Nanostructured carbon electrode modified with N-doped graphene quantum dots–chitosan nanocomposite: a sensitive electrochemical dopamine sensor

A highly selective and sensitive dopamine electrochemical sensor based on nitrogen-doped graphene quantum dots–chitosan nanocomposite-modified nanostructured screen printed carbon electrode is presented, for the first time. Graphene quantum dots were prepared via microwave-assisted hydrothermal reaction of glucose, and nitrogen doping was realized by introducing ammonia in the reaction mixture. Chitosan incorporation played a significant role towards the selectivity of the prepared sensor by hindering the ascorbic acid interference and enlarging the peak potential separation between dopamine and uric acid. The proposed sensor's performance was shown to be superior to several recently reported investigations. The as-prepared CS/N,GQDs@SPCE exhibited a high sensitivity (i.e. ca. 418 µA mM cm−2), a wide linear range i.e. (1–100 µM) and (100–200 µM) with excellent correlations (i.e. R2 = 0.999 and R2 = 1.000, respectively) and very low limit of detection (LOD = 0.145 µM) and limit of quantification (LOQ = 0.482 µM) based on S/N = 3 and 10, respectively. The applicability of the prepared sensor for real sample analysis was tested by the determination of dopamine in human urine in pH 7.0 PBS showing an approximately 100% recovery with RSD < 2% inferring both the practicability and reliability of CS/N,GQDs@SPCE. The proposed sensor is endowed with high reproducibility (i.e. RSD = ca. 3.61%), excellent repeatability (i.e. ca. 0.91% current change) and a long-term stability (i.e. ca. 94.5% retained activity).

will be assessed against previously reported works in the literature. Finally, real sample analysis will be carried out in order to judge the applicability of the presented sensor, and its repeatability, reproducibility and stability characteristics will be investigated.
All solutions were prepared with ultrapure water (18.2 MΩ cm) from a Milli-Q water purification system, Millipore (www.merckmillipore.com).
A screen printed three-electrode system (SPCE) from Orion High Technologies (www.orion-hitech. com) comprising a nanostructured carbon working electrode (φ = 4 mm), a Ag|AgCl reference electrode and a carbon counter electrode was employed in all electrochemical measurements.

Quantum dots preparation
In a typical synthesis protocol, 2 g glucose was dissolved in 20 ml of a four times diluted ammonia solution then transferred into a Discover SP microwave synthesizer, CEM Corp. (www.cem.com) and irradiated under 300 W for 5 min. The solution colour changes from colourless to light brown indicating the formation of nitrogen-doped graphene quantum dots which will be denoted as N,GQDs in this work. The same experiment was repeated in the absence of ammonia for comparison and the prepared graphene quantum dots are denoted as GQDs in this case.

Graphene quantum dots-chitosan nanocomposite preparation
An adequate amount of chitosan (CS) was dissolved in 1% acetic acid solution in order to prepare a 0.5% CS solution in 1% acetic acid, which was subsequently stirred for 2 h until a clear solution was obtained. Then 1 ml of this CS solution was mixed with 1 ml of the as-prepared N,GQDs solution and stirred for a further hour, resulting in the formation of CS/N,GQDs nanocomposite solution.

Electrochemical sensor preparation
The SPCE was rinsed with Milli-Q water, and then electrochemically cleaned by repetitive potential cycling in a 0.5 M H 2 SO 4 solution at 100 mV s −1 scan rate between −1 V and 1 V. The SCPE was then dried under high purity nitrogen atmosphere and 10 µl of the CS/N,GQDs nanocomposite was dropcasted onto the working electrode surface and dried for 1 h at 40°C. The obtained CS/N,GQDs@SPCE was thoroughly washed with a copious amount of Milli-Q water then dried under high purity nitrogen atmosphere.

