Reduced Graphene Oxide and Gold Nanoparticles-Modified Electrochemical Aptasensor for Highly Sensitive Detection of Doxorubicin

Doxorubicin (DOX) is the most clinically important antibiotic in cancer treatment, but its severe cardiotoxicity and other side effects limit its clinical use. Therefore, monitoring DOX concentrations during therapy is essential to improve efficacy and reduce adverse effects. Here, we fabricated a sensitive electrochemical aptasensor for DOX detection. The sensor used gold wire as the working electrode and was modified with reduced graphene oxide (rGO)/gold nanoparticles (AuNPs) to improve the sensitivity. An aptamer was used as the recognition element for the DOX. The 5′ end of the aptamer was modified with a thiol group, and thus immobilized to the AuNPs, and the 3′ end was modified with methylene blue, which acts as the electron mediator. The combination between the aptamer and DOX would produce a binding-induced conformation, which changes the electron transfer rate, yielding a current change that correlates with the concentration of DOX. The aptasensor exhibited good linearity in the DOX concentration range of 0.3 μM to 6 μM, with a detection limit of 0.1 μM. In addition, the aptasensor was used for DOX detection in real samples and results, and showed good recovery. The proposed electrochemical aptasensor will provide a sensitive, fast, simple, and reliable new platform for detecting DOX.


Introduction
Cancer, also known as a malignant tumor, has become a severe public health problem and a primary cause of death worldwide with population growth and global aging [1][2][3]. The application of anticancer drugs is one of the most widely used therapeutic options [4,5]. Doxorubicin (DOX) is an anthracycline antibiotic with broad spectra of chemotherapeutic applications and anti-neoplastic applications [6][7][8], widely used in the treatment of lung cancer, liver cancer, Hodgkin lymphoma, acute leukemia, and many other types of cancer [9][10][11]. DOX facilitates cancer treatment through several mechanisms, such as the disruption of DNA replication by inserting DNA base pairs and the disruption of DNA repair by interacting with topoisomerase II [12][13][14][15][16]. However, the effects of DOX are not cancer cell-specific; it also affects the DNA functioning of normal cells, resulting in irreversible toxicity to organs such as the heart, brain, and kidneys, which can be lifethreatening in severe cases [17,18]. Based on the dangerous side effects of DOX and the individual differences of patients, monitoring DOX levels in patients' biological samples during treatment is vital to improve the effectiveness of therapy, reduce adverse effects, and promote personalized medicine [19][20][21].

Apparatus
We used Gamry Reference 600 electrochemical instruments (Philadelphia, PA, USA) to perform the chronocoulometry (CC) experiment. Other electrochemical experiments, including electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV) and square wave voltammetry (SWV), were carried out in an Autolab PGSTAT302N electrochemical workstation (Herisau, Switzerland). The deionized water used for the experiments was generated with the Michem ultrapure water apparatus (Chengdu, China) with a resistance of 18 MΩ/cm. Scanning electron microscope (SEM) images were obtained using a Hitachi S-3500 scanning electron microscope (Tokyo, Japan). A Hechuang KH2200E ultrasonic generator (Kunshan, China) was used in homogenizing the solution.
The 200 µm diameter gold wire was ordered from Alfa Aesar (Ward Hill, MA, USA). Heat-shrink polytetrafluoroethylene insulation was obtained from ZEUS (Branchburg Township, CA, USA).
The DOX aptamer was chosen according to prior reported works in the literature [52] and synthesized by Sangon Biotechnology (Shanghai, China) with the following sequence: 5 -SH-(CH 2 ) 6 -ACC-ATC-TGT-GTA-AGG-GGT-AAG-GGG-TGG-T-Methylene Blue-3 . Upon receipt, we diluted the aptamer concentration to 200 µM with TE buffer and stored it in 200 µL aliquots at −20 • C until use.

Fabrication of the Sensor Working Electrodes
Gold wire was cut into 3.5 cm lengths to make the sensor working electrode. The actual sensing window was 7 mm long and located at the front end of the gold wire. There was a 1.5 cm conductive part at the other end for the electrochemical instrument connection. The rest of the middle part was insulated with polytetrafluoroethylene tube. The insulated sensors were sonicated in 2 M NaOH solution, anhydrous ethanol solution, 2 M H 2 SO 4 solution, and deionized water, in turn, for 10 min to initially remove impurities from the electrode surface. After that, the electrode was electrochemically cleaned by CV in 0.5 M H 2 SO 4 solution. Electrochemical cleaning was performed on the gold wire at a scan rate of 100 mV s −1 over a voltage range of −0.35 V to 1.5 V for 8 to 10 cycles.

