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

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.

Anahtar Kelimeler:

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

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

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,

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