Electrochemical measurements
A computer-controlled Autolab PGSTAT 128N potentiostat/galvanostat (www.metrohm-autolab.com) was used in all electrochemical experiments that were conducted in a miniaturized 4 ml electrochemical cell. All potentials are reported with respect to the Ag|AgCl reference electrode.
Cyclic voltammetry (CV) measurements were conducted in unstirred, air-saturated conditions and further specific experimental details will be given when appropriate. Electrochemical impedance spectroscopy (EIS) experiments were realized using the integrated FR32 frequency response analyser employing a single sine wave with 10 −2 V amplitude and a maximum integration time of 0.125 s while maintaining the potential at 200 mV and scanning the frequency in the interval 5 × 10 −2 Hz-10 5 Hz. As for differential pulse voltammetry (DPV) measurements, the potential was scanned at a rate of 50 mV s −1 with 50 mV modulation amplitude, 5 mV step potential and 50 ms modulation time.

Characterization techniques
Morphological characterization was conducted using a JEM-1400 transmission electron microscope (TEM), JEOL Co. (www.jeol.co.jp), operating at acceleration voltage of 98 kV. The spectroscopic characterization was done by recording the absorbance spectra with an Evolution 201 UV-visible spectrophotometer, Thermofisher Scientific Inc. (www.thermofisher.com), and the photoluminescence (PL) spectra were measured by means of an RF-5301PC spectrofluorophotometer from Shimadzu Scientific Instruments (www.ssi.shimadzu.com).

N,GQDs characterization
The prepared quantum dots showed a monodispersed, spherical shape with a size ranging between approximately 3 and 4 nm as revealed from the TEM image shown in figure 1 which is in the typical range of nitrogen-doped graphene quantum dots [49].
On the other hand, the formation of graphene quantum dots is further confirmed by UV-Vis spectra (cf. figure 2a) showing two absorbance peaks at 227 nm and 383 nm, typical of an aromatic system, associated to π → π * and n → π * electron transitions of C=C and C=O, respectively [50,51]. Moreover, the successful nitrogen incorporation into the graphene quantum dots matrix is also confirmed by inspection of the spectra shown in figure 2b, exhibiting three adsorption peaks. The first two situated at 207 nm and 271 nm are respectively ascribed to π /n → π * electron transitions of C=C and C=O while the third one occurring at 302 nm corresponds to the C=N π → π * electron transition [52,53].
It is very well reported in the literature that an enhanced photoluminescence of nitrogen-doped graphene quantum dots is exhibited compared with their nitrogen-free counterparts [54,55], therefore the prepared GQDs and N,GQDs PL spectra were recorded and results are shown in figure 3. The presented data are for the maximum emission that was obtained with an excitation wavelength λ ex = 450 nm. The drastically increased photoluminescence (blue line) confirms again the successful nitrogen doping of the prepared quantum dots. It is noteworthy that figure 3 shows a PL emission's 10 nm blue shift for the N,GQDs compared to GQDs (i.e. emission peak shift from 531 to 521 nm) attributable to the increased electron affinity upon incorporation of nitrogen atoms [56].