Preparation of rGO/AuNPs-Modified Electrode
The rGO/AuNPs composites were modified on the surface of the working electrode by a one-step electrochemical deposition technique to increase its surface area and obtain a larger response current. Our plating solution was obtained by mixing 2 mg mL −1 GO solution with 10 mg mL −1 HAuCl 4 solution at a volume ratio of 9:1 and ultrasonicating it for 30 min. Then, CV was conducted in the potential ranges of −1.5 to 0.6 V for 15 cycles at a scan rate of 50 mV s −1 .

Frabriction of DOX Aptasensor
The thiol group of the aptamer and the gold nanoparticles on the electrode formed a Au-S bond, thus fixing the aptamer to the electrode. Firstly, an aliquot of the DOX aptamer solution (200 µM, 2 µL) was thawed and then reduced with 10 µL of 100 µM TCEP solution for 1 h at room temperature to reduce the 5 -disulfide bone. Subsequently, the reduction-treated aptamer was diluted to 300 nM with PBS, and the rGO/AuNPs-modified electrode was immersed in the aptamer solution for 2 h at room temperature. Subsequently, the electrode was immersed in the MCH solution (20 mM) at 4 • C for 12 h to remove nonspecifically adsorbed DNA and produce a self-assembly well-aligned monolayer (SAM) of thiol modified aptamer. Then, the electrode was rinsed thoroughly with deionized water to remove residual MCH and stored in PBS at 4 • C for future use.

Electrochemical Measurements
A three-electrode system consisting of a nanomaterial-modified gold wire as the working electrode, a 0.6 mm diameter platinum electrode as the counter electrode, and a commercial Ag/AgCl (3 M KCl) electrode as the reference electrode was used in all electrochemical experiments. CC experiments used to calculate the electroactive areas of the working electrodes were carried out in 5 mM K 3 Fe(CN) 6 solution containing 0.1 M KCl as the supporting electrolyte, with a pulse width of 1 V and a pulse period of 1 s. The other CC experiment, conducted in 10 mM Tris-HCl buffer (pH = 7.4) in the absence and presence of 50 µM RuHex, with a pulse width of 0.5 V and a pulse period of 0.5 s, was used to measure aptamer surface density. EIS measurements were performed in a 5 mM K 3 Fe(CN) 6 /K 4 Fe(CN) 6 mixture containing 0.1 M KCl as the supporting electrolyte, with a frequency range from 10 5 Hz to 0.1 Hz and a sinusoidal voltage perturbation of 5 mV. SWV measurements were carried out to obtain the response of the aptasensor to different concentrations of DOX. The SWV curves were performed in the PBS solution (PH = 7.4) with a potential range of −0.5 V to 0 V, a potential step width of 1 mV, a pulse amplitude of 50 mV, and a frequency of 300 Hz. It was worth noting that the DOX and aptasensor were allowed to incubate for 20 min before SWV measurement to allow the sufficient binding of DOX and aptamer. All the above experiments were carried out at room temperature.

Characterization of rGO/AuNPs Nanomaterials and Constructed rGO/AuNPs/Apt Interface
The working electrodes were modified with rGO/AuNPs nanomaterials for the sensitive and fast detection of DOX. The schematic diagram of the modification of the working electrode was shown in Scheme 1.
The rGO/AuNPs were electrodeposited together on the electrode by CV. During the negative voltage scan, some oxygen-containing groups of GO in contact with the electrode were irreversibly reduced. The resultant rGO was less soluble, and thus directly attached to the electrode surface. At the same time, AuCl 4 − was also reduced to obtain AuNPs. After electrodeposition, the rGO/AuNPs composite was formed on the electrode surface, and its SEM images are shown in Figure 1. Figure 1a shows the overall morphology of the electrochemically deposited nanomaterials. In the figure, the rGO/AuNPs nanocomposite shows a rough porous-like structure, which greatly increased the specific surface area of the electrode. Figure 1b,c shows the enlarged morphology of rGO/AuNPs; the outer layer of rGO was muslin-like, and the AuNPs, appearing spherical, attached to the wrinkled structure formed by the underlying rGO. The SEM image of the bare gold electrode is shown in Figure S1a,b. The surface of the electrode was flat and smooth, without any material attached to it. The unique morphology of rGO/AuNPs can immobilize more aptamers and accelerate electron transfer, thus amplifying the current signal. The energy dispersive X-Ray spectrometer (EDS) of the rGO/AuNPs-modified electrode is provided in Figure S1c.
of rGO was muslin-like, and the AuNPs, appearing spherical, attached to the wrinkled structure formed by the underlying rGO. The SEM image of the bare gold electrode is shown in Figure S1a,b. The surface of the electrode was flat and smooth, without any material attached to it. The unique morphology of rGO/AuNPs can immobilize more aptamers and accelerate electron transfer, thus amplifying the current signal. The energy dispersive X-Ray spectrometer (EDS) of the rGO/AuNPs-modified electrode is provided in Figure S1c.

