Doxorubicin Anticancer Drug Monitoring by ds-DNA-Based Electrochemical Biosensor in Clinical Samples

In this research, glassy carbon electrode (GCE) amplified with single-wall carbon nanotubes (SWCNTs) and ds-DNA was fabricated and utilized for voltammetric sensing of doxorubicin with a low detection limit. In this technique, the reduction in guanine signal of ds-DNA in the presence of doxorubicin (DOX) was chosen as an analytical factor. The molecular docking study revealed that the doxorubicin drug interacted with DNA through intercalation mode, which was in agreement with obtained experimental results. The DOX detection performance of ds-DNA/SWCNTs/GCE was assessed at a concentration range of 1.0 nM–20.0 µM. The detection limit was found to be 0.6 nM that was comparable and even better (in many cases) than that of previous electrochemical reported sensors. In the final step, the ds-DNA/SWCNTs/GCE showed powerful ability for determination of the DOX in injection samples with acceptable recovery data.


Introduction
Cancer is regarded as one of the most important diseases in the world as well as the primary cause of mortality for many individuals [1,2]. The use of chemotherapy and anticancer drugs is the best and most important way to deal with this deadly disease [3,4]. Breast cancer is one of the most important and prevalent kinds of cancer that has been diagnosed [5]. Doxorubicin is one of the most important and extensively used drugs with an appropriate response for the treatment of breast cancer, which has been used for this purpose for many years [6,7]. This drug is one of the initial options of doctors in the treatment of breast cancer [8,9]. However, due to the drug's adverse effects, its regulated dosage is essential in chemotherapy [10]. Therefore, its continuous measurement in biological environments following treatment is crucial and required [11][12][13][14][15]. Numerous methods have been used for this purpose, such as high-performance liquid chromatography (HPLC), spectroscopic methods, and electrochemical sensors [11,[16][17][18][19].
Electrochemical strategies showed many advantages for sensing of biological, anticancer drugs and a wide range of electroactive materials due to easy operation, fast response, high sensitivity, and portable ability [20][21][22][23][24][25]. Furthermore, the electrochemical sensors are considered to be more efficient and sensitive than other methods due to the opportunity to be boosted using various modifiers [26][27][28][29]. As a result of these considerations, numerous teams have reported electrochemically modified sensors for DOX monitoring [15,[30][31][32]. However, the low selectivity issue is still one of the most important obstacles of electrochemical sensors for the detection of DOX. Due to the specific reaction, biological recognition elements, such as DNA, can be used to overcome this issue [33,34].
The intercalation of DOX with ds-DNA inhibits macromolecular biosynthesis, thereby it helps in the progression of topoisomerase II, an enzyme that relaxes supercoils in DNA The intercalation of DOX with ds-DNA inhibits macromolecular biosynthesis, thereby it helps in the progression of topoisomerase II, an enzyme that relaxes supercoils in DNA for transcription [35]. On the other hand, intercalation between ds-DNA bases and doxorubicin allows the formation of a highly sensitive and selective strategy for electrochemical monitoring of DOX, which has also been confirmed by different groups [36].
Nanotechnology is now considered to be an important part of engineering processes in the world [37][38][39][40][41][42]. The unique properties of nanomaterials have led to their widespread application in industry and the design of engineering systems and have led to significant changes in various scientific fields [43][44][45][46][47][48][49]. SWCNTs, meanwhile, have been surprisingly improved as a one-dimensional nanomaterial with high electrical conductivity in improving the properties of electrochemical sensors [50]. Bearing all points in mind, herein, it was aimed to design an SWCNTs-modified sensor to be utilized in DOX analysis. The schematic illustration of the proposed work was given in Scheme 1. The findings revealed that the ds-DNA/SWCNTs/GCE showed good selectivity and high sensitivity for sensing of doxorubicin in pharmaceutical samples. Scheme 1. The schematic illustration of the present work.