Electrochemical behaviour of the prepared CS/N,GQDs@SPCE
In the present work, the redox couple [Fe(CN) 6 ] 3−/4− was used as an electrochemical probe for monitoring the interfacial properties variations upon modification of bare SPCE and the preparation of the CS/N,GQDs@SPCE electrochemical sensor. A general overview is obtained from CV experiments (cf. figure 4) showing a clear enhancement of the electrochemical properties of the modified electrode. For instance, as compared to bare SPCE, the CS/N,GQDs@SPCE voltammogram shows a significant improvement of the anodic and cathodic peak currents with a very-close-to-unity ratio in addition to smaller peak separation owing to an improved electron transfer rate, a larger electroactive surface area and probably a better conductivity of the electrode surface.   A more effective electrochemical characterization was conducted by EIS and typical Nyquist plots are given in figure 5 showing two portions at high and low frequencies. The former (i.e. a semicircular shape) reveals the electron transfer limited process, while the latter (i.e. a linear branch) is related to the diffusion controlled process [57]. The experimental data were best fitted to the equivalent circuit given in the inset of figure 5 [58].
R s represents the solution resistance, R ct represents the charge transfer resistance which is inversely proportional to the electron transfer rate and corresponds to the diameter of the semicircular loop of the plot, C dl represents the double layer capacitance and W is a Warburg impedance used to fit the linear portion of the Nyquist plot and is an indication of the diffusion process. An eye-catching feature is the impressive decrease of the semicircle loop's diameter in the case of CS/N,GQDs@SPCE, which not only confirms the surface modification and successful attachment of the CS/N,GQDs nanocomposite but also reveals a significant increase of the charge transfer rate as a consequence of the great decrease of R ct . In this respect, the fitting results show a great decrease in charge transfer resistance from ca. 2631 Ω to ca. 159 Ω which goes in line with a tremendously increasing value of C dl from ca. 1.9 µF to 44 µF, confirming the excellent improvement of electrical conductivity. It is worth mentioning here that in the absence of N,GQDs, the modification of the SPCE with CS resulted in a significant improvement of the electrode conductivity lying in between that of the bare SPCE and CS/N,GQDs@SPCE (cf. CS @SPCE plot in figure 5). In this respect, the obtained values of R ct and C dl were 1637 Ω and 22 µF, respectively. Such results prove that the observed enhancement of the electrochemical properties of the prepared CS/N,GQDs@SPCE are indeed a combination of CS and N,GQDs induced effects.

Cyclic voltammetry behaviour of dopamine at the prepared CS/N,GQDs@SPCE
In the following, all electrochemical measurements were conducted in 0.1 M pH 7.0 PBS solution. Figure 6 shows   sight, this nanostructured electrode seems to perform quite well with the appearance of well-defined peaks for the oxidation and reduction of DA as per the reaction equation outlined in scheme 1. Dopamine is oxidized during the positive going scan to dopamine-o-quinone and the latter is reduced back to dopamine in the course of the negative going scan.
This nice-looking CV is completely altered upon addition of 100 µM uric acid (UA) and/or ascorbic acid (AA). Knowing that these are the most common species interfering with DA [59], this represents a serious drawback when it comes to DA sensing in body fluids. Although UA itself shows almost no redox behaviour in the present work conditions and AA shows insignificant oxidation current at the bare SPCE (cf. figure 6), their presence with DA results in a huge decrease of both oxidation and reduction currents which would lead to erroneous DA detection.
On the other hand, when SPCE was modified with the prepared CS/N,GQDs nanocomposite, a totally different behaviour was observed as displayed in figure 7. In detail, AA redox reaction is completely hindered against a clear improvement of DA both anodic and cathodic currents in addition to a nice peak separation of DA and UA. The observed change in electrochemical activities can be explained  3), AA (pK a = 4.1) and UA (pK a = 5.8) are negatively charged while DA (pK a = 8.9) is positively charged [60,61]. This would lead to a mutual electrostatic attraction between DA and CS in contrast to an electrostatic repulsion between CS-UA and CS-AA [61], the latter being perhaps stronger, most probably due to larger pK a values difference (i.e. 2.2 versus 0.5). (ii) A significantly different π-π interaction between the sp 2 conjugated carbon-carbon bonds in GQDs and their counterparts in DA, UA and AA. Arranging these π-π interactions in an increasing order of strength would be GQDs-AA < GQDs-UA < GQDs-DA according to the respective structures of these compounds. For instance, the strongest π-π interaction in the case of GQDs-DA is due to the phenyl moiety of DA while the weakest is expected for GQDs-AA since AA structure comprises less π bonds compared to UA [62]. This synergy is supported by comparison of the CVs of CS@SPE and CS/N,GQDs@SPCE shown in the inset of figure 7. We can clearly notice the significant enhancement of DA redox peaks at CS/N,GQDs@SPCE that are otherwise hardly noticeable in the case of CS@SPE due to interfering UA and AA. This comes in addition to a distinguishable oxidation peak separation of DA and UA.

