A Sensitive Sensor for Nano-Molar Detection of 5-Fluorouracil by Modifying a Paste Sensor with Graphene Quantum Dots and an Ionic Liquid

Document Type: Research Paper


1 Department of Chemistry, Mashhad Branch, Islamic Azad University, Mashhad, Iran

2 Department of Chemical Engineering, Laboratory of Nanotechnology, Quchan University of Advanced Technology, Quchan, Iran

3 Department of Applied Chemistry, University of Johannesburg, Johannesburg 17011, South Africa

4 School of Resources and Environment, University of Electronic Science and Technology of China, Xiyuan Ave, Chengdu, P.R. China



5-fluorouracil is a widely used anticancer drug with many side effects on humans, and hence its analysis in biological samples is very important. Accordingly, a novel sensitive electrochemical approach was fabricated by incorporating graphene quantum dots (GCD) and 1-butylpyridinium bromide (BPBr) in the formulation of a carbon paste electrode (GQD/BPBr/CPE). The GQD was synthesized and characterized TEM method and results confirmed them as being spherical with D~ of 5.0 nm. The applicability of the GQD/BPBr/CPE in voltammetric analysis of 5-fluorouracil was evaluated. The relations of oxidation currents and potentials of 5-fluorouracil with pH at the surface of GQD/BPBr/CPE were investigated and the results confirmed the involvement of electrons and protons in the electro-oxidation mechanism of 5-fluorouracil. In square wave voltammetry (SWV) analyses, the GQD/BPBr/CPE showed good sensitivity for 5-fluorouracil over a wide linear range of 0.001–400 μΜ and a detection limit of 0.5 nΜ was achieved. The GQD/BPBr/CPE was successfully applied for the determination of 5-fluorouracil in pharmaceutical samples and acceptable results were obtained.


The determination of pharmaceutical components and especially anticancer drugs such as doxorubicin, epirubicin, 5-fluorouracil is very important due to the adverse effects of these compounds on human body [1-5]. Although, some analytical methods such as spectroscopy, chemiluminescence, flow injection systems, high performance liquid chromatography and electrochemical sensors have been suggested as efficient tools for the determination of drug compounds [6-10], electrochemical methods have shown better potentials in this respect due to advantages of simplicity, low cost, fast response and ease of operation [11-18]. Electrochemists have recently introduced modified electrodes as powerful substitutes for conventional electrodes offering improved selectivity and sensitivity for trace and simultaneous determination of drugs or other biological species [19-23].
Ionic liquids, carbon nanotubes, conductive polymers, dendrimers and DNA are the most important electrode surface modifiers suited for electrochemical sensors [24-31], the application of which in electroanalytical sensors can greatly improve the performance [32-38].
5-Fluorouracil is an antimetabolite fluoropyrimidine analog, prescribed as a chemotherapy drug [39]. The medicine is widely used for breast, stomach, pancreatic, skin, gullet and bowel cancers. The consumption of 5-fluorouracil can cause many side effects such as nausea, diarrhea and increase the risks of infection. Consequently, controlling the dose of this drug in biological samples and studying the purity of its pharmaceutical forms can help manage its side effects. Due to the above-mentioned and the potentials of electrochemical methods, several reports have been published on preparing electrochemical sensors for 5-fluorouracil during recent years [39-44].
Fallah-Shojaei et al. used the synergic effect of 1,3-dipropylimidazolium bromide and ZnFe2O4 magnetic nanoparticles for modification of an electrode as a sensor for the electrochemical determination of 5-fluorouracil and reached a detection limit of 0.07 μM [1].
Bukkitgar et al. used a glucose modified electrode as a sensor for the determination of 5-fluorouracil and achieved a detection limit of 5.17 nM in pharmaceutical and urine samples [45].
Bukkitgar et al. used methylene blue to modify the surface of a carbon paste electrode to develop a sensor for the determination of 5-fluorouracil and reported a detection limit of 2.04 nM [39].
In this investigation, the synergic effect of graphene quantum dots and 1-butylpyridinium bromide was used for modifying a carbon paste electrode. The resulting electrode, i.e. GQD/BPBr/CPE, was found to be a powerful tool for the electrochemical determination of 5-fluorouracil in pharmaceutical samples. The results showed better detection limits as compared to previous reports on the electrochemical sensors modified with GQD and BPBr as conductive binders.

