1 Introduction

To create a novel class of biosensors for detecting dsDNA interactions with other chemicals like contaminants or pharmaceuticals, nucleic acids have been electrochemically coupled with transducers. Given that these substances interact with the dsDNA, this detection aids in screening them as well [1]. Small molecules of drugs, chemicals, ligands, or other compounds can bind to DNA through covalent or non-covalent interactions. Covalent interactions between compounds and DNA can lead to irreversible chemical alterations of the DNA, leading to cell death. In contrast, non-covalent interactions between compounds and DNA occur with intermolecular forces weaker than covalent interactions and occasionally reversible [2,3,?4]. Different electrochemical approaches have been used to look for interactions between DNA and anticancer drugs [5,6,7,?8]. Other techniques, including molecular modeling, UV?Vis Spectroscopy, Fluorescence Spectroscopy, Nuclear Magnetic Resonance, Footprinting, Gel Electrophoresis, Mass Spectrometry, FTIR, Circular Dichroism Spectroscopy, Raman Spectroscopy, Capillary Electrophoresis, and Surface Plasmon Resonance (SPR), have been used for detection of DNA interaction with other compounds. These techniques have the benefits of being sensitive and selective. Still, they also have disadvantages. They require vast amounts of analytes for analysis, are difficult to use, take a long time, and require an extended period for sample pretreatment before analysis. Electrochemical methods have several advantages over more conventional ones, including inexpensive preparation costs, high sensitivity, equipment usability, selectivity, and a minimal requirement for analytes when comparing DNA and pharmacological molecules [2, 4,5,6,7,8,9,10,11,12,13,?14].

The antibiotic adriamycin (ADR), a member of the anthracycline family (Fig. 1), has a wide range of uses in chemotherapy and has anti-neoplastic properties. The drug?s anticancer properties have been known for about 30 years [15, 16], but its mode of action in vivo has yet to be thoroughly characterized [17]. Understanding anticancer properties and their mechanisms of action are still crucial since doing so will help improve how it is administered. According to experimental data, ADR causes the DNA of malignant cells to have oxidative damage by producing reactive oxygen species [17,18,19,?20]. It has been determined that the DNA?s guanine oxidation product is highly mutagenic and contributes to cell malfunction [21]. In 1972, X-ray crystallography and other techniques were used as proof to demonstrate that ADR and other analogs in the family of anthracycline interact with the DNA intercalatively [15, 22,23,24,25,?26]. The portion of the ADR that intercalates with the DNA base pairs when interacting with dsDNA is represented by the rings on its B and C sides (Fig. 1) [24, 25, 27, 28]. The DNA?s double helix minor groove still contains the ADR?s portion, which has amino sugar and its carbonyl side chain that binds to A. The DNA?s double helix main groove side is where the ring D extends [22, 27, 28]. The amino sugar has a positively charged amino group portion that interacts with the phosphate group of the DNA to perform its function and is crucial for intercalation and binding affinities to the dsDNA [23].

Fig. 1
figure 1

Schematic representation of the intercalation activity of Adriamycin and dsDNA shown in the space-filing model. The Adriamycin is first depicted in 2D (top) and then in a 3D sticks model (down) with carbon atoms colored cyan inserted manually on the dsDNA. The dsDNA was retrieved from the Protein Data Bank (PDB) file 2MG8, and the Adriamycin was retrieved from the PubChem database (PubChem CID: 51,066,577)