Scheme 1.
Step-by-step modification process of the electrochemical aptamer sensor, and the detection principle. Step-by-step modification process of the electrochemical aptamer sensor, and the detection principle.
of rGO was muslin-like, and the AuNPs, appearing spherical, attached to the wrinkled structure formed by the underlying rGO. The SEM image of the bare gold electrode is shown in Figure S1a,b. The surface of the electrode was flat and smooth, without any material attached to it. The unique morphology of rGO/AuNPs can immobilize more aptamers and accelerate electron transfer, thus amplifying the current signal. The energy dispersive X-Ray spectrometer (EDS) of the rGO/AuNPs-modified electrode is provided in Figure S1c.

Scheme 1.
Step-by-step modification process of the electrochemical aptamer sensor, and the detection principle.
where Q is the charge (C), n is the number of electrons transferred, F is the Faraday constant (96,485 C eq −1 ), A is the electrode area (cm 2 ), C is the molar concentration of the active species (mol cm −3 ), D is the diffusion coefficient (7.6 × 10 −6 cm 2 s −1 for 5 mM K 3 Fe(CN) 6 solution [53]), and t is time (s). There is a linear relation between Q and t 1/2 . According to the slope of the plot of Q vs. t 1/2 , we found that the electroactive areas of bare electrodes and rGO/AuNP modified electrodes were 0.049 cm 2 and 0.065 cm 2 , respectively.
where Q is the charge (C), n is the number of electrons transferred, F is the Faraday constant (96,485 C eq −1 ), A is the electrode area (cm 2 ), C is the molar concentration of the active species (mol cm −3 ), D is the diffusion coefficient (7.6 × 10 −6 cm 2 s −1 for 5 mM K3Fe(CN)6 solution [53]), and t is time (s). There is a linear relation between Q and t 1/2 . According to the slope of the plot of Q vs. t 1/2 , we found that the electroactive areas of bare electrodes and rGO/AuNP modified electrodes were 0.049 cm 2 and 0.065 cm 2 , respectively. EIS was used to study the impedance changes during the stepwise modification of the electrode surface. The EIS responses of bare, rGO/AuNPs-modified electrodes, rGO/AuNPs/Apt, and the rGO/AuNPs/Apt/MCH interface in the 5 mM K3Fe(CN)6/K4Fe(CN)6 mixture are shown in Figure 2b,c. The Nyquist plot in Figure 2b consists of a semicircle at the high frequency and a linear section at the low frequency. The semicircle in the high frequency region represents the charge transfer impedance (Rct) of the electrode. The Rct of the bare electrode and the rGO/AuNPs-modified electrode were 599.15 Ω and 58.24 Ω, respectively, indicating that the modification of nanomaterials enhanced the transfer of electrons between the electrode surface and electrolyte solution, thereby increasing the sensitivity of the sensor. When the rGO/AuNPs-modified electrode was successively incubated with DOX aptamer and MCH, the Rct increased to 415.93 Ω and 645.21 Ω, respectively, which was consistent with the principle, indicating that the sensor was successfully fabricated. The Bode plot of the electrode impedance was shown in Figure 2c. We focused on the impedance at 300 Hz, which was the frequency used at EIS was used to study the impedance changes during the stepwise modification of the electrode surface. The EIS responses of bare, rGO/AuNPs-modified electrodes, rGO/AuNPs/Apt, and the rGO/AuNPs/Apt/MCH interface in the 5 mM K 3 Fe(CN) 6 / K 4 Fe(CN) 6 mixture are shown in Figure 2b,c. The Nyquist plot in Figure 2b consists of a semicircle at the high frequency and a linear section at the low frequency. The semicircle in the high frequency region represents the charge transfer impedance (R ct ) of the electrode. The R ct of the bare electrode and the rGO/AuNPs-modified electrode were 599.15 Ω and 58.24 Ω, respectively, indicating that the modification of nanomaterials enhanced the transfer of electrons between the electrode surface and electrolyte solution, thereby increasing the sensitivity of the sensor. When the rGO/AuNPs-modified electrode was successively incubated with DOX aptamer and MCH, the Rct increased to 415.93 Ω and 645.21 Ω, respectively, which was consistent with the principle, indicating that the sensor was successfully fabricated. The Bode plot of the electrode impedance was shown in Figure 2c. We focused on the impedance at 300 Hz, which was the frequency used at SWV. The impedances of bare, rGO/AuNPs-modified electrodes, rGO/AuNPs/Apt and the rGO/AuNPs/Apt/MCH interface were 444.2 Ω, 83.38 Ω, 131.50 Ω, and 374.83 Ω, respectively, which were consistent with the changing trends of R ct , indicating the successful fabrication of the sensor from the other aspects.