Reagents and Instrumentation
Diethyl ether, citric acid, 5-fluorouracil, phosphoric acid and 1-butylpyridinium bromide were purchased from Sigma-Aldrich. Graphite powder was obtained from ACROS Company.
The electrochemical experiments were performed using a potentiostat/galvanostat system (Autolab). An Ag/AgCl/KClsat was used as the references electrode in all voltammetric experiments.

Synthesis of GQD nanoparticles
A pyrolysis approach was used for preparing GQD. The method was based on using citric acid as the carbon source. In the first step, 2 g of citric acid was transferred to a beaker and heated for 30 min at 250 ˚C to convert the citric acid to a liquid phase with orange color (GQD).

Fabrication of GQD/BPBr/CPE
GQD/BPBr/CPE was prepared by mixing of 0.12 g of 1-butylpyridinium bromide, 0.88 g of paraffin oil, 0.04 g of GQD, and 0.96 g of graphite powder in mortar and pestle. The mixture was hand mixed for ~ 2 h and a portion of the obtained paste was packed into one end of a glass tube, while a copper wire was inserted into the tube and the paste from the other opening of the tube.

Characterization of GQD
The TEM images of GQD were recorded (Fig. 1A), indicating the presence of spherical particles of less than 5 nm in diameter. The UV-Vis spectra of GQD (e.g. Fig. 1B) contained an absorbance band at~ 350 nm relative to GQD [46].

Electrochemical behavior of 5-fluorouracil
The oxidation behavior of 5-fluorouracil was studied in the pH ranges of 5.0-9.0, through square wave voltammetry analyses (Fig. 2 insert). A linear relation between the oxidation potential of 5-fluorouracil and pH with a slope of 60.2 mV/pH was observed for the electro-oxidation of 5-fluorouracil at GQD/BPBr/CPE (Fig. 2), confirming the equal number of electrons and protons involved in the electro-oxidation of 5-fluorouracil (Fig. 3). In addition, the maximum oxidation current was observed at pH=7.0, and hence this value was applied in the next experiments.
The SW voltammograms of a 100.0 μM solution of 5-fluorouracil was recorded using GQD/BPBr/CPE (Fig. 4 curve a), BPBr/CPE (Fig. 4 curve b), GQD/CPE (Fig. 4 curve c) and CPE (Fig. 4 curve d) as the working electrodes. 5-fluorouracil produced oxidation signal at potentials of 1006, 1026, 1061 and 1071 mV with oxidation currents 21.7 μA, 14.6 μA, 10.7 μA and 5.26 μA at the surfaces of GQD/BPBr/CPE, BPBr/CPE, GQD/CPE and CPE, respectively. Moving from CPE to GQD/BPBr/CPE, the oxidation potential of 5-fluorouracil decreased and the oxidation current of the drug increased, confirming the high conductivity of GQD and BPBr at the carbon paste matrix.
In addition, the data obtained from the current density confirmed the trends in the previous results (good electrical conductivity of mediators) (Fig. 4, insert). The respective active surface areas of GQD/BPBr/CPE, BPBr/CPE, GQD/CPE and CPE were determined to be 0.26 cm2, 0.27 cm2, 0.26 cm2 and 0.21 cm2.
The electro-oxidation behavior of 5-fluorouracil was investigated at in the scan rate range of 20-200 mV/s using GQD/BPBr/CPE (Fig. 5). The linear relation between Ipa and ν1/2, observed for the electro-oxidation of 5-fluorouracil, indicated the electro-oxidation of 5-fluorouracil at the modified electrode to be diffusion controlled in nature.
The diffusion coefficient (D) of 5-fluorouracil was determined by recording the chronoamperometric (applied potential of 1100 mV) signals of 1.0, 2.0 and 3.0 mM 5-fluorouracil at the surface of GQD/BPBr/CPE (see Fig. 6A). The Cottrell plots of GQD/BPBr/CPE in the presence of 1.0, 2.0 and 3.0 mM 5-fluorouracil can be seen in Fig. 6B, based on the slopes of which the D value was determined to be 2.18 × 10-6 cm2 s-1.
The analytical factors influencing the determination of 5-fluorouracil by GQD/BPBr/CPE were investigated by square wave voltammetry (Fig. 7). The GQD/BPBr/CPE showed two linear dynamic ranges of from 0.001 to 10.0 μM with a regression equation of I = 1.004423 C5-fluorouracil + 0.77 (r2 = 0.9916); and of 10.0 to 400 μM with a regression equation of I = 0.0987 C5-fluorouracil + 10.6280 (r2 = 0.9936). The GQD/BPBr/CPE showed a detection limit of 0.5 nM (S/N=3) for 5-fluorouracil, which is better than those of previously reported electrochemical sensors for this anticancer drug (table 1). This high sensitivity was attributed to the presence of GQD and BPBr at surface of CPE.

