Electrochemical immunosensors fabrication for oral cancer detection in patient samples based on carbon based screen printed electrode

Abstract

Oral cancer is a type of carcinoma which starts as a growth in the mouth and affects different regions involving lips, tongue, cheeks, floor of the mouth, and pharynx (throat). World-wide, it is rated sixth most prevalent of all types of cancer and reports second number of highest cancer cases. In India, the reported global incidence of oral cancer cases are 77,000 new cases and 52,000 deaths [1]. It is a life-threatening disease which requires biomarker availability for its timely detection and accurate prognosis. Therefore, there is an urgent need to develop specific, non-invasive tools for oral cancer diagnosis.

Biomarkers (CYFRA 21-1, IL-8, P-53) have certain clinical significance in detection of Oral Squamous Cell Carcinoma (OSCC). The most common form of oral cancer, OSCC causes impairment in oral epithelial cells occurred due to accumulation of genome alteration in the cells. IL-8 promotes angiogenesis, chemotaxis of granulocytes as well as macrophages which is highlighted in the OSCC’s stroma cells. IL-8 works as an indicator for oral cancer transformation from pre-cancerous lesion. CYFRA 21-1 is one of the diagnostic markers which studies telomerase activity in the tumor cells. CYFRA 21-1 also maintains the telomere length during replication of chromosome. A report presented by Sawant et al. mentioned sensitivity and specificity of CYFRA 21-1 biomarker as 84 % and 93 % respectively in oropharyngeal cancer patients [2][3][4][5][6][7]. P-53 is a genomic biomarker for OSCC detection. Loss of heterozygosity (LOH) is also observed in chromosomes (such as 9p, 3q, 13q, and 17p) which serves as an early marker in oral carcinoma-genesis. P-53 biomarker’s potential use in OSCC diagnostics was given by Liao et al. due to presence of mutated p-53 gene on the chromosome 17p in the DNA. P-53 is involved in arresting cell cycle and initiate apoptosis in case of DNA damage [8][9]. As these biomarkers have different clinical significance therefore, it is beneficial for oral cancer diagnostics from different perspective and also contributes in accurate prognosis of cancer condition.

Biomarkers are detected using laboratory made carbon ink-based screen-printed electrodes (SPEs). SPEs are based on three-electrode system consisting of working electrode, auxiliary electrode and reference electrode, where reference electrode is modified with silver paste and both other electrodes are made of carbon material. SPEs are cost-effective, disposable, sensitive and bio-compatible electrode for biomolecules. Conductive carbon ink is fabricated using gum Arabic as a binder, water as a carrier molecule and graphite as a carbon source. All the three components are mixed in an appropriate manner to obtain viscous ink for screen printing using stencil (of three electrode system) and screen-printing machine.

In this work, we report conductive ink synthesis by using graphite powder and gum Arabic; thereafter printing of that ink on the substrate with the help of screen-printing machine and stencil. Subsequently, the substrates were cured at 80℃ for 15 minutes. Here, three immunosensors were prepared for detection of CYFRA 21-1, IL-8 and P-53 biomarkers. SPEs, working electrodes were immobilized with different antibodies of 10 µg/mL concentration for 5 h in a humid chamber followed by washing with phosphate buffer saline (PBS). Then non-binding sites on the working electrodes were blocked using BSA for which BSA immobilization was carried out for 1.5 h in a humid chamber followed by washing with PBS (pH=74.). Sensing was done with respective antigen and dilutions were prepared for all three biomarkers. Electrochemical technique was employed for the sensing purpose (shown in Figure 1, 2 & 3). Lower detection of all the three immunosensors were calculated from the calibration plot (Peak current v/s concentration of antigen), shown in Figure 1(b), 2(b) & 3(b). CYFRA 21-1 immunosensor showed detection limit of 9.79 ng/mL, Il-8 showed detection limit of 0.371 ng/mL and P-53 showed detection limit of 2.6 ng/mL. Thereafter, patient bodily fluids (serum and saliva, N=25) were also tested for all the three biomarkers and results were verified with the standard technique ELISA.

Figure 1: (a) Cyclic voltammogram of BSA/anti-CYFRA 21-1/carbon ink/SPE immunosensor response in sensing range from 0.1 ng to 20 ng; (b) Calibration plot of BSA/anti-CYFRA 21-1/carbon ink/SPE immunoelectrode between peak current v/s concentration of CYFRA 21-1.

Figure 2: (a) Cyclic voltammogram of BSA/anti-IL-8/carbon ink/SPE immunosensor response in sensing range from 100 pg to 2000 pg; (b) Calibration plot of BSA/anti-IL-8/carbon ink/SPE immunoelectrode between peak current v/s concentration of IL-8.

Figure 3: (a) Cyclic voltammogram of BSA/anti-P-53/carbon ink/SPE immunosensor response in sensing range from 1 pg to 5000 pg; (b) Calibration plot of BSA/anti-P-53/carbon ink/SPE immunoelectrode between peak current v/s concentration of P-53.