Introduction

Adriamycin is an antibiotic of the family of anthracyclines with a wide spectrum of chemotherapeutic applications and anti-neoplasic action widely used as an antitumor agent, and exhibits antitumor activity against solid tumors, such as breast and lung cancers [1]. Its strong DNA binding properties lead the discoverers of the antibiotics to suggest that the drugs receptor site is DNA [2, 3]. Most of the available evidence indicates that it binds to double helix mainly at CG-GC steps, the aminosugar being determinant for intercalation to occur [4].

Up to now, many techniques for the determination of the drug and DNA have been developed, for example, high performance liquid chromatography method [5]; microbiological assay method [6]; chemiluminescent [7]; spectrophotometric [8]; photochemical-fluorimetric [9]; electroanalytical techniques [10–13] etc. Among these techniques, electrochemical techniques are becoming increasingly important in the determination of compounds of pharmaceutical interest due to its simple, rapid, and low cost. Recently, nano-materials have attracted much attention and have been widely used in material, electronic, physical fields for various purposes due to their unique properties. Carbon nanotubes (CNT), discovered in 1991 by Iijima [14], represent an important group of nanoscale materials. It had been widely used as a new support for catalysts [15–19]. In recent years, many attempts have been conducted to enhance the electron transfer between electroactive species and electrodes based on deposit various metal particles onto the surface of CNTs, such as platinum, palladium, copper, nickel, ruthenium, silver, gold and so on, have been deposited on the CNTs successfully [20–28].

In this work, a silver nanoparticles (Agnano)/multi-walled carbon nanotubes (MWCNTs-COOH) modified electrode was fabricated and first applied for detecting ADM. The experiments results showed the proposed electrode exhibited a large enhancement of the differential pulse voltammetric response to ADM and had a low detection limit and a good sensitivity.

Experimental

Apparatus and reagents

Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were performed with a CHI650C electrochemical workstation (Shanghai Chenhua Instruments Co, China, http://chi.instrument.com.cn). The three-electrode system was used in the experiment with a bare GCE or modified electrode as working electrode, a saturated calomel electrode (SCE) as reference electrode, and a platinum wire as counter electrode. All electrochemical measurements were carried out in a 10 mL cell at room temperature, where oxygen was removed with high-purity nitrogen for 30 min. All measurements were performed under a nitrogen atmosphere. All the potentials given in this paper were referred to SCE.

The UV/Vis absorbance spectra were obtained by using U-3010 spectrofluorometer (Hitachi, Japan). All of the spectroscopic work was performed at room temperature.

Calf thymus DNA (ctDNA) (sodium salt, type I) was obtained from Sigma (USA, http://www.sigmaaldrich.com) and was used without further purification, and its purity was confirmed by UV/Vis absorption, which produced A260/A280 of approximately 1.8–1.9, suggesting the DNA sample was free of proteins. The stock solution of DNA was directly dissolved in water and stored at 4 °C. The DNA concentration per nucleotide (DNA-P) was determined spectrophotometrically [29] at 260 nm by using the extinction coefficient 6,600 cm−1 M−1. Adriamycin was purchased from Alfa Aesar (Tianjing, China, http://alfaaesar1.blog.sohu.com), sliver nitrate (AgNO3) was purchased from Sigma Aldrich (http://www.sigmaaldrich.com), multi-walled carbon nanotubes with carboxy groups (20–30 nm diameters, 30 mm length and >95% purity) were purchased from the Chengdu Institute of Organic Chemistry (http://www.timesnano.com). 0.1 M phosphate buffer solutions with different pH values were prepared by mixing the stock standard solution of Na2HPO4 and NaH2PO4 and the pH was adjusted with H3PO4 or NaOH sulution. All chemicals were of analytical reagent grade. All solutions were prepared with doubly distilled water.

