Electrochemical investigation was performed by electrochemical workstation model Ivium-Vertex connected to an electrochemical Cell (Azar electrode Company, Iran). Moreover, the I-V signals were displayed based on the Ag/AgCl/KCl_sat reference electrode's potential. Mitoxantrone hydrochloride, ZIF-8, BMIMF, and DNA (Calf Thymus) were purchased from Sigma-Aldrich. Also, carbon powder and paraffin oil were obtained from Merck Company. In addition, phosphoric acid, boric acid, acetic acid, and Tris hydrochloride were purchased from Across Company. Notably, the stock solution of mitoxantrone hydrochloride (0.001 M) was prepared by dissolving 0.517 g mitoxantrone hydrochloride in 100 mL distillated water under the stirring conditions.

The BMIMS/ZIF-8/CPE was prepared by mixing ZIF-8 with carbon powder as the powder components in the ratio 5:95 (w/w), and paraffin oil and BMIMS as the liquid binders in the ratio 8:2 (v/v). Accordingly, these ratios of powder and binder components were optimized by recording voltammograms of solution containing 1.0 mM [Fe(CN)₆]^{3−, 4−} at the surface of electrodes with different ratios of the components. Also, the stability of ZIF-8 as aqueous solution helps for a repeatable electrochemical response in electroanalytical systems.

DNA/BMIMS/ZIF-8/CPE was perpetrated by the addition of 10 μL of ds-DNA solution (50 mg/L) prepared into acetate buffer (0.5 M, pH 4.8) using a dropwise strategy. Notably, this value was optimized by recording ds-DNA at the surface of DNA/BMIMS/ZIF-8/CPE in the concentration ranged between 10 and 60 mg/L.

To study the intercalation of mitoxantrone hydrochloride with ds-DNA at the surface of DNA/BMIMS/ZIF-8/CPE, the electrode was immersed into a solution containing Tris-HCL buffer solution (pH = 7.4) with mitoxantrone hydrochloride and then remained for 12 min under the stringing condition. Afterward, the electrode was washed with the acetate buffer solution and the differential pulse voltammograms of electrode was then recorded in acetate solution (0.5 M, pH 4.8).

In this work, the molecular docking study is performed to evaluate the affinity of mitoxantrone drug in the active site of DNA hexamer d(CGATCG)2 containing an intercalation gap (PDB ID:1Z3F). For a comprehensive investigation of the binding orientation analysis, the best conformer with the lowest root mean square deviation (RMSD) value of 0 Å and the highest binding energy value is selected. The docking of mitoxantrone into the DNA sequence suggests the intercalation of the aromatic rings of mitoxantrone drug between cytosine and guanine base pairs of DNA with a binding energy of −6.7 kcal/mol as shown in Figure 1, curve a. The docked model reveals that the hydrogen and oxygen atoms of mitoxantrone drug have participated as the donor and acceptor to form four intermolecular hydrogen bonds (HBs) with base pairs of DNA (see Figure 1, curve b). It is found that the interaction of oxygen (O5) atoms of drug molecule with H22 atom of deoxyguanosine (DG6 of chain B) of DNA leads to O5…H22-N2 conventional HB with distance of 2.6 Å. Also, the H36 atom of drug as the proton donor interacts with O4′ atom of deoxyribose sugar moiety linked to guanine (DG6 of chain B) as a proton acceptor with a distance of 2.3 Å. Furthermore, the hydrogen atom of the hydroxyl terminal group of drug molecule is bonded to the second and forth oxygen atoms of deoxycytosine (DC5 of chain B) of hexamer of DNA, i.e., O2…H60-O6 and O4′…H60-O6 with the O…H distances of 2.4 and 2.2 Å, respectively. The hydrogen bond angles are 109.5°, 110.5°, 134.5°, and 163.6° for O5…H22-N2, O2…H60-O6, O4′…H60-O6 and O4′…H36-N8, respectively. In addition, the intermolecular interactions between carbonyl groups of mitoxantrone drug and oxygen atoms of DNA sequence, i.e., O2 atom of DC5 chain B, O4′ atom of DG6 chain B and O4′ atom of DG2 chain A with the respective O…O bond lengths of 3.3, 3.5 and 3.4 Å are observed. The docking study approves the interaction between mitoxantrone drug and guanine residues of DNA contributes in the formation of the stable mitoxantrone-DNA complex.

Mitoxantrone (12.5 mg/12.5 mL) was purchased from a local pharmacy and then used as a real sample with no pretreatment. The standard addition method was used for analyzing the mitoxantrone concentration in the injection sample using DNA/BMIMS/ZIF-8/CPE.