Preparation of ds-DNA/SWCNTs/GCE
GCE (A = 0.314 cm 2 , Azar Electrode, Iran) was polished via alumina powder and washed by distilled water solution. Subsequently, 10 mL of SWCNTs-COOH suspension Scheme 1. The schematic illustration of the present work.

Materials and Instrument
Calf Thymus DNA (double-stranded), boric acid, phosphoric acid, Tris hydrochloride, acetic acid, doxorubicin hydrochloride, and SWCNTs-COOH (>90% carbon basis, D × L 4-5 nm × 0.5-1.5 µm) were purchased from Across and Sigma-Aldrich Companies. Stock solution of doxorubicin hydrochloride (0.001 M) was prepared by dissolving 0.0058 g doxorubicin hydrochloride in 10 mL buffer solution. An Ivium-Vertex machine connected to the three-electrode experimental setup consisting of Ag/AgCl/KCl sat reference, Pt wire (Azar Electrode, Tehran, Iran) and ds-DNA/SWCNTs/GCE were employed for differential pulse voltammetric (DPV) (pulse height of 50 mV and a pulse width of 5 mV) analysis.

Preparation of ds-DNA/SWCNTs/GCE
GCE (A = 0.314 cm 2 , Azar Electrode, Iran) was polished via alumina powder and washed by distilled water solution. Subsequently, 10 mL of SWCNTs-COOH suspension was dropped at the surface of clean GCE and dried for 10 min. The ds-DNA was prepared into acetate buffer (0.5 M, pH 4.8). Afterwards, 10 µL of ds-DNA solution (50 mg/L) was dropped at the surface of SWCNTs/GCE.

Preparation of Clinical Samples
Injection samples of doxorubicin (2 mg/mL) and dextrose saline were purchased from a local pharmacy and used for clinical samples analysis without any pretreatment. Samples were diluted in tris-HCl buffer solution pH 7.4 and transferred to electrochemical cell for clinical sample analysis by standard addition strategy. Different concentrations of doxorubicin were spiked in dextrose saline and used for real sample analysis.

Experimental Procedure
Doxorubicin solution was prepared in tris-HCl buffer solution pH 7.4. Intercalation of doxorubicin and ds-DNA was checked in tris-HCl buffer solution at pH 7.4 for 20 min. Finally, the electrode was transferred into another electrochemical cell and relative voltammograms were recorded in an acetate solution (0.5 M, pH 4.8). All of the experiments were repeated four times. Electrochemical impedance spectroscopy (EIS) was employed in the frequency range of 100 kHz to 1.0 Hz.

Molecular Docking
Molecular docking was conducted to predict the favorable DOX-DNA complexes using AutoDock Vina [51] and AutoDockTools (ADT) V.1.5.6 [52]. 3D structures of DNA dodecamer (PDB ID: 1BNA) and DNA hexamer containing an intercalation gap (PDB ID:1Z3F) were extracted from RCSB protein data bank [53]. Moreover, the 3D structure of DOX drug was downloaded from PubChem database [4] (CID 31703). The partial atomic charges for both DNA receptor and DOX were assigned using the Kollman united atom [54] and Gasteiger-Marsili [55] methods, respectively. The parameters of DOX-DNA complexes were prepared using ADT, which were denoted in the PDBQT file. The grid maps of dimensions 72 × 72 × 122 Å (80 × 68 × 82 Å) with a grid-point spacing of 0.375 Å were created around the center of 1BNA (1Z3F) so that the whole structure was included. In addition, the grid box centers for x, y and z coordinates of 1BNA receptor (1Z3F) are 14.779 Å, 20.976 Å, and 8.804 Å (2.303 Å, 15.703 Å, and 37.592 Å). During the docking process, DOX molecule was allowed to move freely, whereas the receptor molecule was kept rigid and fixed. The structures of DOX-DNA docked models were visualized using PyMol software [56].

