values indicated good linearities (R2=0.9813 and 0.9875, respectively). The relative standart deviation (RSD %) for precision was calculated as less than 10%. In conclusion, it was found that the detection of homocysteine can be done both in nano- and micro-molar concentration levels. Additionally, designed biosensor showed desired stability and reproducibility. Finally, a new method different from the present methods for the use in the analysis of Hcy was proposed and developed which detects homocysteine by designed QCM technique with a rapid, cheeper and less pretreatment processes. Additionally, homocysteine detection was performed in nano- and micro- molar concentration values.

Keywords

Homocysteine, anti-Homocysteine antibody, Quartz Crystal Microbalance (QCM), Surface modifikation

Supporting Institution

THE SCIENTIFIC AND TECHNOLOGICAL RESEARCH COUNCIL OF TURKEY

Project Number

105S052

Thanks

Special thanks to Prof. Dr. Hakan Ayhan and Prof. Dr. İ. Cengiz Koçum for their scientific support.

Homosistein tayini amaçlı QCM-temelli biyosensör

Abstract

Yüksek plazma homosistein düzeyleri, kardiyovasküler, serebrovasküler ve peripheral arterial hastalık risklerini arttırabilmektedir. Bunun yanında doğumsal bozukluklar gibi diğer çeşitli patolojiler, Alzheimer hastalığına ve diğer gerilikler, osteoporoz, şeker ve böbrek hastalıkları da yüksek homosistein değerlerinin sonucu olabilmektedir. Sunulan araştırma çalışmasının amacı Kuartz Kristal Mikroterazi (QCM) biosensörü kullanarak Homosisteinin (Hsis) tayinini gerçekleştirmektir. Sıcaklık kontrollü QCM sistemi, yerli olarak tasarlanmış ve üretilmiş bir cihaz olup gümüş elektrodlu kuartz kristal kullanılmıştır. Gümüş elektrod kristal yüzeylerinin modifikasyonu sodium hidroksit, aseton ve methanolün ardışık olarak uygulanarak yapılan yüzey yıkama işlemi ile sağlanmıştır. Daha sonra sisteamin ile kendiliğinden tek tabaka oluşumu ile uzatma kolu/ligand oluşumu gerçekleştirilmesi amacıyla tek tabakanın serbest ucuna glutaraldehitin kimyasal olarak bağlanması sağlanarak yeni bir fonksiyonel yüzey elde edilmiştir. Glutaraldehit bağlı yüzeylere homosisteine özgü ligand olan anti-Homosistein antikoru immobilize edilmiştir. Her modifikasyon basamağı için frekans değerlerinin değişimi ölçülmüştür. Antikor çözeltisinin seyrelme oranı modifiye yüzeyler kullanılarak optimize edilmiştir. En düşük antikor derişimi olan 1/10000 v/v oranı optimize antikor oranı olarak belirlenmiştir. Homosisteinin en düşük tayin sınırı 0.1 µM olarak tespit edilmiştir ve kalibrasyon eğrilerinin doğrusal aralıkları 0.1-2.0 µM ve 10-50 µM olarak bulunmuştur. Belirtilen doğrusal aralıklarda Hsis değerleri oldukça yüksek doğrusallık (sırası ile R2=0.9813 and 0.9875) göstermiştir. Bağıl standart sapmanın duyarlılığı % 10’dan küçük hesaplanmıştır. Sonuç olarak homosistein tayininin hem nano hem mikro molar derişim değerlerinde yapılabileceği bulunmuştur. Ek olarak tasarlanan biyosensör istenen kararlılık ve tekrarlanabilirlik göstermiştir. En son olarak Homosistein analizinde kullanıma yönelik yeni bir yöntem önerilerek geliştirilmiş ve bu hızlı, ucuz ve daha az ön işlemi olan QCM tekniği ile homosistein tayini başarı ile yapılabilmektedir.

Keywords

Homosistein, Anti-Homosistein antikor, Kuartz Kristal Mikrodenge (QCM), Yüzey modifikasyonu