Stability and Selectivity
The stability of GQD/BPBr/CPE through the electrochemical determinations of 5-fluorouracil was studied by recording square wave voltammograms of 100 μM solutions of 5-fluorouracil over a period of time (Fig. 8). As can be seen, the oxidation current of 5-fluorouracil at surface of GQD/BPBr/CPE still showed 92.3% of its original oxidation signal after 14 days. This was considered as confirming the good stability of GQD/BPBr/CPE for determination of 5-fluorouracil. The selectivity of GQD/BPBr/CPE toward the determination of 20.0 μM 5-fluorouracil was found to have an acceptable error of 5%. The results presented in table 2, confirm the high selectivity of GQD/BPBr/CPE for the determination of 5-fluorouracil.

Real sample analysis
The powerful square wave voltammetric analyses were used for the determination of 5-fluorouracil in injection and pharmaceutical samples through the standard addition method. The analytical data are presented in table 3. The obtained results were compared with another electrochemical strategy and F-test and t-test were used to check the accuracy of the method.

A new composite modified electrode (GQD/BPBr/CPE) was successfully designed and used as a powerful voltammetric sensor for the nano-molar determination of 5-fluorouracil. The combination of GQD and BPBr allowed for the sensitive detection of 5-fluorouracil in different samples. Using the GQD/BPBr/CPE, 5-fluorouracil could be measured over a linear calibration range of 0.45–450 μM. The GQD/BPBr/CPE showed an acceptable performance in the analysis of 5-fluorouracil in real samples.

The authors declare that there is no conflict of interests regarding the publication of this manuscript.


