Disposable Nanosensor for the Electrochemical Determination of Temozolomide and İts Interaction with Double-Stranded DNA

Abstract Novel disposable screen-printed electrodes modified with silicon dioxide nanoparticles were developed for the electrochemical determination of temozolomide and its interaction with DNA. The characterization of silicon dioxide nanoparticles (SiNPs) was conducted by transmission electron microscopy and energy-dispersive x-ray spectroscopy (EDX). The EDX measurements show that the silicon nanoparticles consisted of 53.08% oxygen (O) and 46.92% silicon (Si), confirming their purity. Chemical characterization was conducted using Fourier transform infrared spectroscopy. Surface characterization of SiNPs-modified screen-printed electrodes was performed using atomic force microscopy. SiNP modification was demonstrated to increase the electrode surface area by 2.3-folds, from 0.75 × 10−3 cm2 to 1.75 × 10−3 cm2, resulting in an enhanced signal. The detection limit was 5.6 µg/mL for temozolomide and 7.2 µg/mL for DNA, respectively. The linear concentration range was from 18.6 to 200 µg/mL for temozolomide and 23.9 to 80 µg/mL for DNA.


Introduction
DNA, the carrier of genetic information, interacts with small molecules such as drugs, proteins, and toxins.Investigating the interaction between anticancer drugs and DNA is important not only for understanding the mechanism of interaction but also for the design and development of new DNA-targeted anticancer drugs (Chaires 1998;Hajian et al. 2009;Temerk et al. 2018;Muti and Muti 2018).2,3,5]tetrazine-8-carboxamide) is a compound belonging to the imidazotetrazine group, which is an orally administered DNA alkylating chemotherapeutic agent (Newlands et al. 1997;Liu et al. 2010;Castro et al. 2015;Kaina and Christmann 2019;Hwang et al. 2019).It is used in the treatment of various cancer types, particularly glioblastoma.Temozolomide (TMZ) slows the growth of tumor tissue by suppressing DNA synthesis.Due to its lipophilic structure, TMZ is rapidly absorbed and easily crosses the blood-brain barrier, resulting in high bioavailability (Lopes et al. 2013;Chen et al. 2017;Thomas et al. 2017;Almalki et al. 2017;Woo et al. 2019).
In cancer patients, monitoring the administered drug in biological samples using various methods is essential for selecting the dosage of drugs, determining dose intervals, developing new products, and ensuring quality control (Nussbaumer et al. 2011;Bi et al. 2014).Analytical determination of TMZ from biological samples such as blood, urine, tissue, saliva, and cerebrospinal fluid is conducted by high-performance liquid chromatography (HPLC), gas chromatography (GC), liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS), ultra-performance liquid chromatography (UPLC), spectrophotometry, and capillary electrophoresis (CE) (Wolfender 2009;Maduraiveeran et al. 2018;Qian et al. 2021).However, these traditional methods require costly equipment, have long analysis times, and necessitate experienced technical support.This situation has heightened the demand for faster, more cost-effective, easily produced, highly selective, and sensitive methods (Nussbaumer et al. 2011;Ali et al. 2013;Brahman et al. 2017;Sabourian et al. 2020).Therefore, there is a significant interest in electrochemical analysis.Ghalkhani et al. (2010) conducted a study to investigate the electrochemical properties of TMZ using a glassy carbon electrode.They determined that the reduction was irreversible, pH-dependent, and controlled by adsorption.The linear dynamic range, limit of detection (LOD), and limit of quantification (LOQ) were reported to be 2.0 � 10 −6 to 1.3 � 10 −5 M, 1.1 � 10 −6 M, and 3.7 � 10 −6 M, respectively.Altay et al. (2015) utilized pencil graphite electrodes to characterize the interaction between TMZ and DNA.They employed differential pulse voltammetry (DPV), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS) to determine the interaction of TMZ with immobilized double-stranded DNA (dsDNA) and singlestranded DNA (ssDNA) on the electrode surface.The limit of detection was 6.1 mg/mL, with a linear concentration range of 40-100 mg/mL.
Nanomaterials are employed to enhance biosensors, enabling them to conduct more selective, sensitive, rapid, real-time measurements and to produce cost-effective, userfriendly electrodes (Lu et al. 2020;Khanmohammadi et al. 2020;Eskandarinezhad et al. 2022).Nanomaterials positively contribute to biosensor performance by augmenting electrode surface area, surface conductivity, catalytic activity, and lowering the detection limit (Zhu et al. 2015;Lee et al. 2019;Kumar et al. 2019).Nanomaterials facilitate the attachment of biomolecules to the sensor surface as they increase the electrode surface area, enabling precise measurements even with low-concentration samples.Additionally, nanomaterials offer specific binding with biomolecules, allowing the design of more stable and miniature nano-biosensors (Jianrong et al. 2004;Vaseashta and Dimova-Malinovska 2005;Luo et al. 2006;Muti and Muti 2018).Pathak et al. (2018) modified the surface of a screen-printed carbon electrode with a reduced graphene oxide/silver nanocube nanocomposite using the molecular imprinting method for the electrochemical determination of TMZ at trace levels.The linear working range of the prepared nanosensor was from 1.09 to 144.21 ng/mL, and the detection limits in standard solutions, blood plasma, and urine were 0.16, 0.24, and 0.42 ng/mL, respectively.
The electrochemical properties of TMZ in acidic, basic, and neutral solutions were investigated by Reddy et al. (2020) using a carboxylic acid-functionalized multiwalled carbon nanotube (MWCNT-COOH) paste electrode.The detection limits were 0.056, 0.069, and 0.065 mM in acidic, basic, and neutral solutions respectively.Eksin et al. (2022) examined the dsDNA-TMZ interaction on the surface of a pencil graphite electrode modified with graphene oxide (GO).
In this study, the interaction between temozolomide (TMZ) and double-stranded fish sperm DNA was investigated electrochemically on the surface of a screen-printed electrode modified with silicon dioxide nanoparticles (SiNP).To the best of our knowledge, this study is the first in which a silicon dioxide nanoparticle-based disposable electrode was developed for the determination of temozolomide and its interaction with DNA.A lower detection limit and an improved linear working concentration range were achieved compared to previous studies based on nanomaterial-modified sensors.Additionally, these nanosensors, developed using easy, inexpensive, and rapid electrode preparation techniques, remained stable for 1 month, retaining 70% of their performance by the end of the second month.We anticipate that this work, with its advantages of a low detection limit, easy modification, and time-efficient nanosensor preparation, will contribute to the field of drug-DNA interactions.