Full size image

Graphite is advantageous as an electrode material because it combines the characteristics of metals and non-metals [29]. 4% graphite powder mixed with an inorganic resin, clay matrix, and cellulose is used to make pencils [30]. The Graphite-Reinforced Composite (GRC) is widely available commercially and is utilized as disposable material due to its high quality and low content of heavy metal impurities [31]. Graphite is a dark gray, soft, and porous material with adsorption capacity that is naturally abundant and highly conductive with an electrical conductivity for conducting polymer composites and electrical resistivity. The carbon atoms of the graphites are only bonded in two dimensions. It has a layered structure in which each carbon atom in graphite is sp2 hybridized. Three of each carbon atom?s four valence electrons form bonds with three other carbon atoms, while the fourth electron forms delocalized bonds that spread uniformly across all carbon atoms. The carbon atoms are hexagonally arranged in a planar condensed ring. The layers are stacked parallel to one another, with the atoms within the rings covalently bonded and the layers loosely bonded together by Van der Waal?s forces. Graphite?s anisotropic nature is caused by two types of bonding acting in opposite crystallographic directions. These two opposing chemical bonds may explain graphite?s ability to form a solid film lubricant. Because of the weak Van der Waal forces that govern the bonding between the individual layers, and because there are no bonds between the layers, the layers can easily slip off one another, making it an ideal lubricant and resulting in a lower coefficient of friction and, thus, wear. This is the same reason graphite is a good material for pencils ? layers come off and get left on the paper as you write. It conducts heat and electricity well along each layer of graphite but not perpendicular to it. The structure of graphite accounts for its high electrical conductivity. Graphite has been used as electrode material in constructing disposable biosensors for detecting various analytes due to its good electro-catalytic properties [32,33,?34]. With many studies and analyses of organic and inorganic chemicals reported by various academics worldwide in recent years, pencil graphite electrodes have grown in usefulness because of their electrochemical and economic properties. PGEs, which are easily disposable, are prepared from pencil graphite leads. They are easily accessible on the market, inexpensive, and user-friendly. PGEs show lower current signals than other electrodes, their results are reproducible easily and highly sensitive, and their surface encourages adsorption. The electrode surfaces can be cleaned easily through pretreatment or application of a potential to activate the surface, thereby being used for different voltammetric techniques [35].

This study aims to use pencil graphite electrodes to detect fish sperm dsDNA and ADR interaction using voltammetric and impedimetric methods. To the best of our knowledge and after a thorough search, there are few reports on voltammetric and impedimetric techniques on dsDNA and ADR employing PGE (Table 1). Most of the works employed working electrodes such as carbon paste electrodes, glassy carbon electrodes, screen-printed electrodes, and other modified types of electrodes. Voltammetric methods like CV, DPV, and EIS are used in this study, and this work adds a report on the impedimetric analysis of dsDNA-ADR.

Table 1 Comparison of recently published articles on Pencil Graphite Electrodes, drugs, and DNA
Full size table

2 Experimental

2.1 Reagents

Fish sperm dsDNA was purchased from Sigma Aldrich in Istanbul, Turkey. 0.5 M of Acetate buffer solution (ABS) with a pH of 4.8 was combined with 20mM NaCl to create a stock solution of dsDNA at 1000 ppm (5 mg/5ml). Acetate buffer solutions (ABS) were prepared at a concentration of 0.5 M and various pH values ranging from 4.0 to 8.0. They were made according to conventional procedure using 0.5 M K2HPO4 milliliters (mL) and 0.5 M KH2PO4 milliliters (mL). The working solutions were created by diluting the 1.8 mM ADR stock solution with ultra-pure water to various concentrations (Fig. 2).

2.2 Apparatus

The AUTOLAB-PGSTAT204 and the program Nova 2.1.2 were used to perform voltammetric and impedimetric measurements. The analysis was performed using a three-electrode setup connected to the AUTOLAB. The three-electrode system includes the platinum wire-based auxiliary electrode, the Ag/AgCl/3MKCl reference electrode, and the PGEs working electrode.

2.3 Procedures for preparation, pretreatment, and immobilization

The Ultra-Polymer TOMBOW 0.5HB pencil leads were utilized for the analysis. They were purchased from Izmir at a stationery shop. They were divided into two halves with a length of 3.5 cm, and about 1.5 cm was marked with a white marker and used as the transducer (working electrode surface). The surface of the PGEs was cleaned in the pretreatment process by applying a potential at 1.4 volts (V) for 30 s using chronoamperometry to prevent contaminants and noise in the analysis. The procedure was repeated for each PGE.

