Electrochemical activity of glassy carbon electrode modified with ZnO nanoparticles prepared Via Senna Alata L. leaf extract towards antiretroviral drug
Micro and Nano Systems Letters volume 10, Article number: 5 (2022)
The phytosynthesis method was used to prepare ZnO nanoparticles (ZnO NPs) via Senna alata L. leaf extract (SALE) by involving alkaloids, which play an essential role as a source of weak bases during the formation reaction of NPs. ZnO NPs on glassy carbon electrodes (GCE/ZnO NP) have been introduced to investigate its electrochemical activity towards the antiretroviral drug, lamivudine (3TC). Several characterization techniques, such as Fourier Transform Infra-Red (FTIR), X-Ray Diffraction (XRD), Scanning Electron Microscopy (SEM), Energy Dispersive X-Ray Spectroscopy (EDS), and Dynamic Light Scattering (DLS) techniques were employed to analyze the properties of GCE/ZnO NPs. As a result, ZnO NPs in spherical shape showed a high purity crystalline hexagonal wurtzite structure with a particle diameter of 40–60 nm. A Cyclic Voltammetry (CV) measurement confirmed that the electrochemical reduction of 3TC on GCE/ZnO NPs exhibited an excellent linear range of 10–300 µM with a detection limit of 1.902 µM, quantitation limit of 6.330 µM, and sensitivity of 0.0278 µA/µM. Thus, this research suggests a facile method for the preparation of material-based ZnO NPs as a promising antiretroviral drug sensors due to their excellent electrochemical properties.
Lamivudine (2′-deoxy-3′-thiacytidine, 3TC) is a nucleoside analog reverse transcriptase inhibitor (NARTI) drug that is actively against retroviruses, including human immunodeficiency virus type 1 (HIV-1), human immunodeficiency virus type 2 (HIV-2), and hepatitis B virus (HBV) . 3TC inhibits the virus growth through the DNA chain-breaking by forming intracellular triphosphate to prevent the multiplication of viral DNA in the host [2,3,4]. However, the use of 3TC in long-term use leads to the emergence of mutant HBV resistance. Drug-resistant genotypes with mutations at codons rt80, rt173, rt180, and rt204 are rapidly selected by 3TC monotherapy of HBV, occurring in 15% of treated people after one year and up to 80% after 3 years. In addition, selection of mutants not identified by hepatitis B surface antigen (HBsAg) testing may obscure the detection of HBV infection after 3TC treatment. Individuals with these mutations may spread HBV that is resistant to the immunological responses generated by the HBV vaccine .
Based on their medicinal and pharmacological significance, evaluating appropriate levels of 3TC in biological fluids and pharmaceutical preparations is tremendously essential. The analytical methods for assessing 3TC levels have been repeatedly reported, such as spectrophotometry [6, 7], chromatography , electrochemistry [9,10,11,12], etc. The chromatography and spectrophotometry methods have presented good selectivity and detection limits. However, several flaws, such as time-consuming, relatively expensive, requiring a complicated analysis step have been investigated. Therefore, well-qualified personnel are required to develop other analytical methods such as electrochemistry due to its low cost, easy to handle, and better reproducibility [13, 14]. Additionally, in the electrochemical method, modification of the electrode surface using electroconductive materials should be taken into account to optimize electron transfer between the analyte and the sensing surface. Also, electroconductive materials have enhanced the surface area of the electrode to produce superior sensitivity [15, 16].
ZnO NPs are materials with good electrical conductivity, negligible toxicity, high electron transfer kinetics, and excellent electrocatalytic activity against biomolecules . Currently, phytosynthesis has been considered as one of the prospective methods to prepare ZnO NPs compared to the conventional one due to its harmless route  since it uses no toxic chemicals  such as sodium dodecyl sulfate (SDS), tetramethylammonium bromide (CTAB) [20, 21], and polyvinylpyrrolidone (PVP) . Additionally, this method utilizes the secondary metabolite content of plant extracts as a source of weak bases, chelating agents, stabilizing agents, and reducing agents [23, 24].
In this work, ZnO NPs were synthesized using leaf extract of Senna Alata L. (SALE), an angiosperm belonging to the family of Fabaceae , which contains secondary metabolites in the form of alkaloids, flavonoids, and saponins that act as sources of weak bases, chelating agents, and stabilizing agents during the reaction. Furthermore, ZnO NPs were used to modify the surface of the glassy carbon electrode (GCE). The electrochemical activity of GCE/ZnO NPs was analyzed against 3TC. The sensitivity and detection limit were also investigated by electrochemical measurements.