Equipment
Surface characterization of Si nanoparticles (SiNPs) was conducted using transmission electron microscopy (TEM, acceleration voltage: 200 kV, resolution: 0.19 nm) with a JEOL-Jem 2100 EDX. 10 mg of silicon dioxide nanoparticles in powder form was dispersed in ethyl alcohol within an Eppendorf tube.The mixture underwent ultrasonic treatment until it became homogeneous.Subsequently, 5-10 mL of the mixture were deposited onto the carbon-coated surface of the electrode and allowed to dry.
Electrochemical measurements were carried out using the Ivium Compact Stat Plus module.Screen-printed carbon electrodes (SPCEs) (Life Sens-LS-103, 3 mm diameter) were obtained from Life Sense Technology (Turkey).EIS measurements were performed in a Faraday cage (BASi C3 cell stand).

Chemicals
Tetraethyl orthosilicate, chitosan, and double-stranded fish sperm DNA (dsDNA) were obtained from Sigma.The stock solution of dsDNA (1000 mg/mL) was prepared in pH 8.00 Tris-EDTA buffer (Tris-HCl/EDTA 10:1 mM) and stored frozen.The required dilutions of dsDNA were prepared in 0.50 M acetate buffer (ABS) with 20 mM NaCl at pH 4.8.All chemicals used in this study were of analytical reagent grade.
The TMZ was obtained from Sigma-Aldrich.The stock solution of TMZ was prepared at 1000 mg/mL in 0.05 M Phosfate buffer solution with 20 mM NaCl, (PBS, pH¼7.4).Further dilutions of TMZ were prepared in PBS at pH 7.4.Voltammetric measurements were conducted in PBS at pH 7.4.Ultrapure water was obtained from a Millipore system.