2.4 dsDNA and ADR immobilizations on the pencil graphite electrodes? surface

Vial tubes containing 200 µL of fish sperm dsDNA were made as the working solution at a concentration of 100 µg/mL, with the PGEs inserted into the vial tubes. All of the PGEs employed for the study had dsDNA immobilized on them for about 30 min. dsDNA was immobilized on the electrode surface of PGEs using an adsorption technique.

Different concentrations of solutions were prepared and used as working solutions from the stock solution of ADR. The PGEs inserted into the dsDNA solution were removed and inserted into vial tubes containing 74 µM of ADR. The immobilization was done for about 30 min. To get rid of unbound dsDNA and ADR, the PGEs were rinsed with ABS with a concentration of 0.5 M and a pH of 4.8 for 3 s.

For the pH study, the PGEs were immobilized in a 5 ml ADR working solution of 0.92 mM in a beaker for about 30 min, rinsed for 2 s in the ABS, and then used for the study (Fig. 2).

2.5 Electrochemical measurement technique

CV, DPV, and EIS measurements were employed for the study. A minimum of three rounds of experiments were made, with five PGEs being analyzed for each concentration. Their average values were then calculated and shown in the results section. Newly pre-treated PGEs were utilized in every round of repetition of the analysis to study the dsDNA and ADR interaction. The DPV measurements were performed in the acetate buffer solution. The scanning potential range employed for the measurements is +?0.15 V to +?1.4 V, and a scan rate of 10.071 mVs?1 was used.

Then, the EIS measurements were performed using a redox solution of 2.5 mM [Fe (CN) 6]3?/4? produced in 0.1 M KCl. The measurement?s frequency range was between 100 kHz and 0.1 Hz. Using the AUTOLAB-PGSTAT204, the values for charge transfer resistance (Rct) were obtained using the fit and simulation function in Nova 2.1.2 software, which corresponds with the semicircle diameter of the Nyquist plot.

Voltammetric techniques for CV and DPV were employed for the analysis in various buffer solutions prepared with a concentration of 0.5 M ABS at ranges from pH 4.0 to 8.0 for the pH analysis of the ADR. After each investigation, peak current signals for each measurement were recorded. The CV technique was employed with scan rates between 20, 40, 60, 80, 100, 200, 250, 300, 350, 400, and 450 mVs?1, respectively (Figs. 2 and 6).

2.6 Preparation and detection of ADR in real samples

To employ the proposed fabricated biosensor in detecting ADR in real samples, urine samples were collected from a healthy individual and filtered. DPV was used to analyze the ADR in the spiked samples of urine. The voltammograms obtained were recorded using a standard addition method using serial dilutions of known concentrations and volumes of the ADR. A volume of 2 mL of urine was inserted into a beaker, and a known aliquot concentration of 0.03 mM and 0.05 mM of the ADR was added and made to a final volume of 10 mL using 0.5 M ABS at a pH of 4.8. They were then stirred up for 1 min using a magnetic stirrer. The measurements were recorded from the voltammograms of each beaker containing the urine and aliquots concentrations of ADR.

Fig. 2
figure 2

Experimental Procedure of the electrochemical detection of the interaction between dsDNA and Adriamycin immobilized on Pencil Graphite Electrodes and the photo of the sensing setup

Full size image

3 Results and discussion

3.1 Adriamycin and dsDNA electrochemical behavior on PGE

For the study on the interaction between dsDNA and ADR, the Differential Pulse Voltammetry technique was applied, and the guanine oxidation peak current signal of 100 µg/mL dsDNA was recorded at an oxidation potential of 1.0 to 1.1 V (Fig. 3A) [2, 50,51,?52]. All of the results of the dsDNA and ADR interaction investigation repeatedly showed the guanine oxidation at this same potential. This is because guanine is suggested to be the most electroactive base of the dsDNA among other bases of the dsDNA. Thus, guanine is the marker for dsDNA interaction with other compounds. Using DPV measurements in ABS, the behavior of Adriamycin was only examined at 0.46 mM concentration (Fig. 3B). After applying a positive potential, the oxidation potential of ADR was found to be between 0.55 and 0.60 V[53].