Project Number

105S052

References

• Abraham, JM, Cho, L, (2010) The homocysteine hypothesis: Still relevant to the prevention and treatment of cardiovascular disease? (Review). Cleveland Clinic Journal Of Medicine, 77:911-18.
• Alam, S. F., Kumar, S., Ganguly, P., (2019). Measurement of homocysteine: a historical Perspective. J. Clin. Biochem. Nutr. November, 65, 3, 171–177.
• Andersson, M, Andersson, J, Sellborn, A. Berglin, M., Nilsson, B., Elwing, H, (2005). Quartz crystal microbalance-with dissipation monitoring (QCM-D) for real time measurements of blood coagulation density and immune complement activation on artificial surfaces. Biosensors and Bioelectronics, 21: 79-86. Nutrition, 85:329–30.
• Ayhan, F, Gülsu, A, Ayhan, H, (2007) Quartz Crystal Piezo Electrode Surface Modification for Gold Nano-particles Immobilization. Hacettepe J. Biol. & Chem., 35: 203-208.
• Ayhan, F., Kaya, G., Ayhan, H. (2014). Homocysteine-BSA-affinity based biosensor design. Turkish Journal of Biochemistry, 9(3):383–396.
• Bakhshpour, M., Kevser Piskin, A., Yavuz, H., Denizli, A., (2019). Quartz crystal microbalance biosensor for label-free MDA MB 231 cancer cell detection via notch-4 receptor. Talanta, 204 840–845.
• Beitollahi, H., Zaimbashi, R., Mahani, M. T., Tajik, S., (2020). A label-free aptasensor for highly sensitive detection of homocysteine based on gold nanoparticles. Bioelectrochemistry 134, 107497.
• Bunde, RL, Jarvi, .J, Rosentreter, JJ, (1998) Piezo electric quartz crystal biosensor, Talanta, 46: 1223-36.
• Dixon, M. C., (2008). Quartz Crystal Microbalance with Dissipation Monitoring: Enabling Real-Time Characterization of Biological Materials and Their Interactions. Journal of Biomolecular Techniques 19:151–158.
• Erdamar, A, Ayhan, F, Koçum, İC, Ayhan H, (2008) Urease Immobilized Piezoelectric Quartz Crystal for Urea Conversion. Hacettepe J. Biol. & Chem., 36: 173-180.
• Forgacsova, A., Galba, J., Mojzisova, J., Mikus, P., Piestansky, J., (2019). Ultra-high performance hydrophilic interaction liquid chromatography – Triple quadrupole tandem mass spectrometry method for determination of cysteine, homocysteine, cysteinyl-glycine and glutathione in rat plasma. Journal of Pharmaceutical and Biomedical Analysis, 164, 442–451.
• Frantzen, F., Faaren A.L., Alfheim, I., Nordhei, A.K., (1998). .Enzyme conversion immunoassay for determining total homocysteine in plasma or serum. Clin Chem, 44: 311–316.
• Fulgione, A., Cimafonte, M., Ventura, B. D., Iannaccone, M., Ambrosino, C., Capuano, F., Proroga, Y. T. R., Velotta, R., Capparelli, R., (2018). QCM-based immunosensor for rapid detection of Salmonella Typhimurium in food. Scientific Reports, 8:16137. Hankey GJ, Eikelboom JW, (1999). Homocysteine and vascular disease. Lancet, 354:407–13.
• Hasan, T., Arora, R., Bansal, A. K., Bhattacharya, R., Sharma, G. S., Singh, L. R., (2019). Disturbed homocysteine metabolism is associated with cancer. Experimental & Molecular Medicine 51:21.
• Hoffman, M, (2011) Hypothesis: Hyperhomocysteinemia is an indicator of oxidant stres. Medical Hypotheses 77: 1088–1093. Jiang, H., Li, C., Wei, B., Wang, Q., Zhong, J., Lu, J. (2018). Serum homocysteine levels in acne patients. J Cosmet Dermatol. 17:523–526.
• Kang, Y.F., Qiao, H.X., Meng, Y.L., Xin, Z.H., Ge, L.P., Dai, M.Y., Xu, J.J., Zhang, C.H., (2017). Selective detection of cysteine over homocysteine and glutathione by a simple and effective probe. Anal. Methods 9: 1707-1709.
• Karousos, N.G., Aouabdi, S., Way, A.S., Reddy SM, (2002). Quartz crystal microbalance determination of organophosphorus and carbamate pesticides, Analytica Chimica Acta, 469: 189–96.
• Kim, H.J., Lee, K.S., Jeon, Y.J., (2017). Electrochemiluminescent chemodosimeter based on iridium (III) complex for point-of-care detection of homocysteine levels Biosens. Bioelectron. 91, 497-503.
• Kocum, C., Erdamar, A. Ayhan, H., (2010) Design Of Temperature Controlled Quartz Crystal Microbalance System Instrumentation Science & Technology, 38:39-51.
• Kumar, A., Palfrey, H. A., Pathak, R., Kadowitz, P. J., Gettys, T. W., Murthy, S. N. (2017). The metabolism and significance of homocysteine in nutrition and health. Nutrition & Metabolism 14:78.
