A label-free electrochemical platform for the highly sensitive detection of hepatitis B virus DNA using graphene quantum dots

Based on the strong interaction between single-stranded DNA and graphene material, we have designed a simple but smart electrochemical platform to detect HBV-DNA by using a graphene quantum dot (GQD) modified glassy carbon electrode coupled with specific sequence DNA molecules as probes. The probe DNA is designed to be complementary to the HBV-DNA, when the probe DNA is strongly bound to the surface of the GQD modified electrode the transfer of an electron from the electrode to the electrochemically active species K3[Fe(CN)6] will become difficult. Nevertheless, if the target HBV-DNA is found in the test solution, the probe DNA will bind with the target HBV-DNA instead of GQDs. As a result, the obtained peak currents of K3[Fe(CN)6] will have a different degree of increase with the different concentrations of the target HBV-DNA. In particular, the proposed sensor exhibits high sensitivity with a detection limit of 1 nM, and the linear detection range is from 10 nM to 500 nM. Additionally, the sensor could be used in detecting other probe DNA, which may have potential applications in the future.


Introduction
As the main human pathogen, hepatitis B virus (HBV) is very harmful to humans and easily causes infection. There are surveys showing that there are 3 million HBV carriers worldwide. Liver inammation is a common phenomenon in most patients, and some even get cirrhosis and liver cancer. At present, the most reliable and direct marker of HBV replication activity is HBV-DNA. 1 As a result, in order to understand the degree of infection of patients with HBV and evaluate the efficacy of antiviral therapy, it is of great signicance to make quantitative detection of HBV-DNA a realization. At present, many methods have been used to detect some specic genes or gene mutations, such as biological detection bar code technology, the signal amplication of DNA testing technology, 2 etc. Unfortunately, such methods have some drawbacks such as low sensitivity, needing expensive instruments or complex pretreatments, consumption of large sample volumes, time consuming, etc. 3 In contrast, electrochemical methods have outstanding advantages including fast and low-cost, effective, simplicity, quantitative application and possibility of miniaturization. Consequently, it is helpful to nd a simple, quick and sensitive electrochemical method to detect the DNA sequence of HBV.
Recently, based on their unique properties and excellent chemical sensing performances, a large number of nanomaterials have been used to prepare simple biological nanosensors. Graphene quantum dots (GQDs), since found in 2008 by Dai, it has become signicant in research area as a new kind of carbon-based nanomaterial in recent years. 4 It inherits the excellent properties of grapheme such as strength, large specic surface area. 5 Combined with the advantages of quantum dots as the quantum conned effect, size effect and edge effect, GQDs also shows many fascinating properties, such as desirable biocompatibility, low cytotoxicity, excellent solubility, stable photoluminescence, thus, making them potential application in the areas of sensor systems and bio-imaging. At present, there are mainly two developed routes for GQDs synthesis, 'top-down' and 'bottom-up' methods. Top-down methods are different physical or chemical approaches to make bulk carbon materials into GQDs, including ionic liquid assisted grinding, 6 hydrothermal, 7,8 chemical ablation, 9 photofenton oxidation, 10 oxygen plasma treatment, electrochemical oxidation 11 and etc. On the contrary, bottom-up methods mainly converse suitable organic precursors to GQDs by the ways such as microwave, 12 solvothermal treatment, 13-15 thermal pyrolysis, 16,17 etc. Hence, there are obvious advantages of bottom-up methods over top-down methods, because the composition and physical properties of GQDs can be easily adjusted by careful selecting precursors from diversied organic compounds as well as the carbonization conditions. 16,17 In this contribution, an ultra-sensitive label-free electrochemical biosensor using GQDs for detecting HBV-DNA was made. GQDs was synthesized by a safe and simple bottom-up methods, and can be directly modied onto the surface of glassy carbon electrode (GCE) because of the physical adsorption with van der Waals forces. 18,19 And then we chose K 3 [Fe(CN) 6 ] as the electroactive indicator to detect and monitor what changes were happening on the surface of electrode. 20 The changes caused by DNA immobilization and hybridization were detected by directly monitoring the differential pulse voltammetric (DPV) response. Compared with other biosensors, such a sensor is quite convenient, safe and cheap because there is no uorophore labelling or enzyme amplication step, and easy to fabricate. In addition, the high sensitivity is also a remarkable advantage.

Synthesis of GQDs by 'bottom-up' methods
The GQDs were synthesized by pyrolyzing citric acid (CA) based on the previous report, 17 get 2 g of citric acid into a beaker, then heated to 200 C and last for 20 min to get CA melt and pyrolyzed. From the color of the liquid, we can judge whether the formation of GQDs or not. Keep heating until the color of the liquid turned from colorless to faint yellow. Then, dissolved the liquid into 50 mL of 0.25 mol L À1 NaOH solutions and continuous stirring for about 30 min. At last, 0.25 mol L À1 NaOH solution was used to obtained faintly acid GQD solution.