Fig. 3
figure 3

Differential pulse voltammograms in pH 4.8, 0.5 M acetate buffer solution showing the signal of A 100 µg/mL, dsDNA only immobilized on bare PGE without interaction with ADR. B Adriamycin only with a concentration of 0.46 mM immobilized on bare PGE without interaction with dsDNA

Full size image

3.2 Electrochemical behavior of dsDNA and adriamycin interaction on PGE

DPV measurements were used to examine the electrochemical behavior of the interaction between dsDNA and ADR on PGEs (Fig. 4), showing the peak current signal response as ADR?s concentration increases with 100 µg/mL of dsDNA before and after the reaction. The increased concentration of ADR was from 0.46 mM, 0.92 mM, 1.4 mM, and 1.8 mM, respectively, as it interacts with the dsDNA. The peak current signal of the guanine signal is seen to be affected by the increase in ADR?s concentration, thereby gradually decreasing the guanine signal. Three assumptions can be suggested for this electrochemical behavior: (a) The ADR is attached to the dsDNA bases, shielding the oxidizable groups of the electroactive DNA bases; as a result, the peak current signal decreases with increasing ADR concentration. This causes the drug to interact with dsDNA, especially at greater concentrations, and confine to the surface [52], (b) A marker of the ADR?s intercalation activity as a planar molecule into the dsDNA double helix structure is the decrease in the dsDNA guanine current peak signals [54]. The structure of ADR (Fig. 1) depicts the interaction between the drug and dsDNA by intercalation [55][56]. The aminosugar and carbonyl side chain of ADR connects to ring A to interact with the minor groove of the dsDNA. The B and C rings of ADR intercalate between the DNA bases. The positively charged aminosugar of the ADR binds to the phosphate backbone of the dsDNA as ring D extends to interact with the major groove of the dsDNA [22, 23, 27, 28]. (c) A decrease in the strength of the guanine signal oxidized at the PGEs surface could be suggested to be from modifications in the charge transfer characteristics of DNA due to its interaction with the medication [54, 57, 58].

Fig. 4
figure 4

A Differential pulse voltammograms and B bar plots show the different measurements in pH 4.8, 0.5 M ABS using PGEs. The labeled results on the voltammogram and the bar plots, respectively, represent Bare PGEs, 100 µg/mL of dsDNA only, 0.46 mM of ADR, 0.46 mM of ADR with 100 µg/mL dsDNA, 0.92 mM of ADR with 100 µg/mL dsDNA, 1.4 mM of ADR with 100 µg/mL dsDNA, 1.8 mM of ADR with 100 µg/mL dsDNA all immobilized on PGEs

Full size image

3.3 Impedimetric investigation on the interaction between dsDNA and adriamycin on PGE showing their electrochemical behavior

The impedimetric analysis of the electrochemical behavior was conducted in the redox solution of 2.5 mM of [Fe (CN) 6]3-/4- prepared in 0.1 M KCl using a frequency of 100 kHz to 0.1Hz. The spectrum obtained (Fig. 5A) of the EIS measurement is for bare PGEs, 100 µg/mL dsDNA-modified PGEs, 0.46 mM ADR-modified PGEs, and ADR concentrations of 0.46 mM, 0.92 mM, 1.4 mM, and 1.8 mM with dsDNA, respectively, on PGEs. The charge transfer resistance (Rct) values related to the Randles-Sevcik circuit are evaluated in the inset (Fig. 5A). According to the results, the bare PGE ?a? has an Rct value of 170 ?, suggesting a slow electron transfer rate between the redox solution and electrode surface during the electrochemical reaction [59]. The Rct value for the dsDNA immobilized on the PGEs is 250 ? (Fig. 5B), which is increased more than the bare PGE. This suggests successful adsorption of the dsDNA on the surface of the electrode and could also be attributed to the negatively charged dsDNA on the electrode surface and the electrostatic effect of the redox solution [60]. The Rct values of dsDNA and ADR decrease as concentration increases due to their immobilization on the electrode surface. The positive charges on the ADR balance the solution?s negative charges, and this could indicate that the dsDNA and ADR interacted through intercalation activity [58, 60]. The Nyquist diagram contains the analysis?s circuit model, which represents the corresponding impedance measurement, with Cdl as the charge capacitance, Rct as the charge transfer resistance, and W as the Warburg impedance caused by mass transfer to the electrode surface, and Rs as the solution resistance [60].

