The layer-by-layer assembled ERGO+/ERGO− multilayer modified electrode for sensitive detection of dopamine

Graphene materials represented by graphene oxide (GO) have been widely regarded as functional coatings or films to modify surface of the electrode for detecting dopamine molecules. However, interfacial material properties for detection sensitivity, film stability, and applicability to electrodes are still highly desired. Herein, we first present a screen-printing carbon electrode (SPCE) coated with an electrochemically reduced layer-by-layer (LbL) assembled multilayer driven by an electrostatic interaction between positively charged polyethyleneimine-modified GO with amine groups (ERGO+) and negatively charged carboxyl-functionalized GO (ERGO−), which is briefly described as (ERGO+/ERGO−)n/SPCE. Firstly, without using conventional glassy carbon and gold electrodes, SPCE was tried to make coatings adapt to more flexible and unstable electrodes, simultaneously guaranteeing higher detection performance. Secondly, although a variety of electrochemical sensors such as GO−/SPCE and ERGO−/SPCE were obtained through the drop-casting technique, as-prepared (ERGO+/ERGO−)n/SPCE showed much higher electrocatalytic activities with enhanced peak current signals and reduced charge transfer resistance. Finally, excellent electrochemical properties and sensing performances of the (ERGO+/ERGO−)n/SPCE sensor for detection of dopamine were demonstrated, especially having a linear range of 1 μM to 1000 μM. Meanwhile, the detection limit is as low as 0.39 μM and S/N is equal to 3. The present work offers a potential direction to develop GO modified electrodes for sensitive biomolecular detection.