Preparation of the Agnano /MWCNTs-COOH modified electrode

Prior to modification, the bare GCE was mechanically polished to a mirror-like surface with 1.0, 0.3 and 0.05 μm α-Al2O3. The electrode was rinsed with doubly distilled water, and then successively ultrasonically washed with nitric acid (obtained by mixing nitric acid with doubly distilled water according to the volume ration 1:1), ethanol and doubly distilled water for 1 min, respectively, at last allowed to dry at room temperature. The MWCNTs-COOH modified electrode was obtained by dropping 5 μL of the suspension (obtained by dispersing 0.1 mg MWCNTs-COOH into 2 mL alcohol) onto the surface of clean GCE and it was dried at room temperature, the obtained electrode was taken as MWCNTs-COOH/GCE. The Agnano/MWCNTs-COOH electrode was obtained by immersing the MWCNTs-COOH/GCE into AgNO3 solution for 15 min to adsorb and form silver complexes depended on the electrostatic and complexation interaction of silver ion with the carboxy groups of MWCNTs-COOH [30], after rinsing thoroughly with 0.5 M KNO3 solution, the MWCNTs-COOH modified electrode with silver ions was transferred into 0.5 M KNO3 solution and Agnano were grown by a chronoamperometry method according to the previous report [27]. The potential was stepped from −0.5 to 0 V (vs. SCE) for 30 s, where oxygen was removed with high-purity nitrogen for 30 min.

Results and discussion

The characterization of the Agnano/MWCNTs-COOH/GCE

Figure 1 displays the typical morphology of the MWCNTs-COOH (Fig. 1a) and the Agnano/ MWCNTs-COOH modified electrode (Fig. 1b) was characterized by scanning electron microscopy (SEM). For the Fig. 1a, the MWCNTs-COOH distributed homogeneously on the electrode and displayed a special three-dimensional structure. For the Fig. 1b, it can be obviously observed that the Agnano have been successfully covered on MWCNTs-COOH.

Fig. 1
figure 1

The SEM images of the MWCNTs-COOH (a), the Agnano/MWCNTs-COOH (b) modified GCE

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Electrochemical behavior of the Agnano/MWCNTs-COOH/GCE

Figure 2a shows the CVs of the Agnano/MWCNTs-COOH modified electrode with different scan rate in KNO3 (0.5 M). A pair of redox peak was observed at 0.22 and −0.04 V, which was ascribed to the redox of silver. The Fig. 2b showed the peak current was linear with scan rate.

Fig. 2
figure 2

(a) CVs of the Agnano/MWCNTs-COOH/GCE in KNO3 (0.5 M) with different scan rate: 50, 100, 150, 200, 250, 300, 350 and 400 mV·s−1 (a–h). (b) Plots of the peak current vs. the scan rate

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Electrochemical behaviors of ADM at the Agnano/MWCNTs-COOH/GCE

Figure 3 shows the CVs of ADM at the bare GCE, the MWCNTs-COOH/GCE, and the Agnano/MWCNTs-COOH/GCE in 0.1 M PBS (pH 7.0). At the bare GCE (Fig. 3a), the CV response of ADM was poor. When MWCNTs-COOH was modified on GCE (Fig. 3b), the CV response of ADM was enhanced, meanwhile the peak potential negatively shifted due to MWCNTs-COOH having a large specific surface area and good electronic properties. For the Agnano/MWCNTs-COOH/GCE (Fig. 3c), the peak current of ADM was further enhanced and the peak potential further negatively shifted. This may attribute to the Agnano/MWCNTs-COOH composite film can adsorb much more ADM. Meanwhile we can conclude the electrode reaction of ADM at the Agnano/MWCNTs-COOH/GCE was reversible based on the CVs. In addition, the effect of the scan rate on the peak current of ADM at the Agnano/MWCNTs-COOH/GCE was investigated by CV in 0.1 M PBS (pH 7.0). The result showed that the peak currents were proportional to scan rates from 20 to 580 mV·s−1, which suggest that the electrode reaction of ADM at the Agnano/MWCNTs-COOH/GCE is an adsorption-controlled process. The effect of the solution pH on electrochemical response of ADM at the Agnano/MWCNTs-COOH/GCE was also investigated by CV in 0.1 M PBS (pH 7.0) (The figure was not shown). The experiments results showed that the peak potentials shifted negatively with increasing in the solution pH, which indicated that protons take part in the electrochemical process. And the peak currents increased with the solution pH increased in the range of 3.0–7.0. When the pH > 7.0, the peak current decreased. So we selected pH 7.0 PBS as the support electrolyte in this paper.

