Development of a New Disposable Label-free AFP Immunosensor for Electrochemical Detection of Cancer Biomarker Alpha-Fetoprotein

Abstract

In this study, a new disposable label-free immunosensor was developed for the electrochemical determination of alpha-fetoprotein (AFP) that is a cancer biomarker and its application was performed on human blood serum samples. In the work, firstly the screen printed carbon electrodes (SPCE) were modified with reduced graphene oxide (RGO), then the electrode surface was coated with polyneutral red (PNR) by applying the electropolymerization technique and finally, gold nanoparticles (AuNP) were created by electrodeposition method on the surface. Electrochemical characterizations of the SPCE/RGO/PNR/AuNP electrode were performed using cyclic voltammetry (CV), differential pulse voltammetry (DPV), and square wave voltammetry (SWV) methods. Immobilization of Anti-AFP to the surface of SPCE/RGO/PNR/AuNP electrodes was carried out by a covalent binding method using 3-mercaptopropionic acid (3-MPA) and 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide/N-hydroxysuccinimide (EDC/NHS). The optimum working parameters of the prepared AFP immunosensor were determined by DPV and SWV methods. It was calculated that the linear range was 1-500 ng mL-1, the limit of detections was 0.79 ng mL-1, and 0.86 ng mL-1, respectively. The reproducibility of the AFP immunosensor was tested for 50 ng mL-1 AFP concentration and relative standard deviation (R.S.D.) values were calculated as 4.06% (n = 10) and 3.68% (n = 10) for DPV and SWV methods, respectively. The operation stability of the developed AFP immunosensor was examined by voltammetric methods for 60 days, and the storage stability for 12 weeks. The prepared AFP immunosensors were used for the analysis of different concentrations of AFP added to the human serum samples in a known amount and over 95% recoveries were obtained.

Keywords

• Electrochemical AFP immunosensor
• cancer biomarker
• reduced graphene oxide
• polyneutral red
• gold nanoparticle
• screen-printed electrodes

Kanser Biyobelirteci Alfa-Fetoproteinin Elektrokimyasal Tayini İçin Tek Kullanımlık Etiketsiz Yeni AFP İmmünosensörünün Geliştirilmesi

Abstract

Bu araştırmada, bir kanser biyobelirteci olan alfa-fetoproteinin (AFP) elektrokimyasal tayini için tek kullanımlık yeni etiketsiz immünosensör geliştirilmiş ve insan kan serumu numunelerinde uygulaması gerçekleştirilmiştir. Çalışmada yüzey baskılı karbon elektrotlar (SPCE) ilk olarak indirgenmiş grafen oksit (RGO) ile modifiye edilmiş ardından elektropolimerizasyon tekniği uygulanarak elektrot yüzeyi polinötral kırmızı (PNR) ile kaplanmış, son olarak elektrodepozisyon metodu ile altın nanoparçacıklar (AuNP) yüzeyde oluşturulmuştur. SPCE/RGO/PNR/AuNP elektrodunun elektrokimyasal karakterizasyonu dönüşümlü voltametri (CV), diferansiyel puls voltametri (DPV) ve kare dalga voltametri (SWV) metotları kullanılarak yapılmıştır. SPCE/RGO/PNR/AuNP elektrotların yüzeyine Anti-AFP’nin immobilizasyonu 3-merkaptopropiyonik asit (3-MPA) ve 1-etil-3-(3-dimetilamino-propil) karbodiimid/N-hidroksisüksinimid (EDC/NHS) kullanılarak kovalent bağlama yöntemi ile gerçekleştirilmiştir. Hazırlanan AFP immünosensörünün optimum çalışma parametreleri DPV ve SWV metotları ile belirlenmiştir. DPV ve SWV metotları ile belirlenen doğrusal tayin aralıkları 1-500 ng mL-1, tespit limitleri ise sırasıyla 0.79 ng mL-1 ve 0.86 ng mL-1 olarak belirlenmiştir. AFP immünosensörünün tekrarlanabilirliği 50 ng mL-1 AFP konsantrasyonu için test edilmiş ve bağıl standart sapma (R.S.D.) değerleri DPV ve SWV metotları için sırasıyla %4.06 (n=10) ve %3.68 (n=10) olarak hesaplanmıştır. Geliştirilen AFP immünosensörünün uygulama kararlılığı 60 gün boyunca, depolama kararlılığı ise 12 hafta depolama süresince voltametrik metotlarla incelenmiştir. Hazırlanan AFP immünosensörleri insan serumu örneklerine bilinen miktarda ilave edilen farklı konsantrasyonlardaki AFP’nin analizi için kullanılmış ve %95’in üstünde geri kazanımlar elde edilmiştir.