Fig. 5
figure 5

A showing the Nyquist diagrams for the Rct values in the solution containing 5mM K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) with 0.5 M KCl by using Bare PGEs, 100 µg/mL dsDNA, 0.46 mM of ADR, 0.46 mM of ADR with 100 µg/mL dsDNA, 0.92 mM of ADR with 100 µg/mL dsDNA, 1.4 mM of ADR with 100 µg/mL dsDNA, 8?×?10?3 M of ADR with 100 µg/mL dsDNA, all immobilized on PGEs B Bar charts showing the Rct values of the respective analysis performed on bare electrode PGE, dsDNA immobilized on PGE. DsDNA with ADR immobilized on the PGEs

Full size image

3.4 Study of scan rate and its impact on adriamycin peak current

Cyclic voltammetry was used to show the effect of scan rate (Fig. 6A) on the electrochemical behavior of Adriamycin immobilized on the PGEs. The peak current signals of the ADR could be seen on the voltammogram to increase as the applied scan rates increased. From the observed results, a linear plot was done to obtain the equation of peak current Ip versus square root of scan rate ?1/2. This was done to indicate a typical diffusion-controlled reaction of the electrochemical behavior of ADR on the electrode surface (Fig. 6C; Table 2).

The peak currents Ip were plotted linearly against scan rate (?), and a straight line was produced (Fig. 6B; Table 1). The results show a linear relationship where the scan rate increases with anodic and cathodic peak currents, suggesting that the oxidation reaction is quasi-reversible. To determine whether the electrochemical reaction that occurred on the electrode surface whether it is diffusion or adsorption controlled, the logarithm of scan rate and anodic peak current (log v (mVs?1) versus log Ipa) technique was used (Fig. 6D; Table 2). The analysis showed that the slope was almost 0.5, indicating that the electrochemical processes that took place in the ADR analysis were diffusion controlled [59].

Table 2 Linear regression equations for peak current Ip and the square root of scan rate, peak current Ip and scan rate, and the logarithm of anodic peak current and the logarithm of scan rate for ADR on PGEs
Full size table
Fig. 6
figure 6

A Cyclic voltammograms showing the electrochemical oxidation and reduction of ADR immobilized at PGEs in 0.5 M phosphate buffer of pH 4.0 at different applied scan rates of (20, 40, 60, 80, 100, 200, 250, 300, 350, 400, and 450) mVs?1, respectively. B and C show the influence of peak current Ip on the square root of scan rate ?1/2 (mVs?1)1/2 and scan rate ? (mVs?1). D shows the logarithm of anodic peak current and the logarithm of scan rate (log Ip versus log ? (mVs?1))

Full size image

3.5 The effect of a pH study on the oxidation of adriamycin at PGEs

Cyclic voltammetry was applied, and the current response of ADR oxidation at PGE was investigated to identify its electrochemical behavior as a function of the buffer solution?s pH effect (Fig. 7A). The different pH values of the buffer solutions impacted the oxidation activity of the ADR on the surface of PGE, which changed its electrochemical behavior. The ADR?s anodic peak currents (Ipa) and potentials (Epa) indicated a gradual decrease as the pH values increased from 4.0 to 8.0. The cyclic voltammograms of the electrochemical behavior of ADR suggested that the electrochemical alterations to the behavior of ADR at the electrode surface are distinctly pH dependent, causing the anodic peak current to decrease when the pH values are increased. This result causes the peak potentials to shift negatively to the left, demonstrating a linear regression relationship between the pH values and the anodic peak potentials (Epa) (Fig. 7B; Table 3). The Nernstian value of 59 mV/pH is the value for two electrons and two protons involved in oxidation and reduction reaction processes, and 29.5 mV/pH for processes involving two electrons and one proton reaction, the value derived from the result of the slope of Epa and pH relationship when compared to the Nernstian value, indicates that the reaction is a two-electron/two-proton oxidation and reduction reaction process [59].