Fig. 3
figure 3

CVs responses of 5.5 × 10−6 M ADM at different electrodes: the bare GCE (a); the MWCNTs-COOH/GCE (b); the Agnano/MWCNTs-COOH/GCE (c) in 0.1 M PBS (pH 7.0). Scan rate: 100 mV·s−1

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Determination of ADM

DPV was used to determine ADM. The reduction peak current of ADM was used as the analytical signal. The results were shown in Fig. 4, it could be observed that the peak currents increased with the concentration of ADM increased, and were linear with the concentration of ADM in the range of 8.2 × 10−9 M–19.0 × 10−9 M (shown in inset of the Fig. 4), the linear regression equation was \( i\left( {{{10}^{ - 6}}A} \right) = - 0.73 + 1.49c\left( {{{10}^{ - 8}} M} \right) \), with a correlation coefficient r = 0.9957. The detection limit was 1.7 × 10−9 M (S/N = 3).

Fig. 4
figure 4

DPVs responses of the different concentrations ADM at the Agnano /MWCNTs—COOH/ GCE in 0.1 M PBS (pH 7.0); the inset: the DPV reduction peak current vs. the concentration of ADM

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The stability and reproducibility

The stability of the Agnano/MWCNTs-COOH modified GCE was studied by CV in 0.1 M PBS (pH 7.0) containing 1.0 × 10−6 M ADM, and scanning for 30 continuous cycles at the potential between −0.3 and −1.0 V (vs. SCE) with a scan rate of 100 mV/s, the peak heights of the cycle voltammogram showed a negligible change. After the modified electrode was stored in PBS (pH 7.0) for 3 days, the peak current of the same concentration ADM decreased less than 8%. The reproducibility of the Agnano/MWCNTs-COOH modified GCE was also investigated. The fabrication of five modified electrodes, made independently, showed a well reproducibility with a relative standard deviation (R.S.D) of 7.6% for the currents detection of 1.0 × 10−6 M ADM. Therefore, the Agnano/MWCNTs-COOH modified GCE exhibited an excellent stability and reproducibility.

Detection of ctDNA

The UV/Vis absorbance spectrum was used to investigate the interaction between ctDNA and ADM in 0.1 M PBS (pH 7.0). The obtained experiment result is according with the report [12], which indicated the ctDNA can react with ADM. In this work, we used the change of CV of ADM to detect ctDNA. Figure 5 shows the CVs of (3.4 × 10−6 M) ADM in 0.1 M PBS solution in the presence of ctDNA with different concentrations. As it could be seen, with adding ctDNA into ADM solution, the peak currents of ADM gradually decreased, which is the similar with the previous report [12]. Furthermore, the reduction peak currents were linear with the concentrations of ctDNA in the range from 8.60 × 10−6 to 4.3 × 10−5 g·mL−1, the linear regression equation was \( {i_{pc}}\left( {{{10}^{ - 6}}A} \right) = 5.45--1.07{c_{ctDNA}}\left( {{{10}^{ - 5}} M} \right) \), the correlation coefficient (r) was 0.9855, which could be used to determine ctDNA.

Fig. 5
figure 5

CVs of 3.4 × 10−6 M ADM in 0.1 M PBS solution in the presence of ctDNA with different concentrations: 0 (a), 8.6 × 10−6 M (b), 1.7 × 10−5 M (c), 2.6 × 10−5 M (d), 3.4 × 10−5 M (e), 4.3 × 10−5 M (f)

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Conclusions

Herein, we have prepared an advanced Agnano/MWCNTs-COOH modified GCE and investigated the electrochemical behaviors of ADM at the modified electrode. The results showed that the modified electrode could effectively enhance the electrochemical response of ADM, and had high sensitivity for detection of ADM, the detection limit was 1.7 × 10−9 M. In addition, the sensor exhibited an excellent reproducibility and stability. Furthermore, CV signal of ADM can be applied to determine ctDNA.