Performance comparison between multienzymes loaded single and dual electrodes for the simultaneous electrochemical detection of adenosine and metabolites in cancerous cells

• Published in: Biosensors & bioelectronics 2018-06, Vol.109, p.263-271
• Main Authors: Hussain, Khalil K., Akhtar, Mahmood H., Kim, Moo-Hyun, Jung, Dong-Keun, Shim, Yoon-Bo
• Format: Article
• Language: English
• Subjects: A549 Cells; Adenosine - chemistry; Adenosine - isolation & purification; Adenosine Deaminase - chemistry; Adenosine inosine hypoxanthine detection; Biosensing Techniques; Conducting polymer; Dipyridamole; Electrodes; Enzyme sensor; Humans; Hypoxanthine - chemistry; Inosine - chemistry; Inosine - isolation & purification; Limit of Detection; Metabolomics - methods; Neoplasms - diagnosis; Neoplasms - metabolism; Neoplasms - pathology; Purine-Nucleoside Phosphorylase - chemistry; Simultaneous analysis; Xanthine Oxidase - chemistry
• ISSN: 0956-5663; 1873-4235
• DOI: 10.1016/j.bios.2018.03.031

The analytical performance of the multi enzymes loaded single electrode sensor (SES) and dual electrode sensor (DES) was compared for the detection of adenosine and metabolites. The SES was fabricated by covalent binding of tri-enzymes, adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP), and xanthine oxidase (XO) along with hydrazine (Hyd) onto a functionalized conducting polymer [2,2:5,2-terthiophene-3-(p-benzoic acid)] (pTTBA). The enzyme reaction electrode in DES was fabricated by covalent binding of ADA and PNP onto pTTBA coated on Au nanoparticles. The detection electrode in DES was constructed by covalent binding of XO and Hyd onto pTTBA coated on porous Au. Due to the higher amount (3.5 folds) of the immobilized enzymes and Hyd onto the DES than SES, and the lower Michaelis constant (Km) value for DES (28.7 µM) compared to SES (36.1 µM), the sensitivity was significantly enhanced for the DES (8.2 folds). The dynamic range obtained using DES was from 0.5 nM to 120.0 µM with a detection limit of 1.43 nM ± 0.02, 0.76 nM ± 0.02, and 0.48 nM ± 0.01, for adenosine (AD), inosine (IN), and hypoxanthine (Hypo) respectively. Further, the DES was coupled with an electrochemical potential modulated microchannel for the separation and simultaneous detection of AD, IN, and Hypo in an extracellular matrix of cancerous (A549) and non-cancerous (Vero) cells. The sensor probe confirms a higher basal level of extracellular AD and its metabolites in cancer cells compared to normal cells. In addition, the effect of dipyridamole on released adenosine in A549 cells was investigated. •Performance comparison between multienzymes loaded single and dual electrodes sensor.•Electrochemical potential modulated microchannel (EPMM) coupled with dual electrode sensor (DES).•Electrochemical separation analysis of adenosine, inosine, and hypoxanthine in extracellular cancer cells.•Adenosine concentration was monitored with and without an adenosine reuptake inhibitor (dipyridamole) in A549 and Vero cells.