1. Fallah Shojaei A, Tabatabaeian K, Shakeri S, Karimi F. A novel 5-fluorouracile anticancer drug sensor based on ZnFe2O4 magnetic nanoparticles ionic liquids carbon paste electrode. Sensors and Actuators B: Chemical. 2016;230:607-614.
2. Karimi F, Shojaei AF, Tabatabaeian K, Shakeri S. CoFe2O4 nanoparticle/ionic liquid modified carbon paste electrode as an amplified sensor for epirubicin analysis as an anticancer drug. Journal of Molecular Liquids. 2017;242:685-689.
3. Alavi-Tabari SAR, Khalilzadeh MA, Karimi-Maleh H, Zareyee D. An amplified platform nanostructure sensor for the analysis of epirubicin in the presence of topotecan as two important chemotherapy drugs for breast cancer therapy. New Journal of Chemistry. 2018;42(5):3828-3832.
4. Alavi-Tabari SAR, Khalilzadeh MA, Karimi-Maleh H. Simultaneous determination of doxorubicin and dasatinib as two breast anticancer drugs uses an amplified sensor with ionic liquid and ZnO nanoparticle. Journal of Electroanalytical Chemistry. 2018;811:84-88.
5. Mohammadian A, Ebrahimi M, Karimi-Maleh H. Synergic effect of 2D nitrogen doped reduced graphene nano-sheet and ionic liquid as a new approach for fabrication of anticancer drug sensor in analysis of doxorubicin and topotecan. Journal of Molecular Liquids. 2018;265:727-732.
6. Manjunatha JG. Surfactant modified carbon nanotube paste electrode for the sensitive determination of mitoxantrone anticancer drug. Journal of Electrochemical Science and Engineering. 2017:39.
7. Huang SC, Lin CC, Huang MC, Wen KC, Simultaneous determination of magnesium ascorbyl phosphate, ascorbyl glucoside, kojic acid, arbutin and hydroquinone in skin whitening cosmetics by HPLC, Journal of Food and Drug Analysis, 2004; 12: 13-18.
8. Beytur M, Kardaş F, Akyıldırım O, Özkan A, Bankoğlu B, Yüksek H, et al. A highly selective and sensitive voltammetric sensor with molecularly imprinted polymer based silver@gold nanoparticles/ionic liquid modified glassy carbon electrode for determination of ceftizoxime. Journal of Molecular Liquids. 2018;251:212-217.
9. Atar N, Yola ML. Core-Shell Nanoparticles/Two-Dimensional (2D) Hexagonal Boron Nitride Nanosheets with Molecularly Imprinted Polymer for Electrochemical Sensing of Cypermethrin. Journal of The Electrochemical Society. 2018;165(5):H255-H262.
10. Başkaya G, Yıldız Y, Savk A, Okyay TO, Eriş S, Sert H, et al. Rapid, sensitive, and reusable detection of glucose by highly monodisperse nickel nanoparticles decorated functionalized multi-walled carbon nanotubes. Biosensors and Bioelectronics. 2017;91:728-733.
11. Bozkurt S, Tosun B, Sen B, Akocak S, Savk A, Ebeoğlugil MF, et al. A hydrogen peroxide sensor based on TNM functionalized reduced graphene oxide grafted with highly monodisperse Pd nanoparticles. Analytica Chimica Acta. 2017;989:88-94.
12. da Silva WP, de Oliveira LH, Santos ALd, Ferreira VS, Trindade MAG. Sample preparation combined with electroanalysis to improve simultaneous determination of antibiotics in animal derived food samples. Food Chemistry. 2018;250:7-13.
13. Raoof JB, Ojani R, Karimi-Maleh H. Electrocatalytic oxidation of glutathione at carbon paste electrode modified with 2,7-bis (ferrocenyl ethyl) fluoren-9-one: application as a voltammetric sensor. Journal of Applied Electrochemistry. 2009;39(8):1169-1175.
14. Tahernejad-Javazmi F, Shabani-Nooshabadi M, Karimi-Maleh H. Gold nanoparticles and reduced graphene oxide-amplified label-free DNA biosensor for dasatinib detection. New Journal of Chemistry. 2018;42(19):16378-16383.
15. Ensafi AA, Karimi-Maleh H. Voltammetric determination of isoproterenol using multiwall carbon nanotubes-ionic liquid paste electrode. Drug Testing and Analysis. 2011;3(5):325-330.