Determination of Aptamer Surface Density
According to the previously reported literature [54,55], the CC measurements were performed using RuHex acting as a redox marker to determine the aptamer surface density. The general principle is as follows: the negatively charged DNA phosphate skeleton and the positively charged RuHex have electrostatic attraction, and the density of the aptamer can be calculated according to the amount of charge required for the reduction of RuHex absorbed on the electrode surface, with the following equation: where Γ apt is the aptamer surface density (molecules cm −2 ), Q ab is the charge required for the reduction of RuHex adsorbed on the electrode surface (C), N A is the Avogadro's number, n is the number of electrons transferred, F is the Faraday constant (96,485 C eq −1 ), A is the electroactive area of the electrode (cm 2 ), z is the charge of RuHex (z = 3), and m is the number of bases of the aptamer (m = 28). According to the difference between the intercept of the linearly fitted plot in the presence and absence of RuHex (Figure 2d), which refers to the Q ab , the aptamer surface density of the rGO/AuNPs-modified electrode was 2.37 × 10 13 molecules cm −2 .

Electrochemical Properties of the Aptasensor
The properties and stepwise modification of the working electrode were tested through SWV in PBS. Bare and rGO/AuNPs-modified electrodes had no response in the voltage range of −0.5 V to 0 V (Figure 3a). This was because MB had not been fixed to the electrode. After the aptamer was immobilized to the electrode, we observed a significant SWV response near −0.25 V, which was caused by the oxidation of MB modified on the aptamer (Figure 3b). At this time, despite having a high peak current, the current baseline was also very high. However, when the aptasensor was incubated with MCH afterwards, the peak SWV current of the sensor was reduced and the current baseline was also reduced with it. This may be due to the formation of a well-aligned SAM of the thiol-modified aptamers.
fabrication of the sensor from the other aspects.

Determination of Aptamer Surface Density
According to the previously reported literature [54,55], the CC measurements w performed using RuHex acting as a redox marker to determine the aptamer surface d sity. The general principle is as follows: the negatively charged DNA phosphate skele and the positively charged RuHex have electrostatic attraction, and the density of the tamer can be calculated according to the amount of charge required for the reductio RuHex absorbed on the electrode surface, with the following equation: where Гapt is the aptamer surface density (molecules cm −2 ), Qab is the charge required the reduction of RuHex adsorbed on the electrode surface (C), NA is the Avogadro's nu ber, n is the number of electrons transferred, F is the Faraday constant (96,485 C eq −1 is the electroactive area of the electrode (cm 2 ), z is the charge of RuHex (z = 3), and m the number of bases of the aptamer (m = 28). According to the difference between intercept of the linearly fitted plot in the presence and absence of RuHex (Figure which refers to the Qab, the aptamer surface density of the rGO/AuNPs-modified electr was 2.37 × 10 13 molecules cm −2 .