The modification of the CPE surface with BMIMS and ZIF-8 was investigated by recording the cyclic voltammograms of the solution containing 1.0 mM [Fe(CN)₆]^{3−, 4−}. By moving CPE (Figure 1, curve a) to ZIF-8/CPE (Figure 1, curve b), a little improvement was obtained in the oxidation signal of [Fe(CN) ₆]^{3−, 4−} redox solution, which can be related to the creation of a high surface area of ZIF-8 at surface of CPE. After the addition of BMIMS and at a surface of BMIMS/CPE (Figure 1, curve c), the oxidation current of CPE was increase from 14.65 to 24.4 μA that is relative to high conductivity of IL.

After modification of CPE with ZIF-8 and BMIMS, a sharp redox signal with an oxidation current 34.8 μA was observed relative to [Fe(CN)₆]^3−/4− that can be associated with the synergic effects of BMIMS and ZIF-8 at surface of CPE. This amplification can be created under a high sensitivity condition to determine mitoxantrone at surface of DNA/BMIMS/ZIF-8/CPE. In addition, active surface area of CPE, ZIF-8/CPE, BMIMS/CPE and BMIMS/ZIF-8/CPE were calculated about 0.121, 0.163, 0.184, and 0.22 cm² by solution containing 1.0 mM [Fe(CN)₆]^3−/4− and results confirmed that mediators could be increased active surface area of CPE.

Figure 2 displays the ds-DNA signal of DNA/BMIMS/ZIF-8/CPE in the absence (curve a) and in the presence of 35.0 and 80.0 μM mitoxantrone (curves b & c), respectively. According to the data reported, the oxidation signal of ds-DNA decreased from 6.2 to 3.87 μA and 2.02 μA in the presence of 35.0 and 80.0 μM mitoxantrone, respectively. Furthermore, the peak potential related to the guanine base shifted from 816 to 825 mV and 845 mV along with the increase of mitoxantrone concentration confirming the intercalation interaction between mitoxantrone and guanine base in ds-DNA structure.

As can be seen, along with increasing of the mitoxantrone concentration, the oxidation current of ds-DNA decreased. Accordingly, this point can be selected as an analytical factor for the determination of mitoxantrone concentration in the solution.

In order to create the best analytical conditions, it is important to optimize the significant factors in the analytical behavior of the biosensor. Therefore, the initial concentration of dsDNA, temperature, buffer types, and incubation time should be optimized.

Figure 3 displays the oxidation current of ds-DNA compared to the initial concentration of ds-DNA during the modification process.

As can be seen, along with the increase of the initial concentration of ds-DNA, the oxidation signal of DNA/BMIMS/ZIF-8/CPE increased to a concentration of 50 mg/L, which then remained constant. Correspondingly, this point confirms that, in the solution containing 50 mg/L, the electrode surface of BMIMS/ZIF-8/CPE was saturated by ds-DNA, and also the maximum signal can be observed.

Temperature is known as one of the important factors in the fabrication of a DNA biosensor. In this regard, the changes in the ambient temperature of the test can affect the stability of the DNA at the electrode surface. Therefore, in this research, this factor was optimized. As can be seen in Figure 4A, by increasing the ambient temperature from 15 to 25°C, the ds-DNA signal has increased, and then, along with increasing the temperature up to 35°C, this signal has decreased. This point confirms that, in high temperatures, the electrode surface cannot keep ds-DNA at surface of BMIMS/ZIF-8/CPE.

Accordingly, the decreased viscosity of binders in the carbon paste matrix could be considered as one of the main reasons for this point. In addition, in low temperatures, the activity of ds-DNA was low and DNA/BMIMS/ZIF-8/CPE showed a low oxidation signal.

In addition, the type of buffer is one of the main factors in the deposition of ds-DNA at surface of BMIMS/ZIF-8/CPE. Therefore, the effect of acetate buffer, Britton–Robinson buffer, and phosphate buffer solutions on the deposition step of ds-DNA were investigated. As can be seen in Figure 4B, the best oxidation signal relative to ds-DNA can be detected in the solution containing acetate buffer solution and this buffer was selected as the best condition in the next step of the experiment. Moreover, the interference between the phosphate groups of phosphate buffers or Britton–Robinson buffer can be considered as the most important factor in the creation of a weak ds-DNA signal in these buffers.