Synthesize of silicon dioxide nanoparticles
The synthesis of nanoparticles was carried out as described by Guo et al. (2017).1.3 mL of distilled water, 0.025 g of polyethylene glycol (PEG 1000), 75 mL of ethyl alcohol, and 6.3 mL of tetraethyl orthosilicate (TEOS) were sonicated at 45 � C for 2 h.Next, 1 mL of ammonium hydroxide and 1 mL of ethyl alcohol were added, and sonication continued for three additional hours.The solution was transferred to a vacuum oven and maintained at 60 � C for 3 days to form a gel.The vacuum oven temperature was increased to 80 � C, and the resulting gel was dried, pulverized, transferred to a crucible, and heated in a muffle furnace to 800 � C for 8 h.
The SiNPs prepared using PEG were acidified according to the literature (Chen et al. 2012) to enhance the effectiveness of SiNPs in DNA immobilization by making them positively charged.The PEG-SiNPs were activated by the addition to 6 M of HCl stirred for 8 h at room temperature, washed with distilled water until the pH became neutral, and dried at 70 � C for 8 h.The electrodes were modified with a mixture of SiNP and chitosan at concentrations of 4000 and 5000 lg/mL respectively.

Procedure Modification of the SPE with SiNP
Before modification, the SiNPs were dispersed in a chitosan solution at a concentration of 5000 mg/mL for 1 h.This mixture was applied to the surface of the SPE (SiNP-SPE) and maintained under an infrared lamp for 15 min.Due to its gel-like structure, chitosan enables nanoparticles to easily and stably adhere to the surface.

Immobilization of DNA on the SiNP-modified SPE surface
A solution of dsDNA was prepared and deposited onto the SiNP-modified electrode surface, allowed to stand at room temperature in 60 min for immobilization, and washed with PBS to remove unbound or weakly bound DNA from the surface.

Interaction studies
Interaction studies were conducted by depositing TMZ onto the DNA-immobilized SiNP-SPE surface and allowing interaction with DNA.After a specified period, the surface was washed with PBS to remove unbound TMZ.These steps are shown in Scheme 1.

Voltammetric transduction
Differential pulse voltammetry (DPV) was conducted in PBS (0.05 M, pH:7.4) by scanning from 0 V to þ1.5 V vs. a Ag/AgCl reference electrode.The pulse amplitude was

Surface morphology
The surface morphology of SiNPs was characterized by TEM as shown in Figure 1A and B. The nanoparticles show weak aggregation.The elemental composition of the synthesized SiNPs was investigated by energy-dispersive X-ray spectroscopy (EDX) as shown in Figure 1C.Silicon and oxygen make up 46.92% and 53.08%, respectively.This composition confirms that the nanoparticles consist of pure SiO 2 .These findings are consistent with the literature of silica nanoparticles (El Messaoudi et al. 2022;Periakaruppan et al. 2023).
The infrared spectrum of SiNPs was recorded from 4000 to 450 cm −1 (Figure 1D).The band at 803 cm −1 is due to the Si-O bending vibration.The sharp peak at 1075 cm −1 demonstrates the asymmetric stretching of the Si-O-Si bond.Due to the calcination during the SiNP synthesis, the FTIR spectrum shows there is no water and the nanoparticles are composed of pure SiO 2 .

Scheme 1. Electrode preparation for the determination of temozolomide (TMZ).
The surface morphology of bare, CHIT-modified, and SiNP-CHIT-modified SPCEs was examined by atomic force microscopy (AFM), as shown in Figure 2. The recessed state of the bare electrode surface is observed in Figure 2A.After modification with chitosan, the surface roughness is significantly reduced due to the gel-like structure of chitosan filling the pores (Figure 2B).Following the modification of the surface with SiNPs dispersed in chitosan, the surface becomes rough again due to the nanoparticles (Figure 2C), but without the pronounced roughness of the bare electrode because of the presence of chitosan.