Senna Alata L. leaves were obtained from Cirebon district, West Java, Indonesia, and further determined at the Research Center for Biology, LIPI, Indonesia. Zinc nitrate hexahydrate (Zn(NO3)2.6H2O), potassium hydrogen phosphate (K2HPO4), potassium dihydrogen phosphate (KH2PO4), methanol, and n-hexane were purchased from Merck. Lamivudine was obtained from the Indonesian Pharmacopoeia Comparative Standard (BPFI). The double distilled water used in this research was purified with Millipore Direct-Q® 5 UV. All chemicals were used as received without purification.
Preparation of SALE
Senna Alata L. leaf was washed, dried, and mashed to obtain powder. 50 g of powders were macerated in 250 mL of methanol for 7 days by continuously stirring. Afterward, the filtrate was filtered using Whatman no. 40 filters and fractionated in n-hexane with a volume ratio of 1:1. The result was evaporated using a vacuum rotary evaporator and dissolved into 100 mL of distilled water. Finally, the obtained SALE was stored at 4 ℃ for the preparation of ZnO NPs by the phytosynthesis method.
Phytosynthesis of ZnO NPs
ZnO NPs were fabricated via phytosynthesis method through a sol–gel process. SALE was added to Zn(NO3)2 0.015 M to obtain 100 mL of the mixture with a volume ratio of 1:9 (v/v). The SALE sol was formed by heating at 60 ℃ for 2 h. The sol was further heated at 120 ℃ for 6 h and centrifuged to form a gel. Finally, the gel was calcined at 500 ℃ for 4 h to obtain white powders of ZnO NPs.
Fabrication of the GCE/ZnO NPs
The GCE surface was manually polished with alumina slurry for a minute and washed with ultrapure water. Then, the GCE was sonicated in methanol for 10 min, then rinsed with ultrapure water. ZnO NPs were dispersed in ethanol (4 mg/ml). Afterward, it was deposited on the surface of GCE (2 mm diameter) by the drop-casting technique. Finally, GCE/ZnO NPs were dried at 60 ℃.
The three-electrode system was used for the electrochemical study of prepared electrodes towards 3TC. The GCE/ZnO NPs, platinum wire, and Ag/AgCl served as the working, counter, and reference electrodes, respectively. The electrolyte was a 0.1 M phosphate buffer solution (PBS) at pH ranging from 5 to 9 prepared by mixing quantitative amounts of KH2PO4 and K2HPO4 in ultrapure water. The electrochemical measurements were performed using CV techniques at a range potential of − 1.2 to 0.5 V.
An Autolab PGSTAT204 was used to perform a three-electrode system for voltammetric measurements. The morphologies of the sample were characterized by Scanning Electron Microscopy-Energy Dispersive X-ray Spectrometer (SEM–EDS, JEOL JSM 6510 LA). In contrast, the sample structure was analyzed using Powder X-Ray Diffraction (XRD, Miniflex 600-Rigaku X-Ray Analytical Instrument) and Fourier Transform Infra-Red spectroscopy (FTIR, IR Prestige-21 Shimadzu). Malvern Zetasizer Nano ZSP, a particle size analyzer, determined the particle size distribution.
Results and discussion
Characterization of SALE and ZnO NPs
Figure 1a shows that the FTIR spectrum of ZnO NPs exhibit absorption peaks at wavenumbers of 3442, 2911, 1625, 1054, and 489 cm−1, which correspond to the vibration of O–H stretching, C–H (sp3) stretching, N–H bending, C–N stretching, and Zn–O stretching, respectively [26, 27]. The O–H stretching vibration is assigned to the residual secondary metabolites of SALE in the form of flavonoids, saponins, and polyphenols and the adsorption of H2O from the atmosphere [1, 23]. The N–H bending and C–N stretching vibrations indicate the presence of alkaloids that play an essential role as a source of weak bases in the phytosynthesis of ZnO NPs [23, 28]. Also, Zn–O stretching vibrations indicate that ZnO NPs have been successfully formed [29,30,31,32] through the phytosynthesis method using SALE.
Moreover, the XRD diffraction pattern in Fig. 1b clearly shows that all the diffraction peaks at (100), (002), (101), (102), (110), (103), (200), (112), and (201) planes were indexed to the crystalline ZnO hexagonal wurtzite structure (JCPDS Card No. 36–1451). The average crystallite size of ZnO NPs was calculated from the following Scherrer’s formula :
where, D is the crystallite size average, λ is the wavelength of the X-rays (0.15406 nm), ß is the FWHM and θ is the diffraction angle. The average value of the crystallite size of ZnO NPs was found to be 18.87 nm.
The SEM images in Fig. 2a depict the ZnO NPs prepared from SALE in a spherical shape with a particle size range of 40–60 nm. The presence of zinc and oxygen by elemental mapping was studied using EDS with an atomic composition ratio of 53.6: 46.4. No other elements from the impurities were observed, which indicates the formation of ZnO NPs with high purity in a homogeneous spherical shape (Fig. 2a,b). In addition, the particle distribution curve obtained by the dynamic light scattering technique shows that the hydrodynamic size of the ZnO NPs was determined to be 68.061 nm (Fig. 2c).