16. Rohani A, Sanghavi BJ, Salahi A, Liao K-T, Chou C-F, Swami NS. Frequency-selective electrokinetic enrichment of biomolecules in physiological media based on electrical double-layer polarization. Nanoscale. 2017;9(33):12124-12131.
17. Sanghavi BJ, Wolfbeis OS, Hirsch T, Swami NS. Nanomaterial-based electrochemical sensing of neurological drugs and neurotransmitters. Microchimica Acta. 2014;182(1-2):1-41.
18. Sanghavi BJ, Srivastava AK. Simultaneous voltammetric determination of acetaminophen, aspirin and caffeine using an in situ surfactant-modified multiwalled carbon nanotube paste electrode. Electrochimica Acta. 2010;55(28):8638-8648.
19. Liu M, Wen Y, Li D, Yue R, Xu J, He H. A stable sandwich-type amperometric biosensor based on poly(3,4-ethylenedioxythiophene)–single walled carbon nanotubes/ascorbate oxidase/nafion films for detection of L-ascorbic acid. Sensors and Actuators B: Chemical. 2011;159(1):277-285.
20. Wen Y, Xu J, Liu M, Li D, Lu L, Yue R, et al. A vitamin C electrochemical biosensor based on one-step immobilization of ascorbate oxidase in the biocompatible conducting poly(3,4-ethylenedioxythiophene)-lauroylsarcosinate film for agricultural application in crops. Journal of Electroanalytical Chemistry. 2012;674:71-82.
21. Zhang L, Wen Y, Yao Y, Xu J, Duan X, Zhang G. Synthesis and Characterization of PEDOT Derivative with Carboxyl Group and Its Chemo/Bio Sensing Application as Nanocomposite, Immobilized Biological and Enhanced Optical Materials. Electrochimica Acta. 2014;116:343-354.
22. Karimi-Maleh H, Hatami M, Moradi R, Khalilzadeh MA, Amiri S, Sadeghifar H. Synergic effect of Pt-Co nanoparticles and a dopamine derivative in a nanostructured electrochemical sensor for simultaneous determination of N-acetylcysteine, paracetamol and folic acid. Microchimica Acta. 2016;183(11):2957-2964.
23. Karimi-Maleh H, Sanati AL, Gupta VK, Yoosefian M, Asif M, Bahari A. A voltammetric biosensor based on ionic liquid/NiO nanoparticle modified carbon paste electrode for the determination of nicotinamide adenine dinucleotide (NADH). Sensors and Actuators B: Chemical. 2014;204:647-654.
24. Xiao F, Ruan C, Li J, Liu L, Zhao F, Zeng B. Voltammetric Determination of Xanthine with a Single-Walled Carbon Nanotube-Ionic Liquid Paste Modified Glassy Carbon Electrode. Electroanalysis. 2008;20(4):361-366.
25. Baghizadeh A, Karimi-Maleh H, Khoshnama Z, Hassankhani A, Abbasghorbani M. A Voltammetric Sensor for Simultaneous Determination of Vitamin C and Vitamin B6 in Food Samples Using ZrO2 Nanoparticle/Ionic Liquids Carbon Paste Electrode. Food Anal. Methods. 2015, 8, 549-557.
26. Eren T, Atar N, Yola ML, Karimi-Maleh H. A sensitive molecularly imprinted polymer based quartz crystal microbalance nanosensor for selective determination of lovastatin in red yeast rice. Food chemistry. 2015, 185, 430-436.
27. Karimi-Maleh H, Tahernejad-Javazmi F, Atar N, Yola ML, Gupta VK, Ensafi AA. A Novel DNA Biosensor Based on a Pencil Graphite Electrode Modified with Polypyrrole/Functionalized Multiwalled Carbon Nanotubes for Determination of 6-Mercaptopurine Anticancer Drug. Industrial & Engineering Chemistry Research. 2015;54(14):3634-3639.
28. Karimi-Maleh H, Ganjali MR, Norouzi P, Bananezhad A. Amplified nanostructure electrochemical sensor for simultaneous determination of captopril, acetaminophen, tyrosine and hydrochlorothiazide. Materials Science and Engineering: C. 2017;73:472-477.
29. Karimi-Maleh H, Tahernejad-Javazmi F, Gupta VK, Ahmar H, Asadi MH. A novel biosensor for liquid phase determination of glutathione and amoxicillin in biological and pharmaceutical samples using a ZnO/CNTs nanocomposite/catechol derivative modified electrode. Journal of Molecular Liquids. 2014;196:258-263.