Characterization of GQDs
Transmission electron microscopy (TEM) was performed with the H-9000 (JEOL Ltd. Japan), and high-resolution transmission electron microscopy (HRTEM) measurements was performed with JEM-2100F (JEOL Ltd. Japan) transmission electron microscopes. X-ray photo electron spectroscopy (XPS) was obtained with aKratos Axis Ultra-DLD XPS System (Kratos Analytical Ltd., Japan). Fourier transform infrared (FTIR) Spectra were measured with an FTIR spectrophotometer (Thermo Scientic Nicolet iN 10MX).

Preparation of GQDs modied GCE
We use 0.3 and 0.05 mm alumina slurries to polish the GCE sequentially. And then went through ultrasonic processing with ethyl alcohol and deionized water for 1 min, respectively. Drip 20.0 mL of the GQDs solution on the polished GCE surface and dried overnight at the room temperature to obtain a uniform lm. The GQD modied GCE electrode was nally prepared by thoroughly rinsed with pure water. For comparison, the GCE without GQDs was also prepared and tested.  6 ] at À0.7 V for 300 s to release the double-stranded DNA (dsDNA) which is produced by hybridization of pDNA and tDNA. [21][22][23][24] Then electrochemical measurements were started on the electrode.

Immobilization and hybridization of DNA
In this paper, we used the DNA oligonucleotides which are synthesized by Shanghai Sangon Biological Engineering Technological Co. Ltd (Shanghai, China). And in this experiment, the HBV-DNA base sequences we used are as below: Probe

Electrochemical measurements
The machine we used to perform electrochemical measurements was a CHI 760E electrochemical workstation (Shanghai CH Instrument Company, China) which had a conventional three-electrode system. A reference electrode (SCE) was the saturated calomel, the auxiliary electrode was a platinum wire and the GQDs modied GCE as the working electrode.
Cyclic voltammetry (CV) experiments were taken at a scan rate of 0.10 V s À1 from 0.8 V to À0.2 V and recorded in KCl solution which contained 0.2 M K 3 [Fe(CN) 6 ]. Also, in the same solution, we made differential pulse voltammetry (DPV) experiments with a pulse width of 0.05 s, a pulse period of 0.5 s, and a pulse amplitude of 0.05 V. Before DPV scanning, the electrode underwent a process of preconditioning at À0.7 V for 300 s. All experiments mentioned above, were carried out under the room temperature.