The electrochemical activity of GCE/ZnO NPs towards 3TC
The electrochemical activity of GCE/ZnO NPs against 3TC in 0.1 M PBS (pH 7) was determined through cyclic voltammetry (CV). Figure 3 demonstrates that GCE and GCE/ZnO NPs both had a reduction peak current of 3TC at a reduction peak potential of − 0.906 and − 1.095 V, respectively, under the same experimental conditions. The improved conductivity and electrochemical characteristics of ZnO NPs resulted in greater electron transport, leading to a higher current response of GCE/ZnO NPs when compared to GCE.
The CV response of 3TC in 0.1 M PBS at the surface of the prepared electrode with different pH from 5 to 9 was performed in Fig. 4a. The highest current response was obtained at an optimal pH level of 7 through a reduction peak of − 1.095 V (Fig. 4b). In addition, Fig. 4c shows that the more alkaline in the pH system, the peak of the cathodic current shifts to a more negative potential, indicating the involvement of protons in the redox reaction . According to the reaction mechanism (Scheme 1), the reduction peak results from the reduction of 3TC in GCE/ZnO NPs, involving two electron transfers. The C = N bond from 3TC is reduced, followed by the deamination process. .
where Ip is the peak current (A), n is the number of electrons transferred peroxidation or reduction of one analyte molecule; A is the electrode surface area (cm3), D is the analyte diffusion coefficient (cm2/s), v is the scan rate (V/ s), c is the concentration of analyte in bulk solution (mol/cm3), R is the ideal gas constant, and T is the temperature (K).
The reduction peak current of 3TC was increased linearly with different scan rates (Fig. 4d). The plot between anodic and cathodic peak currents versus the root of the scan rate in the Randles–Sevcik equation shows a linear relationship, indicating the kinetics of electron transfer controlled by the diffusion process . Figure 4e presents a linear plot between the reduction peak current of 3TC and the root of the scan rate with a correlation coefficient of 0.9903. This result indicates that the electron transfer process at the electrode is diffusion controlled. The plot between the logarithm of the scan rate versus the logarithm of the peak current in the Randles–Sevcik equation was also investigated to understand the possibility of an adsorption process on the electrode surface. If the gradient value in the resulting linear equation is close to 0.5, then the redox reaction is completely diffusion controlled. However, if the gradient value is more than 0.5, it indicates an adsorption process . Figure 4f shows the linear equation between log v and log I at a gradient value of 0.2162. This result suggests that the 3TC reduction process on the surface of GCE/ZnO NPs is not controlled by adsorption.
For the calibration curve, CV measurement was carried out in the 3TC concentration range of 10–300 µM, as seen in Fig. 5a. The calibration curve equation was obtained to be y = − 0.0278×–81 (R2 = 0.9987), indicating excellent linearity of GCE/ZnO NPs towards 3TC in the low concentration range (10–75 µM) to high (300 µM) with a remarkable detection limit of 1.902 µM, a quantitation limit of 6.330 µM, and a sensitivity of 0.0278 µA/µM. The detection limit was obtained from three times the standard deviation devided by the slope of the calibration curve, whereas the limit quantitation was obtained using the ten times. Also, the sensitivity is determined from the slope on the calibration curve .
The repeatability of GCE/ZnO NPs was investigated at 40 µM 3TC using the same fabricated electrode (Fig. 5b). The calculated relative standard deviation (RSD) was found to be 1.26% (n = 6). This result shows the good repeatability with GCE/ZnO NPs for 3TC in 0.1 M PBS (pH 7).
In this work, ZnO NPs have been successfully synthesized by phytosyntesis method using SALE by involving alkaloids which play an important role as a source of weak bases. GCE modified with ZnO NPs showed excellent electrochemical activity toward 3TC at a good linear range of 10–300 µM with a detection limit of 1.902 µM, quantitation limit of 6.330 µM, and sensitivity of 0.0278 µA/µM. The excellent electrochemical activity of the prepared electrode might be attributed to the good electrical conductivity and high electron transfer kinetics of ZnO NPs. Thus, this research suggests a facile method for the preparation of material-based ZnO NPs as promising antiretroviral drug sensors due to their excellent electrochemical properties.
Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
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This work was funded by Rektor Universitas Gunadarma No. 22/SK/REK/UG/2021.
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Ariyanta, H.A., Roji, F. & Apriandanu, D.O.B. Electrochemical activity of glassy carbon electrode modified with ZnO nanoparticles prepared Via Senna Alata L. leaf extract towards antiretroviral drug. Micro and Nano Syst Lett 10, 5 (2022). https://doi.org/10.1186/s40486-022-00147-6
- ZnO nanoparticles
- Electrochemical sensor
- Green synthesis