Improvement of Laser-Induced Graphene-Based Electrochemical Biosensors through Graphene-Conductive Polymer Ink Coating for Sensitive Detection of Alpha-Fetoprotein

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and is the sixth most commonly occurring cancer worldwide with the second-highest mortality rate [1] [2]. Since the 1970s, alpha-fetoprotein (AFP) has been used as a prominent biomarker for the diagnosis of HCC. But AFP is electrochemically inactive, thus, detecting AFP is unreliable for early detection of HCC [3]. In this study, we demonstrated the highly sensitive detection of alpha-fetoprotein (AFP) by developing an electrochemical biosensor using Kapton film. The electrochemical sensor was fabricated by laser scribing technique which is a one-step method that does not require any oxidative acid synthesis route or chemical vapor deposition (CVD) to obtain graphene structure which reduced the cost tremendously [4]. The laser-induced graphene (LIG) electrodes were modified with developed graphene polyaniline (G-PANI) ink to effectively enhance the detection of electrical signals. The peak potential voltage difference was 600µV for the bare electrode, while after modifying with the ink the difference was reduced to 260 µV which indicates a 56% change. Therefore, the electrocatalytic effect of the electrode surface and electrochemical sensor signal was improved after modifying the electrode with ink. To evaluate the performance of the sensor, differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS) techniques were performed in phosphate buffer saline (PBS) buffer with ferro-ferricyanide as the redox probe. For the DPV method, a significant surge in peak current was observed, increasing from 9.2 microamperes at a concentration of 20 pg/mL to 13.6 microamperes at 400 pg/mL which satisfied the fact that increasing the concentration of AFP, led to a higher number of analytes available for electron transfer, thus resulting in the observed current enhancement. The nearly identical current outputs in the DPV measurements were obtained consecutive 15 times which represents the sensor’s stability with an RSD of 3.69% and exhibits high reproducibility (RSD=12.15%, N=5). The obtained EIS result showed good linearity in charge transfer resistance change (coefficient of determination, R2= 0.87) with the AFP concentration increment in the range of 20 pg/mL - 400 pg/mL, and the limit of detection (LOD) was 146 pg/mL at a signal-to-noise ratio of 3, indicating the sensor's sensitivity. The range was selected due to demonstrate high sensitivity in the picogram range and has the potential to cover the entire physiological range for a healthy adult. The specific selectivity of the sensor was evidenced by obtaining a random current peak when both AFP and estrogen were introduced simultaneously on the surface of the sensor. For both electrochemical techniques, a similar coefficient of determination was found from the calibration curves, suggesting that the fabricated biosensor offers high selectivity to the determination of AFP biomarker.<br/><br/>References:<br/>[1] S. F. Altekruse, K. A. McGlynn, and M. E. Reichman, “Hepatocellular Carcinoma Incidence, Mortality, and Survival Trends in the United States From 1975 to 2005,” <i>J. Clin. Oncol.</i>, vol. 27, no. 9, pp. 1485–1491, Mar. 2009, doi: 10.1200/JCO.2008.20.7753.<br/>[2] L. A. Torre, F. Bray, R. L. Siegel, J. Ferlay, J. LortetTieulent, and A. Jemal, “Global cancer statistics, 2012,” <i>CA. Cancer J. Clin.</i>, vol. 65, no. 2, pp. 87–108, Mar. 2015, doi: 10.3322/caac.21262.<br/>[3] W. M. Hai-yong Wang1 and Li-song Teng, “Correlation analysis of preoperative serum alpha-fetoprotein (AFP) level and prognosis of hepatocellular carcinoma (HCC) after hepatectomy,” <i>2013</i>, vol. 11, pp. 1–7.<br/>[4] R. Ye, D. K. James, and J. M. Tour, “Laser-Induced Graphene,” <i>Acc. Chem. Res.</i>, vol. 51, no. 7, pp. 1609–1620, Jul. 2018, doi: 10.1021/acs.accounts.8b00084.