Electrochemical Properties of the Aptasensor
The properties and stepwise modification of the working electrode were tes through SWV in PBS. Bare and rGO/AuNPs-modified electrodes had no response in voltage range of −0.5 V to 0 V (Figure 3a). This was because MB had not been fixed to electrode. After the aptamer was immobilized to the electrode, we observed a signific SWV response near −0.25 V, which was caused by the oxidation of MB modified on aptamer (Figure 3b). At this time, despite having a high peak current, the current base was also very high. However, when the aptasensor was incubated with MCH afterwa the peak SWV current of the sensor was reduced and the current baseline was also duced with it. This may be due to the formation of a well-aligned SAM of the thiol-m fied aptamers. The detection principle of the sensor was shown in Scheme 1. When DOX was binding to the aptamer, the aptamer was in a "stretched" state and the MB at the en The detection principle of the sensor was shown in Scheme 1. When DOX was not binding to the aptamer, the aptamer was in a "stretched" state and the MB at the end of the aptamer was far away from the electrode surface. When the aptamer was bound to DOX, the conformation of the aptamer changed to form a "folded" state, so that the MB was close to the electrode surface, increasing the rates of electrons transferred from the MB to the electrode. When we used SWV to detect the DOX, we could obtain an increased peak Nanomaterials 2023, 13, 1223 8 of 13 current. As shown in Figure 3b, after combining with DOX the peak currents of the SWV response increased from 20.02 µA to 24.97 µA, in accordance with our detection principle.

Analytical Results of the DOX Aptasensor
We measured the SWV response of the aptasensor to standard DOX samples at different concentrations in PBS (PH = 7.4) to evaluate the detection performance of the aptasensor. The binding event of the aptamer to the target does not involve the transfer of electrons, which means that the current changes we detected arose exclusively from changes in electron transfer rates from MB immobilized at the end of the aptamer to the electrode. As shown in Figure 4a, the peak current of the SWV response rose with increasing DOX concentration, consistent with our detection principle. To eliminate the differences between sensor batches, we normalized the SWV peak current and obtained the relationship between the normalized current and the DOX concentration. As shown in Figure 4b, the normalized SWV peak current (I norm ) and log concentration of DOX (C DOX ) showed good linearity over the range of 0.3 µM to 6 µM for DOX, and the linear response can be described as I norm = 1.298 + 0.385 Lg C DOX (µM). The correlation coefficient was 0.996, and the limit of detection was 1 µM. We fitted the sensor's signal gain to the Langmuir isotherm and calculated the apparent dissociation constant to be 2.55 uM. A list of recently developed electrochemical sensors for DOX detection is presented in Table 1. Our sensor had a more balanced performance, and its detection range basically covered the therapeutic range of drugs in human blood [56].
was close to the electrode surface, increasing the rates of electrons transferred from the MB to the electrode. When we used SWV to detect the DOX, we could obtain an increased peak current. As shown in Figure 3b, after combining with DOX the peak currents of the SWV response increased from 20.02 µA to 24.97 µA, in accordance with our detection principle.

Analytical Results of the DOX Aptasensor
We measured the SWV response of the aptasensor to standard DOX samples at dif ferent concentrations in PBS (PH = 7.4) to evaluate the detection performance of the ap tasensor. The binding event of the aptamer to the target does not involve the transfer o electrons, which means that the current changes we detected arose exclusively from changes in electron transfer rates from MB immobilized at the end of the aptamer to the electrode. As shown in Figure 4a, the peak current of the SWV response rose with increas ing DOX concentration, consistent with our detection principle. To eliminate the differ ences between sensor batches, we normalized the SWV peak current and obtained the relationship between the normalized current and the DOX concentration. As shown in Figure 4b, the normalized SWV peak current (Inorm) and log concentration of DOX (CDOX showed good linearity over the range of 0.3 µM to 6 µM for DOX, and the linear response can be described as Inorm = 1.298 + 0.385 Lg CDOX (µM). The correlation coefficient was 0.996 and the limit of detection was 1 µM. We fitted the sensor's signal gain to the Langmuir isotherm and calculated the apparent dissociation constant to be 2.55 uM. A list of recently developed electrochemical sensors for DOX detection is presented in Table 1. Our sensor had a more balanced performance, and its detection range basically covered the therapeu tic range of drugs in human blood [56].