Fig. 7
figure 7

A Cyclic voltammograms showing the effect of pH on the electrochemical behavior of the ADR on PGE in 0.5 M PBS with pH of 4.0, 4.8, 5.8, 6.0, 7.0, and 8.0 at a scan rate of 200 mVs?1, respectively. B shows a Linear regression plot, anodic peak potentials (Epa) equation, and the pH of ADR for cyclic voltammetry used in analyzing the electrochemical behavior of ADR at the PGE surface

Full size image
Table 3 The equation of anodic peak potentials (Epa) and pH with slope from cyclic voltammetry used in analyzing the pH of ADR at the PGE surface
Full size table

3.6 Adriamycin?s LOD and LOQ

Differential pulse voltammetry was applied, and the effect of increasing the concentration of ADR was investigated at PGE using oxidation signals (Fig. 8). Based on the findings of the voltammetric method used, the limit of detection (LOD), and limit of quantification (LOQ) were determined. The ADR was studied from 0.018 µM to 1.8 µM in concentration. The LOD and LOQ for ADR were calculated using the peak currents using Eqs. (1) and (2), respectively.

$${\text{LOD}}\,{\text{ = }}\,{\text{3}}\, * \,{\text{Sa}}/{\text{b}},$$
(1)
$${\text{LOQ}}\, = \,{\text{1}}0\, * \,{\text{Sa}}/{\text{b}}.$$
(2)

The variables ?Sa? and ?b? stand for our calibration curve?s slope and standard deviation, respectively. The detected ADR oxidation signals increased as the drug?s concentration gradually increased. Voltammogram studies revealed a linear association between peak currents signals and increased ADR concentration. The resulting equation, Ip?=?3.3357 C?+?1.646 (Ip: µA, C: mol L?1, and R2?=?0.9937), shows a linear regression. The LOD and LOQ results reported by other authors were compared with the computed detection limits of the drug ADR (Table 4).

Fig. 8
figure 8

The calibration curve and histogram of the different concentrations of the ADR in the range of 9.19 µM, 7.36 µM, 5.5 µM, 3.67 µM, and 1.84 µM immobilized on the PGEs in 0.5 M PBS pH 4.0 used for obtaining the LOD and LOQ in the main work

Full size image
Table 4 The LOD and LOQ of ADR compared to other methods used to determine ADR.
Full size table

3.7 Application of the fabricated biosensor in real samples

The detection of the presence of ADR in urine samples was carried out using PGE. The detection was first done with diluted 10 mL urine samples, which were blanks. DPV of the urine samples was analyzed without the presence of the ADR. The biosensor detected that the urine samples did not contain the presence of ADR. Aliquots of known concentration of ADR added to the urine samples were observed using DPV (Table 5). The oxidation peaks of the spiked ADR in the urine samples increased as the concentration of the ADR was increased (Fig. 9). The PGE detected the presence of the added standard ADR in the urine samples, with recoveries at almost 100%.

Fig. 9
figure 9

DPV was obtained using PGE in a mixture of 0.03 mM and 0.05 mM ADR in urine samples and 0.5 M ABS pH 4.8

Full size image
Table 5 Determination of ADR in the real sample of urine
Full size table

4 Conclusions

The results from voltammetric techniques demonstrated that the interaction between dsDNA and ADR on the surface of PGE resulted in a reduction in the guanine signals. ADR is pH dependent, as shown by the electrochemical behavior of ADR adsorbed on the PGEs under various pH values. According to the EIS study, PGE showed decreasing charge transfer resistance when our dsDNA and ADR concentrations increased between the electrode surface and the redox solution. As there is little to no research on the electrochemical impedance spectroscopy (EIS) of dsDNA and ADR using PGE, this voltammetric and impedimetric study also adds a report on the interaction of dsDNA and ADR that is employing pencil graphite electrodes. The DNA biosensor proved easy to make, affordable, and time-efficient. The LOD and LOQ results were compared to other biosensors reported in different literature and were close to or less than the reported biosensors. The proposed sensor was applied in real sample of urine to determine ADR in the sample of urine with 100% recoveries.