Notably, incubation time is an important factor in the final step of ds-DNA biosensor application in determining anticancer drugs. Also, the low time of the intercalation step does not allow the biosensor to have a proper interaction between the guanine base and the anticancer drugs. Also, in the long term, it is possible to release the ds-DNA-drug into solution and saturation guanine sites in ds-DNA by anticancer drugs. In this regard, the recorded data showed that 12 min is a suitable incubation time for this study (Figure 5).

To investigate the repeatability of the DNA biosensor, four different DNA/BMIMS/ZIF-8/CPE which were fabricated by the same procedure and oxidation signal of guanine were also recorded at the surface of fabricated electrodes (Figure 6). The obtained results showed the relative standard deviation of about 2.7 and 3.1% in the current and potential of guanine signal for four electrodes that are acceptable values for a novel DNA-biosensor.

In this research, the molecular docking study was performed to evaluate the affinity of Mitoxantrone (MTX) drug in the active site of DNA hexamer d (CGATCG) 2 containing an intercalation gap (PDB ID: 1Z3F). For conducting a comprehensive investigation on the binding orientation analysis, the best conformer with the lowest root mean square deviation (RMSD) value of 0 Å and also with the highest binding energy value, was selected. Docking of MTX into DNA sequence suggests the intercalation of the aromatic rings of Mitoxantrone drug between the cytosine and guanine base pairs of DNA with a binding energy of −6.7 kcal/mol, as shown in Figure 7A. The docked model revealed that, the hydrogen and oxygen atoms of MTX drug are participating as the donor and acceptor, to form four intermolecular hydrogen bonds (HBs) with base pairs of DNA, respectively (see Figure 7B). It was found that the interaction of oxygen (O5) atoms of the drug molecule with H22 atoms of deoxyguanosine (DG6 of chain B) of DNA leads to O5…H22-N2 conventional HB with a distance of 2.6 Å. Also, the H36 atom of the drug, as the proton donor, interacts with O4′ atom of deoxyribose sugar moiety linked to guanine (DG6 of chain B), as a proton acceptor with a distance of 2.3 Å. Furthermore, the hydrogen atom of the hydroxyl terminal group of drug molecule is bonded to the second and forth oxygen atoms of deoxycytosine (DC5 of chain B) of hexamer of DNA, i.e., O2…H60-O6 and O4′…H60-O6 with the O…H distances of 2.4 and 2.2 Å, respectively. Moreover, the hydrogen bond angles are 109.5°, 110.5°, 134.5°, and 163.6° for O5…H22-N2, O2…H60-O6, O4′…H60-O6, and O4′…H36-N8, respectively. In addition, the intermolecular interactions between carbonyl groups of MTX drug and oxygen atoms of DNA sequence, such as O2 atom of DC5 chain B, O4′ atom of DG6 chain B, and O4′ atom of DG2 chain A with the respective O…O bond lengths of 3.3, 3.5, and 3.4 Å were observed. The docking study approves the interaction between mitoxantrone drug and guanine residues of DNA that contributes in the formation of the stable MTX-DNA complex.

Using a decreasing trend in DNA signals and its relationship with the concentration of mitoxantrone, a linear dynamic range from 8.0 nM to 110 μM with the equation of ΔI_pa = 0.052 C_mitoxantrone + 0.589 (R² = 0.991) and a detection limit of 3.0 nM was calculated to determine mitoxantrone at surface of DNA/BMIMS/ZIF-8/CPE (Figure 8).

Binding energy between guanine and mitoxantrone can be determined by equation 1 as follows:

Where m is the binding number and K_a is the association equilibrium constant. Using the slope of recording plot in Figure 9 and equation 1, the values of m and K_a were determined to be 0.334 and 1.737 × 10³ M⁻¹, respectively.

The selectivity of DNA/BMIMS/ZIF-8/CPE, as a new biosensor, was investigated in the presence some usual organic and inorganic interference with an acceptable error of 5% in current and potential. The obtained results showed 1,000-fold of ions such as Na⁺, Li⁺, Br⁻, and NO3-, 500-fold of methionine, alanine, and phenylalanine, and also 400-fold of vitamin C and vitamin B₂ had no interference in determination of 20 μM mitoxantrone using the suggested biosensor.

In addition, the ability of DNA/BMIMS/ZIF-8/CPE was checked as a new biosensor for determination of mitoxantrone in the injection samples using the standard addition method. The results are presented in Table 1 and recovery data between 99.18 and 102.08% confirmed the high performance ability of DNA/BMIMS/ZIF-8/CPE for determination of mitoxantrone in real samples.