• Likogianni, V., Janel, N., Ledru, A., Beaune, P., (2006) Thiol compounds metabolism in mice, rats and humans: Comparative study and potential explanation of rodents protection against vascular diseases, Clinical Chimica Acta, 372: 140-6.
• Liu, Y, Zhang, W, Yu, X, Zhang, H, Zhao, R, Shangguan, D., Li, Y., Shen, B., Liu, G., (2004). Quartz crystal biosensor for real-time kinetic analysis of interaction between human TNF- and monoclonal antibodies, Sensors and Actuators: B, 99: 416–24. Liu, Y., Wang, Q., Li, M., (2019). Aldehyde group functionalized iridium(III) complexes for the selective sensing of homocysteine. Journal of Organometallic Chemistry, 898, 120874.
• Madasamy, T., Santschi, C., Martin, O. J., (2015). A miniaturized electrochemical assay for homocysteine using screen-printed electrodes with cytochrome c anchored gold nanoparticles. Analyst, 140: 6071–6078.
• Marx, KA, (2003) Quartz Crystal Microbalance: A Useful Tool for Studying Thin Polymer Films and Complex Biomolecular Systems at the Solution-Surface Interface, Biomacromolecules, 4 : 1099 -1120.
• McCully, K. S. (2007). Homocysteine, vitamins, and vascular disease prevention. Am J Clin Nutr; 86:1563S–8S.
• Nilsson, K., Gustafson, L., Hultberg, B., (2013). Elevated Plasma Homocysteine Level in Vascular Dementia Reflects the Vascular Disease Process. Dementia and Geriatric Cognitive Disorders Extra. 3:16–24.
• Özkan, Y., Akaydın, S., Fırat, H., Çalışkan-Can, E., Ardıç, S., Şimşek, B., (2007). Usefulness of Homocysteine as a Cancer Marker: Total Thiol Compounds and Folate Levels in Untreated Lung Cancer Patients.Anticancer Research 27: 1185-1190.
• Refsum, H, Helland, S, Ueland, M, (1985) Radioenzymatic determination of homocysteine in plasma and urine. Clin Chem 31:624-628.
• Refsum, H, Ueland, M, Svardal, AM, (1989) Fully Automated Fluorescence Assay for Determining Total Homocysteine in Plasma, Clinical Chemistry, 35: 1921-7.
• Smith, A. D., Refsum, H., Bottiglieric, T., Fenech, M., Hooshmand, B., McCaddon, A., Miller, J. W., Rosenberg, I., H., Obeid, R., (2018). Homocysteine and Dementia: An International Consensus Statement. Journal of Alzheimer’s Disease. 62, 561–570.
• Sönmezler, M., Özgür, E., Yavuz, H., Denizli, A., (2019), Quartz crystal microbalance based histidine sensor. Artificial Cells, Nanomedicine, and Biotechnology, 47 (1) 221–227.
• Tewari, P.C., Zhang, B., Bluestein, B.I., (2004). Analytical and clinical evaluation of the Bayer ADVIA Centaur homocysteine assay. Clin Chim Acta; 342: 171–178.
• Ubbink, JB, (2000) Assay Methods for the Measurement of Total Homocyst(e)ine in Plasma. Seminars in Thrombasis and Hemostatis, 26:233-41.
• Ueland, PM, Refsum, H, Stabler, SP, Malinow MR, Andersson A, Allen RH, (1993) Total Homocysteine in Plasma or Serum: Methods and Clinical Applications (Review), Clinical Chemistry, 39: 1764-79.
• Wada, M., Hirose, M., Kuroki, M., Ikeda, R., Sekitani, Y., Takamura, N., Kuroda, N., (2013). Simultaneous determination of homocysteine, methionine and cysteine in maternal plasma after delivery by HPLC‐fluorescence detection with DBD‐F as a label. Biomedical Chromatograhpy, 27, (6) 708-713.
• Wang, J., Ma, L., Liu, G., Ding, H., Pu, S., (2016). Cysteine and homocysteine chemosensor based on photochromic diarylethene with fluorine. Tetrahedron, 72, 8479-8485.
• Xia, Y., Zhanga, H., Zhu, X., Zhang, G., Yang, X., Li, F., Zhang, X., Fang, M., Yu, J., Zhou, H., (2018). A highly selective two-photon fluorescent chemosensor for tracking homocysteine via situ reaction. Dyes and Pigments, 155, 159–163.
• Zappacosta, B., Persichilli, S., Minucci, A., et al. (2006). Evaluation of a new enzymatic method for homocysteine measurement. Clin Biochem; 39: 62–66.
• Zhang, D., Wen, X., Wu, W., Guo, Y., Cui, W., (2015). Elevated Homocysteine Level and Folate Deficiency Associated with Increased Overall Risk of Carcinogenesis: Meta-Analysis of 83 Case-Control Studies Involving 35,758 Individuals. PLOS ONE 18, 1-16.
• Zinellu, A., Sotgia, S., Scanua, B., Pisanua, E., Sanna, M., Sati, S., Deiana, L., Sengupta, S., Carru, C., (2010). Determination of homocysteine thiolactone, reduced homocysteine, homocystine, homocysteine–cysteine mixed disulfide, cysteine and cystine in a reaction mixture by overimposed pressure/voltage capillary electrophoresis. Talanta, 82, 1281–1285.