Results and discussion
The GQDs were obtained by 'bottom-up' methods which carbonized CA at an appropriate degree. During the pyrolysis In Fig. 2a, we can see the TEM image with the low magnication and the corresponding size distribution histogram of GQDs. The as-prepared GQDs are with mean diameters of 2.6 nm and are well dispersed in narrow size distributions. In Fig. 2b, we can see a representative high resolution TEM image of an individual GQDs. The distinct crystal lattice indicates the crystallinity of the GQD, and the lattice parameter of 0.33 and 0.25 nm represents the (002) and (1120) lattice fringe of graphene, respectively.
As shown in Fig. 3a, the XPS spectra illustrates that the GQDs mainly consist of carbon (at ca. 284.8 eV) and oxygen (at ca. 531.4 eV). As shown in C1s high-resolution XPS spectra of GQDs (Fig. 3b), large number of oxygen functional groups can be found, which indicated the incomplete carbonization during the pyrolysis of citric acid. 17 Note that C]O was the main oxygen functional groups, which was formed by CA molecules self-assembled and carboxyl group on the edges of GQDs.
To check whether the probe DNA can be immobilized onto the GQDs, FT-IR spectra were also measured to conrm the surface functional groups of GQDs and pDNA-GQDs (Fig. 4). As shown in the FT-IR spectrum of GQDs, the absorption band at 3460 and 1721 cm À1 were attributed to the stretching vibration of O-H and     We evaluated the feasibility of combining the GQDs and GCE as an electrochemical sensing platform by using K 3 [Fe(CN) 6 ] as the electrochemical active species. The cyclic voltammetry (CV) was carried out in above KCl solution which contained 0.2 M K 3 [Fe(CN) 6 ] solution and was used to characterize the modication of the GCE and the DNA xation on the modied GCE. Fig. 5 shows the CV curves of GQDs modied GCE, bare GCE, and 100 mM probe DNA immobilized GQDs/GCE before and aer adding tDNA, which demonstrates that the GQDs were absorbed on the surface of the GCE, and the pDNA was immobilized onto the surface of the GQD modied electrode successfully. The electron transfer between the electrode and the electro-active species K 3 [Fe(CN) 6 ] will be inhibited due to the electrostatic repulsion which is caused by bind of immobilized pDNA and GQD lm on the GCE electrode surface. 25 Thus, we can observe an evidently decreased peak current of K 3 [Fe(CN) 6 ] obtained at the pDNA-GQD modied electrode. However, aer adding the target HBV-DNA (tDNA), the probe DNA will bind with the target instead of GQDs. For instance, the electrostatic repulsion to the electroactive species K 3 [Fe(CN) 6 ] resulted from the immobilized pDNA will be removed. As a result, the obtained peak currents of K 3 [Fe(CN) 6 ] will have a recovery with the different concentration of the target HBV-DNA. Fig. 6 exhibits the comparison of DPV curves. It shows the DPV signals of the GQDs modied GCE aer different concentration (1 nM to 500 nM, 10 mL) HBV-DNA hybridizing with the pDNA. The DPV experiment was made in the above KCl solution which contains 0.2 M K 3 [Fe(CN) 6 ]. As shown in Fig. 6a, when the concentration of probe DNA is xed (500 nM), the peak current of K 3 [Fe(CN) 6 ] obtained at the modied electrode increases along with the concentration of HBV-DNA, which illustrates that the amount of remaining pDNA had decreased, because the dsDNA formed by the hybridization of pDNA and tDNA had been released from the electrode surface by the electrochemical pretreatment (À0.7 V, 300 s). The more HBV-DNA molecules are in the test solution, the more dsDNA formed and escaped from the GQDs modied electrode, thus higher electrochemical response can be observed. The linear relationship between the peak current and the concentration of HBV-DNA was shown in Fig. 6b. As the plot of 1 nM deected too much, the linear relationship used the data from 10 nM to 500 nM. It could be expressed as y ¼ 0.55x + 0.67, where R 2 ¼ 0.99. The established method for HBV-DNA detection has a broad linear range of 10- Fig. 7 Differential pulse voltammetry obtained at an unmodified GCE electrode, the unmodified electrode further immobilized with pDNA, and the cases that pDNA has been incubated with HBV-DNA (tDNA).  Pyrolysis method Electrochemistry HBV-DNA 10-500 nM 1 nM This study 500 nM with a detection limit of 1 nM DNA. And the limit of quantication (LOQ) is 10 nM. In order to conrm the role of GQDs, we tested the DPV curves based on the GCE without the GQDs modied. As we can see in Fig. 7, aer adding HBV-DNA, there is no increase of the peak currents of K 3 [Fe(CN) 6 ]. Compared with the DPV curves based on the GQDs modied GCE, we can know that GQDs can immobilized DNA and allow double-stranded DNA to escape from electrode in time. Without GQDs modied, bare GCE could not effectively adsorb probe DNA and detect HBV-DNA. Fig. 8 compares three percentages of DIpc/Ipc in different ssDNA hybridization with the probe DNA, including the target complementary DNA (tDNA), one-middle-base mismatched DNA (1MT DNA), and non-complementary DNA (ncDNA) sequence. In order to simulate the real situation, this selectivity experiment was based on the actual situation of the blood in the ratio of the target complementary DNA and non-complementary DNA sequence. From the gure we can see that the highest DIpc is the complementary DNA sequence followed by 1MT DNA and the non-complementary DNA successively. Compared with the DIpc of the complementary DNA, the signal increasement of the non-complementary DNA was much lower and could be neglected. From this result we can conclude that the specicity of the DNA sensor is high selectivity and could distinguish most mismatched DNA sequences.
This DNA detection approach possesses a superior linear range and detection limit which is remarkable compared with that of relative previously report. As shown in Table 1, these results show that the GQDs modied GCE has an ultrahigh sensitivity for the DNA biosensing.

Conclusions
In summary, a new ultra-sensitive and effective label-free electrochemical HBV-DNA sensing platform has been established based on the prepared GQDs. The material was made by pyrolysis method, which is quite convenient, safe and cheap. The limit of detection could get 1 nM and detection range is from the 10 nM to 500 nM. It provides a universal method which can be used for DNA detection. This sensing system can distinguish complementary and mismatched nucleic acid sequences with high sensitivity and good reproducibility. Also, the proposed sensor could be used in detecting other DNA. Since all the materials involved in the sensing system are of excellent biocompatibility, it is expected that this DNA detection method would be used in vitro and can be extended for the detection of more molecules.

Conflicts of interest
There are no conicts to declare.