30. Atta NF, El-Kady MF, Galal A. Palladium nanoclusters-coated polyfuran as a novel sensor for catecholamine neurotransmitters and paracetamol. Sensors and Actuators B: Chemical. 2009;141(2):566-574.
31. Goyal RN, Gupta VK, Chatterjee S. Voltammetric biosensors for the determination of paracetamol at carbon nanotube modified pyrolytic graphite electrode. Sensors and Actuators B: Chemical. 2010;149(1):252-258.
32. Simultaneous determination of dopamine and uric acid using a glassy carbon paste electrode modified with copper- para red complex. Eurasian Chemical Communications. 2019;1(5):559-569.
33. Mansori G, Gholivand MB, Es’haghi, Z. In-situ preconcentration, and electrochemical sensing of zinc(II) and copper(II) based on ionic liquid mediated hollow fibermodified pencil graphite electrode using response surface methodology, Iran. Chem. Commun. 2019; 7: 556-573.
34. Karimi-Maleh H, Fakude CT, Mabuba N, Peleyeju GM, Arotiba OA. The determination of 2-phenylphenol in the presence of 4-chlorophenol using nano-Fe3O4/ionic liquid paste electrode as an electrochemical sensor. Journal of Colloid and Interface Science. 2019;554:603-610.
35. Ershad S, Mofidi Rasi R. Electrocatalytic oxidation of sulfite ion at the surface carbon ceramic modified electrode with prussian blue, Iran. Chem. Commun. 2019; 7: 43-52.
36. Babaei A, Soleimani Babadi S, Sohrabi M. Simultaneous Electrochemical Determination of Acetaminophen and Codeine Based on a MWCNT/MCM48 Nanocomposite Modified Glassy Carbon, J Nanostruct 2019; 9(2): 190-201.
37. Tahernejad-Javazmi F, Shabani-Nooshabadi M, Karimi-Maleh H. 3D reduced graphene oxide/FeNi3-ionic liquid nanocomposite modified sensor; an electrical synergic effect for development of tert-butylhydroquinone and folic acid sensor. Composites Part B: Engineering. 2019;172:666-670.
38. Sarafraz S, Rafiee-Pour HA, Khayatkashani M, Ebrahimi A. Electrochemical Determination of Gallic Acid in Camellia sinensis (L.) Kuntze, Viola odorata L., Commiphora wightii (Arn.) Bhandari, and Vitex agnus-castus L. by MWCNTs-COOH Modified CPE, J. Nanostruct 2019; 9(2): 384-395.
39. Bukkitgar SD, Shetti NP. Electrochemical behavior of an anticancer drug 5-fluorouracil at methylene blue modified carbon paste electrode. Materials Science and Engineering: C. 2016;65:262-268.
40. Fouladgar M. CuO-CNT Nanocomposite/Ionic Liquid Modified Sensor as New Breast Anticancer Approach for Determination of Doxorubicin and 5-Fluorouracil Drugs. Journal of The Electrochemical Society. 2018;165(13):B559-B564.
41. Hadi M, Mollaei T, Ehsani A. Graphene oxides/multi-walled carbon nanotubes hybrid-modified carbon electrodes for fast and sensitive voltammetric determination of the anticancer drug 5-fluorouracil in spiked human plasma samples. Chemical Papers. 2017;72(2):431-439.
42. Lima D, Calaça GN, Viana AG, Pessôa CA. Porphyran-capped gold nanoparticles modified carbon paste electrode: a simple and efficient electrochemical sensor for the sensitive determination of 5-fluorouracil. Applied Surface Science. 2018;427:742-753.
43. Pattar VP, Nandibewoor ST. Electroanalytical method for the determination of 5-fluorouracil using a reduced graphene oxide/chitosan modified sensor. RSC Advances. 2015;5(43):34292-34301.
44. Abbar JC, Nandibewoor ST. Voltammetric oxidation and determination of atorvastatin based on the enhancement effect of cetyltrimethyl ammonium bromide at a carbon paste electrode. Colloids and Surfaces B: Biointerfaces. 2013;106:158-164.
45. Bukkitgar SD, Shetti NP. Electrochemical Sensor for the Determination of Anticancer Drug 5- Fluorouracil at Glucose Modified Electrode. ChemistrySelect. 2016;1(4):771-777.
46. Jin L, Wang Y, Yan F, Zhang J, Zhong F. The Synthesis and Application of Nitrogen-Doped Graphene Quantum Dots on Brilliant Blue Detection. Journal of Nanomaterials. 2019;2019:1-9.