Modification of Electrode Surface
The EIS technique was used to examine the modification of the bare electrode by SWCNTs. The signal of 1.0 mM [Fe(CN)6] 3−,4− + 1.0 mM KCl was selected as a reference signal for this goal. The charge transfer resistances were obtained as 2.1 KΩ, 1.4 KΩ, and 1.85 KΩ for GCE and GCE modified with SWCNTs and GCE modified with SWCNTs and ds-DNA, respectively, confirming the higher conductivity of SWCNTs at the surface of GCE. be observed in guanine signal after the addition of doxorubicin, confirming the intercalation between ds-DNA at the surface of the sensor and doxorubicin.

Optimization of ds-DNA Concentration
ds-DNA concentration is one of the most important factors in DNA-based biosensor applications. Therefore, the ds-DNA concentration was optimized before linear dynamic range investigation and clinical sample analysis. The differential pulse voltammograms of ds-DNA/SWCNTs/GCE prepared with different initial concentrations of ds-DNA was also recorded at the same experimental conditions (not shown). The current concentration diagram relative to DP voltammograms is given in Figure 2A. As can be seen, with increasing the initial concentration of ds-DNA, the oxidation current of guanine base increased and then reached a stable condition at the concentration of 30 mg/L. After this value, the oxidation current of dsDNA/SWCNTs/GCE did not change with increasing ds-DNA, indicating that the electrode surface was saturated. Therefore, 30 mg/L ds-DNA was selected as the optimum concentration for monitoring ds-DNA/SWCNTs/GCE to sensing of doxorubicin.
In addition, intercalation time between 30.0 mg/L ds-DNA and 7.0 µM anticancer drug was optimized as one of the main factors in the ability of the sensor for trace-level analysis of doxorubicin in the period time 0-25 min. As can be observed, with increasing the time until 20 min, the oxidation current of guanine reduced and then remained stable ( Figure 2B). This referred that 20 min was sufficient for intercalation between analytes and ds-DNA.

Optimization of ds-DNA Concentration
ds-DNA concentration is one of the most important factors in DNA-based biosensor applications. Therefore, the ds-DNA concentration was optimized before linear dynamic range investigation and clinical sample analysis. The differential pulse voltammograms of ds-DNA/SWCNTs/GCE prepared with different initial concentrations of ds-DNA was also recorded at the same experimental conditions (not shown). The current concentration diagram relative to DP voltammograms is given in Figure 2A. As can be seen, with increasing the initial concentration of ds-DNA, the oxidation current of guanine base increased and then reached a stable condition at the concentration of 30 mg/L. After this value, the oxidation current of dsDNA/SWCNTs/GCE did not change with increasing ds-DNA, indicating that the electrode surface was saturated. Therefore, 30 mg/L ds-DNA was selected as the optimum concentration for monitoring ds-DNA/SWCNTs/GCE to sensing of doxorubicin.
In addition, intercalation time between 30.0 mg/L ds-DNA and 7.0 µM anticancer drug was optimized as one of the main factors in the ability of the sensor for trace-level analysis of doxorubicin in the period time 0-25 min. As can be observed, with increasing the time until 20 min, the oxidation current of guanine reduced and then remained stable ( Figure 2B). This referred that 20 min was sufficient for intercalation between analytes and ds-DNA. Micromachines 2021, 12, x 5 of 12

Linear Dynamic Range and Limit of Detection
DP voltammograms of ds-DNA/SWCNTs/GCE in the presence different concentrations of doxorubicin were recorded and represented in the inset of Figure 3. As can be seen, the oxidation current of the guanine base displayed a linear dynamic range in the concentration range 1.0 nM-20.0 µM for sensing of doxorubicin at the optimum conditions ( Figure 3). Moreover, the findings indicated that the ds-DNA/SWCNTs/GCE could be successfully utilized for the determination of doxorubicin with a limit of detection (LOD) of 0.6 nM (YLOD = 3Sb/m; Sb is the standard deviation of blank and m is the slope of linear dynamic range). The linear dynamic range and limit of detection of the proposed biosensor were comparable with previous electrochemical suggested sensors for monitoring of DOX (see Table 1).