Keywords

• Elektrokimyasal AFP immünosensörü
• kanser biyobelirteci
• indirgenmiş grafen oksit
• polinötral kırmızı
• altın nanopartikül
• yüzey baskılı karbon elektrotlar

References

1. Altun M, Bilgi Kamaç M, Bilgi A, Yılmaz M, 2020. Dopamine biosensor based on screen-printed electrode modified with reduced graphene oxide, polyneutral red and gold nanoparticle. International Journal of Environmental Analytical Chemistry, 100(4): 451-467.
2. Attar A, Ghica ME, Amine A, Brett CM, 2014. Poly (neutral red) based hydrogen peroxide biosensor for chromium determination by inhibition measurements. Journal of hazardous materials, 279: 348-355.
3. Aydın EB, Sezgintürk MK, 2017. A sensitive and disposable electrochemical immunosensor for detection of SOX2, a biomarker of cancer, Talanta 172: 162-170.
4. Aydın M, Aydın EB, Sezgintürk MK, 2017. A highly sensitive immunosensor based on ITO thin films covered by a new semi-conductive conjugated polymer for the determination of TNFα in human saliva and serum samples, Biosensors and Bioelectronic 97:169-176.
5. Bahadır EB, Sezgintürk, MK, 2015. Applications of electrochemical immunosensors for early clinical diagnostics, Talanta, 132: 162-174.
6. Bilgi Kamac M, Kiymaz Onat E, Yilmaz M, 2020a. A novel non-enzymatic amperometric H2O2 sensor based on screen-printed electrode modified with reduced graphene oxide, polyneutralred and gold nanoparticles, International Journal of Environmental Analytical Chemistry, 100(4): 408-418.
7. Bilgi Kamac M, Kiymaz Onat E, Yilmaz M, 2020b. A new disposable amperometric NADH sensor based on screen-printed electrode modified with reduced graphene oxide/polyneutral red/gold nanoparticle, International Journal of Environmental Analytical Chemistry, 100(4): 419-431.
8. Bilgi M, Ayranci E, 2018. Development of amperometric biosensors using screen-printed carbon electrodes modified with conducting polymer and nanomaterials for the analysis of ethanol, methanol and their mixtures, Journal of Electroanalytical Chemistry, 823: 588-592.

