Direct electrochemical determination of methotrexate using functionalized carbon nanotube paste electrode as biosensor for in-vitro analysis of urine and dilute serum samples

Chemotherapy is a widely used effective method for the treatment of cancer in humans. Severity of dose levels and treatment duration necessitate frequent continuous monitoring of active drug concentration in human serum and urine. Development of an electrochemical biosensor for direct determination of methotrexate (MTX), a chemotherapy agent, from physiological fluids has been investigated. Functionalized multi-walled carbon nanotube paste electrodes (f-CNTPE) have been fabricated for simple, cost-effective and reusable electrochemical sensors. Scanning electron microscopy studies revealed that the fabricated electrode surface is comprised of nano-streaks of independent segregated CNTs with high porosity. Cyclic voltammetric analysis showed that f-CNTPE had an enhanced electrocatalytic activity with sensitivity as much as three times and less over potentials by 70 mV compared with a conventional carbon paste electrode. Under optimized experimental parameters, differential pulse voltammograms (DPVs) exhibited a gradual increase in peak current with concentration, and the plot of peak current against MTX concentration was linear in the range of 0.4–5.5 μM. Square-wave voltammetry analysis of methotrexate exhibited a linear determination range from 0.01 to 1.5 μM, and the low-detection-limit was 2.9 × 10 ⁻⁹ M. Steady-state current–time analysis mimic to hydrodynamic flow-cell experiments demonstrated a low-detection-limit of 10 nM (S/N ratio ~8) and an analysis time of mere 10 s. Selective determination of MTX at micromolar concentrations in the presence of a mixture of possible electroactive interferents, ascorbic acid, dopamine, etc. (1.5 μM each) is successfully demonstrated. The electrochemical biosensor allows the detection of MTX in-vitro directly from pharmaceutical drug, human serum and undiluted urine samples at very low concentration limits of as low as 5.0 × 10 ⁻⁷ M with recovery error limits of 4.5% at maximum.