Linear Dynamic Range and Limit of Detection
DP voltammograms of ds-DNA/SWCNTs/GCE in the presence different concentrations of doxorubicin were recorded and represented in the inset of Figure 3. As can be seen, the oxidation current of the guanine base displayed a linear dynamic range in the concentration range 1.0 nM-20.0 µM for sensing of doxorubicin at the optimum conditions ( Figure 3). Moreover, the findings indicated that the ds-DNA/SWCNTs/GCE could be successfully utilized for the determination of doxorubicin with a limit of detection (LOD) of 0.6 nM (Y LOD = 3 S b /m; S b is the standard deviation of blank and m is the slope of linear dynamic range). The linear dynamic range and limit of detection of the proposed biosensor were comparable with previous electrochemical suggested sensors for monitoring of DOX (see Table 1). Micromachines 2021, 12, x 6 of 12

Analysis of the Clinical Samples
The ability of ds-DNA/SWCNTs/GCE for sensing of doxorubicin was checked in various clinical samples, including injection and dextrose saline. Dextrose saline was selected as a complex matrix that usually has injections added in it. The standard addition results for the determination of doxorubicin were tabulated in Table 2. The recovery range of 97.2-104.9% offered the high-performance ability of ds-DNA/SWCNTs/GCE, implying the potential for utilization as a new biosensor for the determination of doxorubicin in clinical samples.

Analysis of the Clinical Samples
The ability of ds-DNA/SWCNTs/GCE for sensing of doxorubicin was checked in various clinical samples, including injection and dextrose saline. Dextrose saline was selected as a complex matrix that usually has injections added in it. The standard addition results for the determination of doxorubicin were tabulated in Table 2. The recovery range of 97.2-104.9% offered the high-performance ability of ds-DNA/SWCNTs/GCE, implying the potential for utilization as a new biosensor for the determination of doxorubicin in clinical samples.

Molecular Docking Investigation
The binding affinity of doxorubicin (DOX) drug inside the DNA receptor was theoretically assessed in two manners: Groove interaction and intercalation.

Molecular Docking Investigation
The binding affinity of doxorubicin (DOX) drug inside the DNA receptor was theoretically assessed in two manners: Groove interaction and intercalation.

Minor Groove Docking Study
Molecular docking revealed that the binding mode of DOX drug to DNA dodecamer was via minor groove manner with the binding affinity of −9.1 kcal/mol (see Figure 4).
As shown in this figure, doxorubicin drug was located in the vicinity of cytosine and guanine base pairs of DNA in such a way DOX forms O…H-O and O…H-N conventional

Minor Groove Docking Study
Molecular docking revealed that the binding mode of DOX drug to DNA dodecamer was via minor groove manner with the binding affinity of −9.1 kcal/mol (see Figure 4).
As shown in this figure, doxorubicin drug was located in the vicinity of cytosine and guanine base pairs of DNA in such a way DOX forms O…H-O and O…H-N conventional

Intercalation Docking
It was found that doxorubicin was stabilized in the intercalating site of the hexamer of DNA in which drug was surrounded by nitrogenous cytosine (DC-1 and DC-5) and guanine (DG-2 and DG-6) base pairs of DNA with a binding affinity of −9.6 kcal/mol (see Figure 5). In addition, it was observed that the hydrogen and oxygen atoms of DOX drug were participated as the donor and acceptor moieties to form three O . .

Conclusions
In this study, a highly sensitive and powerful electroanalytical biosensor was fabricated and utilized for sensing of doxorubicin. ds-DNA/SWCNTs/GCE offered an intercalation reaction between ds-DNA and doxorubicin and used as analytical approach for sensing of doxorubicin with a detection limit of 0.6 nM. The presence of single-wall carbon nanotubes at the surface of GCE boosted the sensitivity of the sensor for trace-level analysis. Moreover, the docking investigation confirmed the satisfactory intercalation between adenine and guanine bases of ds-DNA and validated the experimental part. Funding: This research received no external funding.

Conflicts of Interest:
The authors declare no conflict of interest.