Evaluation of Repeatability, Selectivity and Stability of the Aptasensor
Reproducibility is an essential evaluation indicator of sensor performance. We evaluated the reproducibility of the aptasensor by measuring the SWV response of three independent sensors in solutions containing DOX. The responses of the aptasensors in 1 µM and 2 µM DOX are shown in Figure 5a. Their normalized peak currents were 1.290, 1.289, and 1.292 for 1 µM DOX, and 1.423, 1.430, and 1.446 for 2 µM DOX, respectively. Based on the above experimental results, the coefficients of variation were calculated to be 0.11% and 0.81%, respectively, indicating the excellent repeatability of the aptasensor. Gold wire/rGO/AuNPs/aptamer 300-6000 100 This work CPE: carbon paste electrode; MWCNTs: multiwalled carbon nanotubes; GCE: glassy carbon electrode; PR: polyrhodanine; GO: graphene oxide; K: kombucha solvent; GQD: graphene quantum dot; BBD: boron-doped diamond electrode.

Evaluation of Repeatability, Selectivity and Stability of the Aptasensor
Reproducibility is an essential evaluation indicator of sensor performance. We evaluated the reproducibility of the aptasensor by measuring the SWV response of three independent sensors in solutions containing DOX. The responses of the aptasensors in 1 µM and 2 µM DOX are shown in Figure 5a. Their normalized peak currents were 1.290, 1.289, and 1.292 for 1 µM DOX, and 1.423, 1.430, and 1.446 for 2 µM DOX, respectively. Based on the above experimental results, the coefficients of variation were calculated to be 0.11% and 0.81%, respectively, indicating the excellent repeatability of the aptasensor.
Selectivity is another important characteristic of the aptasensor. We measured the SWV response of the sensor to 50 µM of TET, DTIC, CPL, and 4 µM of DOX, respectively, to evaluate the selectivity of the sensor. The results are shown in Figure 5b. The normalized current responses of the sensor to the interferents were 3.37%, −10.96%, and −11.48%, respectively, which were much smaller than 49.55% of DOX, indicating the excellent selectivity of the aptasensor.
In addition, we also investigated the stability of this aptasensor. The sensor was soaked in PBS (PH = 7.4) and stored in a refrigerator at 4 °C. We measured the aptasensor's SWV response in PBS solution (PH = 7.4) weekly. The results are shown in Figure 5c. After three weeks, compared with the first week, the sensor's SWV peak current decreased to 80.32% of the original.  Selectivity is another important characteristic of the aptasensor. We measured the SWV response of the sensor to 50 µM of TET, DTIC, CPL, and 4 µM of DOX, respectively, to evaluate the selectivity of the sensor. The results are shown in Figure 5b. The normalized current responses of the sensor to the interferents were 3.37%, −10.96%, and −11.48%, respectively, which were much smaller than 49.55% of DOX, indicating the excellent selectivity of the aptasensor.
In addition, we also investigated the stability of this aptasensor. The sensor was soaked in PBS (PH = 7.4) and stored in a refrigerator at 4 • C. We measured the aptasensor's SWV response in PBS solution (PH = 7.4) weekly. The results are shown in Figure 5c. After three weeks, compared with the first week, the sensor's SWV peak current decreased to 80.32% of the original.

Real Sample Analysis
Normal human serum samples were used to evaluate the performance of the aptasensor in real samples. The serum was diluted 10 times with PBS (PH = 7.4), and the DOX sample at a determined concentration was added. The spiked human serum samples were measured using the proposed aptasensors. As is shown in Table 2, the recoveries of the sensors in human serum samples ranged from 94.8% to 109.0%, indicating that the aptasensor has the potential for clinical application.

Conclusions
In this work, we fabricated an rGO/AuNPs-modified aptamer sensor for the electrochemical detection of DOX. Compared with previous research, this work has the following highlights: (1) rGO/AuNPs were successfully modified on the working electrode by onestep electrochemical deposition. They enlarged the surface area and decreased the electron transfer impedance of the electrode, thus improving the sensitivity of the sensor. (2) The signal of the sensor came from the conformational change of DOX binding to the aptamer, so it can reduce the interference caused by the non-specific adsorption of other substances.
(3) No other reagents needed to be added during the assay, which makes the operation simple and convenient. We believe that this new aptamer sensor can provide a timely, convenient, simple, and sensitive platform for the detection of DOX.

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