Dopamine electrochemical sensing performance at the prepared CS/N,GQDs@SPCE
Further investigations were carried out in the quest for a possible electrochemical sensing application of the as-prepared CS/N,GQDs@SPCE. For this, DPV was used as a fast, effective and a more sensitive electrochemical technique compared to CV [63]. Figure 8 shows the DPV results of the as-prepared CS/N,GQDs@SPCE for the same electrolytic composition as in the previous section with a narrow focusing on the oxidative voltammograms only as per the discussed CV data. One can clearly notice the complete hindrance of AA oxidation current (i.e. nearly identical to the blank) and the very well defined peaks for both DA and UA oxidation that are clearly separated (i.e. E p = ca. 171 mV). Interestingly, when all three analytes are present together, DA peak current retained its original value (i.e. ca. 0.4% change) while the peak potential shifted to less anodic values against an opposite shift of UA peak resulting in ca. 46 mV enlargement of the peakto-peak separation (i.e. reaching ca. 217 mV). On the other hand, the peak current for UA oxidation decreased noticeably (i.e. ca. 12%) in the presence of interfering DA and AA. The obtained results reveal the high selectivity of the prepared system to DA, which is of prime importance for electrochemical sensing.
The second wing of the sensor is the sensitivity, which is evaluated through determining the relationship between the DA concentration and the DPV peak current in an extended concentration range. The obtained data are shown in figure 9 in the concentration range 1-200 µM, exhibiting a steady peak current increase with increasing DA concentrations. Plotting

Application to real sample analysis
The practicability of the prepared electrochemical sensor for the detection of DA in real samples was tested using urine samples from a healthy individuals. The collected samples were hundred-times  diluted in PBS solution to overcome the urine matrix effect interferences [12]. Initially, no DA was detected, then consecutive DA additions were performed and recovery percentages were calculated based on the determined DA concentrations. The results are summarized in table 2. The displayed results show very good recovery (i.e. approx. 100%) and very reasonable relative standard deviations (i.e. less than 2%), indicating a promising applicability with a good reliability of the prepared sensor for the determination of DA in real samples.
3.6. Reproducibility, repeatability and stability of the prepared CS/N,GQDs@SPCE sensor Using six different CS/N,GQDs@SPCE sensors revealed highly reproducible results with very reasonable relative standard deviation (i.e. RSD = ca. 3.61%). The prepared electrochemical sensor showed an excellent repeatability with only ca. 0.91% current change after twelve consecutive electrochemical runs, proving the reliability of the current signal.
We also investigated the stability and long-term reusability of the proposed dopamine sensor by monitoring its electrochemical activity over a one-month period. The results were very impressive as we found that the sensor retained ca. 94.5% of its peak current intensity.

Conclusion
In the present work, a highly selective dopamine electrochemical sensor was developed based on the modification of a nanostructured carbon screen printed electrode with a chitosan/nitrogen-doped graphene quantum dots nanocomposite. The successful preparation of N,GQDs was confirmed by TEM surface analysis and UV-Vis spectrophotometry and supported by the shown high photoluminescence compared to N-free GQDs. Chitosan addition showed a significant impact in avoiding the commonly reported interferences with ascorbic acid and uric acid. EIS confirmed the superior electrochemical properties of the prepared CS/N,GQDs@SPCE in comparison to bare SPCE. For instance there was a marked decrease of charge transfer resistance (i.e. from ca. 2631 Ω to ca. 159 Ω) and by consequence a promoted electron transfer kinetics. The high sensitivity (S = ca. 418 µA mM −1 cm −2 ) of the proposed sensor in addition to its low detection limit (LOD = 0.145 µM) and large dynamic range (1-200 µM) puts it in the forefront of the very recently developed dopamine sensors. In addition, real samples analysis showed very promising results based on DA detection in urine samples with high recovery and low relative standard deviation percentages (i.e. approximately 100% and less than 2%, respectively).
Data accessibility. All data relevant to this work are presented throughout the paper. Competing interests. The author has no competing interests. Funding. The author received no funding for this study.