Surface area after modification
The electrode surface area after modification was calculated by cyclic voltammetry (CV) of K 3 [Fe(CN) 6 ].The electrode surface area was determined using the Randles-Sevcik   equation for reversible processes: I p ¼ 2.69 � 10 5 n 3/2 AD 1/2 CV 1/2 where A is the electrode surface area (cm 2 ), D is the diffusion coefficient of K 3 [Fe(CN) 6 ] (cm 2 /s), I p is the peak current (A), n is the number of electrons transferred (n ¼ 1), C is the concentration of the electroactive species (mol/cm 3 ), and V is the scan rate (V/s).
The cyclic voltammograms of the [Fe(CN) 6 ] 3-/4-system were measured by applying different scan rates.The slope obtained by plotting I p vs. V 1/2 (Figure 3A and B) was used to calculate the surface area of the bare and SiNP-modified electrodes.The substitution of these values into the Randles-Sevcik equation indicates the electroactive surface areas of the bare and SiNP-modified SPE were 7.5 � 10 −4 cm 2 and 1.75 � 10 −3 cm 2 , respectively.There is an approximately 2.3-fold increase in the active surface area following SiNP modification.

Signal enhancement after SiNP modification
The impact of the SiNP modification on biosensing was characterized by cyclic voltammetry and electrochemical impedance spectroscopy.The cyclic voltammograms were obtained using bare, CHIT-modified, and CHIT-SiNP-modified SPEs of 0.1 M KCl and 5 mM K 4 [Fe(CN) 6 ] as shown in Figure 3C.The peak currents due to the oxidation and reduction of the redox probe were significantly higher using chitosan-coated electrodes compared to the bare SPE.Remarkably, the highest oxidation and reduction peak currents, accompanied by well-defined reversible peaks, were achieved following the modification with SiNPs dispersed in chitosan.
Additionally, the influence of SiNP modification upon the electron transfer resistance was characterized by measuring the charge transfer resistance of the [Fe(CN) 6 ] 3-/4-system for the bare and CHIT-SiNP modified electrodes (Figure 3D).The electron transfer resistance decreased for the electrodes modified with SiNPs dispersed in chitosan.

Detection limit of SiNP-SPE
The detection limits of SiNP-modified SPEs were evaluated by conducting TMZ and DNA concentration measurements.The oxidation signals at various concentrations of DNA immobilized onto the surface of SiNP-SPE and the oxidation signal from 20 to 200 mg/mL were used to determine the detection limits (Figure 4).The limits of detection were evaluated by y ¼ 0.0216x þ 0.3919 (R 2 , 0.9995) and y ¼ 0.0402x − 0.0902 (R 2 , 0.9948) for TMZ and DNA, respectively.Using the procedure of Miller and Miller (2000), the limits of detection for TMZ and DNA were determined to be 5.6 and 7.2 mg/mL.These values are lower than the literature values for TMZ and DNA (Altay et al. 2015;Reddy et al. 2020;Eksin et al. 2022).The linear concentration ranges were from 18.6 to 200 mg/mL for TMZ and 23.9 to 80 mg/mL for DNA, longer than the previously reported values (Altay et al. 2015;Eksin et al. 2022).
A comparison of the literature for the determination of TMZ is presented in Table 1.

Stability
The sensor stability was tested by intra-day and inter-day measurements.The sensor performance was stable for 1 month.After two months, the signal only decreased by 30% (Supplementary Figure S1).