9. Bilgi M, Ayrancı E, 2016. Biosensor application of screen-printed carbon electrodes modified with nanomaterials and a conducting polymer: Ethanol biosensors based on alcohol dehydrogenase, Sensors and Actuators B: Chemical, 237: 849-855.
10. Demirbakan B, Sezgintürk MK, 2020. A novel ultrasensitive immunosensor based on disposable graphite paper electrodes for troponin T detection in cardiovascular disease, Talanta, 213: 120779.
11. Diaconu I, Cristea C, Harceaga V, Marrazza G, Berindan-Neagoe I, Sandulescu R, 2013. Electrochemical immunosensors in breast and ovarian cancer, Clinica Chimica Acta, 425: 128-138.
12. Fan F, Shen HY, Zhang GJ, Jiang XY, Kang XX, 2014. Chemiluminescence immunoassay based on microfluidic chips for α-fetoprotein, Clinica Chimica Acta, 431: 113–117.
13. Fang X, Li XQ, Wang H, Wu XM, Wang GL, 2018. Tuning surface states to achieve the modulated fluorescence of carbon dots for probing the activity of alkaline phosphatase and immunoassay of alpha-fetoprotein, Sensors and Actuators B: Chemical, 257: 620–628.
14. Fanjul-Bolado P, Queipo P, Lamas-Ardisana PJ, Costa-Garcia A, 2007. Manufacture and evaluation of carbon nanotube modified screen-printed electrodes as electrochemical tools, Talanta, 74: 427-433.
15. Idris OA, Mabuba N, Arotiba OA, 2018. An alpha-fetoprotein electrochemical immunosensor based on a carbon/gold bi-nanoparticle platform, Analytical Methods, 10: 5649-5658.
16. Jothi L, Jaganathan SK, Nageswaran G, 2020, An electrodeposited Au nanoparticle/porous graphene nanoribbon composite for electrochemical detection of alpha-fetoprotein, Materials Chemistry and Physics, 242: 122514.
17. Lan Q, Shen H, Li J, Ren C, Hu X, Yang Z, 2020. Facile synthesis of novel reduced graphene oxide@ polystyrene nanospheres for sensitive label-free electrochemical immunoassay, Chemical Communications, 56(5): 699-702.
18. Li G, Li S, Wang Z, Xue Y, Dong C, Zeng J, Huang Y, Liang J, Zhou Z, 2018. Label-free electrochemical aptasensor for detection of alpha-fetoprotein based on AFP-aptamer and thionin/reduced graphene oxide/gold nanoparticles, Analytical Biochemistry, 547: 37-44.
19. Liang XL, Bao N, Luo XL, Ding SN, 2018. CdZnTeS quantum dots based electrochemiluminescent image immunoanalysis, Biosensors and Bioelectronics, 117:145–152.
20. Liang Y, Zhao X, Wang N, Wang J, Chen H, Bai L, Wang W, 2019. A label-free immunosensor based on PHEMA/graphene oxide nanocomposite for simultaneous electrochemical determination of alpha fetoprotein, RSC Advances, 9(30): 17187-17193.
21. Liu Q, Yang T, Ye Y, Chen P, Ren X, Rao A, Wan Y, Wang B, Luo Z, 2019. A highly sensitive label-free electrochemical immunosensor based on an aligned GaN nanowires array/polydopamine heterointerface modified with Au nanoparticles, Journal of Materials Chemistry B, 7(9): 1442-1449.
22. Pauliukaite R, Brett CM, 2008. Poly (neutral red): Electrosynthesis, characterization, and application as a redox mediator. Electroanalysis: An International Journal Devoted to Fundamental and Practical Aspects of Electroanalysis, 20(12): 1275-1285.
23. Preechakasedkit P, Siangproh W, Khongchareonporn N, Ngamrojanavanich N, Chailapakul O, 2018. Development of an automated wax-printed paper-based lateral flow device for alpha-fetoprotein enzyme-linked immunosorbent assay, Biosensors and Bioelectronics, 102:27–32.
24. Putzbach W, Ronkainen, NJ, 2013. Immobilization Techniques in the Fabrication of Nanomaterial-Based Electrochemical Biosensors: A Review, Sensors 13: 4811-4840.
25. Sezgintürk MK, 2011. A new impedimetric biosensor utilizing vegf receptor-1 (flt-1): Early diagnosis of vascular endothelial growth factor in breast cancer, Biosensors and Bioelectronics, 26:10, 4032-4039.
26. Sonuç Karaboğa MN, Sezgintürk MK, 2019. Cerebrospinal fluid levels of alpha-synuclein measured using a poly-glutamic acid-modified gold nanoparticle-doped disposable neurobiosensor system, Analyst 144: 611-621.
27. Şahin M, Ayrancı E, 2015. Electrooxidation of NADH on modified screen-printed electrodes: effects of conducting polymer and nanomaterials, Electrochimica Acta, 166: 261-270.
28. Tang Z, Ma Z, 2017. Multiple functional strategies for amplifying sensitivity of amperometric immunoassay for tumor markers: A review, Biosensors and Bioelectronics, 98: 100-12.
29. Tudorache M, Bala C, 2007. Biosensors based on screen-printing technology, and their applications in environmental and food analysis”, Analytical and Bioanalytical Chemistry, 388: 565-578.
30. Wang AJ, Zhu XY, Chen Y, Yuan PX, Luo X, Feng JJ, 2019. A label-free electrochemical immunosensor based on rhombic dodecahedral Cu3Pt nanoframes with advanced oxygen reduction performance for highly sensitive alpha-fetoprotein detection, Sensors and Actuators: B. Chemical 288: 721–727.
31. Wangkam T, Boonperm K, Khomkrachang P, Srikhirin T, Praphanphoj V, Sutapan B, Somboonkaew A, Amarit R, 2016. Hepatocellular carcinoma biomarker detection by surface plasmon resonance sensor, In Advanced Materials Research, 1131: 84–87.
32. Wu Y, Wang Y, Wang X, Wang C, Li C, Wang Z, 2019. Electrochemical Sensing of α-Fetoprotein Based on Molecularly Imprinted Polymerized Ionic Liquid Film on a Gold Nanoparticle Modified Electrode Surface, Sensors, 19(14): 3218.
33. Yang SH, Zhang FF, Wang ZH, Liang QL, 2018. A graphene oxide-based label-free electrochemical aptasensor for the detection of alpha-fetoprotein, Biosensors and Bioelectronics, 112:186–192.
34. Yuan X, Longa W, Liu J, Zhang B, Zhou W, Jiang J, Yu B, Wang H, 2019. Associations of serum markers screening for Down's syndrome with pregnancy outcomes: A Chinese retrospective cohort study, Clinica Chimica Acta, 489:130–135.
35. Zhou J, Zhang C, Chen Y, Wang Z, Lan L, Wang Y, Han B, Pan M, Jiao J, Chen Q, 2019. A simple immunosensor for alpha-fetoprotein determination based on gold nanoparticles-dextran-reduced graphene oxide, Journal of Electroanalytical Chemistry, 833: 126-132.
36. Zhou Q, Xue H, Zhang Y, Lv Y, Li H, Liu S, Shen Y, Zhang Y, 2018. Metal-free all-carbon nanohybrid for ultrasensitive photoelectrochemical immunosensing of alpha-fetoprotein, ACS Sensors, 3(7):1385–1391.