Interaction with DNA
The guanine oxidation signal after interaction with different concentrations of TMZ was measured using differential pulse voltammetry as shown in Figure 5A and B. The most significant decrease in the guanine oxidation signal occurred after interaction with 60 mg/mL TMZ.This concentration was determined to be the highest concentration of TMZ interacting with 80 mg/mL DNA.The interaction time is also an important parameter.To determine this parameter, the oxidation signal of 60 mg/mL TMZ with 80 mg/mL DNA was recorded for various time intervals.Figure 5C shows the greatest decrease in the guanine oxidation signal occurred after 15 min of interaction corresponding to a 40% decrease.The change in the guanine peak indicates DNA modification and is expressed by S% which provides information about toxicity (Bagni et al. 2006): S % ¼ S s S b � 100 where S s and S b represent the guanine signal after and before interaction with TMZ, respectively.The S% value was determined to be 60.3%, indicating moderate toxicity.
The decrease in electron transfer resistance after 15 min of interaction was measured by EIS as shown in Figure 5D.Following DNA immobilization, the charge transfer resistance (R ct ) increased due to the repulsion of the negatively charged phosphate groups of DNA by the negatively charged [Fe(CN) 6 ] 3-/4-couple on the surface.However, the R ct decreased again after 15 min of interaction with TMZ.

Conclusion
Anticancer drugs are commonly used in treatment, making it highly important to develop methods that are inexpensive and fast, with good accuracy and reproducibility, and offer low detection limits.Temozolomide, an alkylating DNA chemotherapeutic agent, is used in the treatment of various cancers.However, the electrochemical determination of temozolomide is limited in the literature.
This study electrochemically determined temozolomide using a nanosensor and investigated its interaction with DNA.By modifying a screen-printed electrode surface with silicon dioxide, the surface area increased by 2.3-folds, resulting in a lower detection limit compared to the literature (Altay et al. 2015;Reddy et al. 2020).These electrodes involve easier and less time-consuming preparation compared to other nanomaterialbased studies (Pathak et al. 2018;Eksin et al. 2022).These disposable nanoelectrodes are anticipated to allow the determination of various biomolecules with low detection limits, good reproducibility, and easy preparation.Although a lower limit of quantification was obtained compared to some literature studies of the TMZ-DNA interaction, the inability to offer a limit of quantification at the ng/mL level is a disadvantage.
50 mV, and the scan rate was 50 mV/s.Cyclic voltammetry was carried out in 0.1 M KCl and 5 mM K 4 [Fe(CN) 6 ] from −0.5 to þ1.0 V vs. the Ag/AgCl reference electrode.Electrochemical impedance spectroscopy (EIS) measurements were performed in 0.1 M KCl and 2.5 mM K 3 [Fe(CN) 6 ]/K 4 [Fe(CN) 6 ] from 10 5 to 10 −1 Hz at an open circuit potential of þ0.23 V vs. Ag/AgCl, with a voltage amplitude of 10 mV.The charge-transfer resistance (R ct ) was calculated using the Ivium Compact Stat Plus software.

Figure 3 .
Figure 3. Cyclic voltammograms of 5 mM K 4 [Fe(CN) 6 ] and 0.1 M KCl at various scan rates on the (A) bare SPE and (B) SiNP-modified SPE.(C) Cyclic voltammograms of 5 mM K 4 [Fe(CN) 6 ] and 0.1 M KCl on the bare, CHIT-modified and CHIT-SiNP-modified SPE.(D) Charge transfer resistance (Rct) values before and after modification of SiNP.The inset shows equivalent circuit elements used to fit the impedance results.

Figure 4 .
Figure 4. (A) Voltammograms and (B) calibration relationship for the oxidation of temozolamide as a function of concentration.(C) Voltammograms and (D) calibration relationship for the oxidation of guanine as a function of concentration.The measurements were performed in pH 7.4 PBS (n ¼ 3).

Figure 5 .
Figure 5. (A) Differential pulse voltammograms and (B) histograms of the guanine oxidation signal before and after interaction with various concentrations of temozolamide.(C) Histograms showing the guanine oxidation signal before and after interaction with temozolamide as a function of interaction time.(D) Charge transfer resistance (R ct ) values obtained before interaction (bare and DNA immobilized) and following 15 min of interaction with temozolamide.The inset shows the equivalent circuit elements used to fit the impedance results.

Table 1 .
Analytical methods for temozolomide determination.