Introduction
Electrochemical biosensor is a kind of biosensor that detects analyte through converting the concentration information of target molecules into the electrical signals [1].Due to the huge sensing and manufacturing features, such as superior selectivity, rapid analysis, high sensitivity, low cost, easy integration, and device miniaturization, it has a wide range of applications to biomedical engineering [2], food safety [3], and environmental monitoring [4].Generally, the electrode with functional interfaces has been regarded as a main component of the prepared electrochemical biosensors, thereby playing an important role in electrochemical reaction and electrical signal transmission [5].However, the electrocatalytic or electron transfer performance for bare electrodes was significantly limited, thereby hindering practical applications of electrochemical biosensors [6].In order to solve this problem, versatile functional nanomaterials endowing the electrode with sensing properties by means of surface modification have been of great interest [7].Due to the extremely small nanomaterials or molecules exhibiting the ultra-high specific surface area, unique physicochemical properties like high electrocatalytic activity can be performed.Therefore, using nanomaterials to modify the electrode surface can make the best of size effect to enlarge surface area and significantly enhance electrocatalytic performance [8].
Carbon-based materials and metal nanoparticles were commonly used, mainly involving carbon nanotubes, graphene oxide (GO), graphene, carbon quantum dots, gold nanoparticles, and platinum nanoparticles.In particular, two-dimensional GO not only has thin thickness and large specific surface area, but also has high biocompatibility, good water solubility, and high electrochemical activity due to special structures [9][10][11].Although the conductivity of GO was greatly reduced due to the rupture of large π bonds in the six-membered carbon ring, this issue can be readily addressed by electrochemically reducing GO by removing oxygencontaining groups by cyclic voltammetry (CV) [12,13].In addition, GO can also use π-π stacking or electrostatic interaction to adsorb various aromatic ring molecules and immobilize biologically active enzymes.Thus, electrochemical biosensors based on GO and electro-reduced graphene oxide (ERGO) were extensively utilized to monitor various representative molecules, including glucose [14], hydrogen peroxide (H 2 O 2 ) [15], dopamine (DA) [16], ascorbic acid (AA) [17], and uric acid (UA) [18], exhibiting high performance for molecular detection.However, GO films or coatings modified electrodes by a conventional drop casting technique was widely created and moved to practical detection, which was limited to some degree [19,20].Specifically, as-prepared GO films can be readily detached from the electrode surface or decomposed due to the weak intermolecular interactions.Also, morphology, thickness, and other interfacial parameters cannot be accurately controlled as well.Meanwhile, building blocks and structures of the nano-films can affect the catalytic performance of electrochemical biosensors [21].Therefore, except a drop casting method for preparing GO-based nano-films to modify electrodes for detection of dopamine, promising techniques were urgent to be developed to overcome issues on poor stability, poor reproducibility, and low sensitivity.
For decades, layer-by-layer (LBL) self-assembly for constructing nano-films has brought hope to solve this problem [22,23].The principle of LBL technology is to use strong interaction forces (such as chemical bonds, etc.) or weak interaction forces (such as electrostatic attraction, hydrogen bond, coordination bond, etc.) between groups on a suitable template to drive the target compound to spontaneously form a film with complete structures, stable performances, and unique functions on the template [24][25][26][27].The LBL technology has no limitation on the substrate, and can achieve controllable surface parameters and film thickness.In addition, with the advantages of simple coating process, low cost, scalability, and green fabrication, it has been widely used to construct multifunctional nanomaterial composite films recently.And the self-assembled nano-films for building multifunctional surfaces/interfaces of electrodes can be well applied, thus detection performance of the electrochemical biosensor can be greatly improved [28][29][30].
In the present work, LBL-based multilayer nano-films containing aminated graphene oxide (GO+) and single-layer graphene oxide (GO−) were successfully prepared on a commercially available screen-printing carbon electrode (SPCE).Then multilayered reduced GO nano-films modified SPCE was simply fabricated integrating an electrochemical process for the first time, which was briefly described as (ERGO+/ERGO−) n /SPCE.The nano-films were formed through intermolecular electrostatic interactions between GO+ with positive amine groups and GO− with negative carboxyl groups, and this method has not been reported before for sensitive detection of dopamine.The SPCE used in this work not only has good sensing performance, but also does not require complicated pre-treatment process, and has the advantage of low production cost, low sample demand, high sensitivity, good repeatability, and simple modification of the electrode surface.It can be discarded after one use, which avoided sample contamination caused by multiple uses [31,32].In contrast to bare glassy carbon electrode (GCE) and drop-coated ERGO/SPCE, (ERGO+/ERGO−) n /SPCE can exhibit higher electrocatalytic activities with enhanced peak current signals and the decreased charge transfer resistance, as well as higher stability and reproducibility stability.That indicated that electrochemical sensors with multilayers film on the electrode surface by the LBL technique exhibit better characteristics than the sensors based on the same materials-coated electrode by the conventional drop casting.In addition, (ERGO+/ERGO−) n multilayer films-based electrochemical sensors were used for dopamine (DA) detection with high sensitivity.

Experimental
2.1.Materials GO+ suspension, GO− suspension, AA, DA, and UA can be commercially available from Nanjing XFNANO and Sigma, respectively.PBS buffer was from Shanghai Sangon.Other reagents can be available commercially as well or self-prepared.
2.2.Preparation of modified SPCE by (GO+/GO−) n and GO nano-films First, the 16-channel SPCE was treated with plasma to make its surface with −OH, so that the bare electrode surface was negatively charged.Then the GO+ suspension (3 μl) with a pH of 6.0 was dripped on the 16-channel SPCE surface by a drop method for 10 min, then treated with water rinsing and nitrogen drying.Subsequently, GO− suspension (3 μl) with a pH of 6.0 was dropped on the 16-channel SPCE surface for10 min, and washed with ultrapure water, dried with nitrogen at last.Repeat the above process to obtain multilayer GO film modified SPCE, denoted as (GO+/GO−) n/ SPCE, which was driven by intermolecular electrostatic interactions between GO+ with positive amine groups and GO− with negative carboxyl groups.The preparation process of GO modified electrode is to drop 5 μl of GO− suspension with pH 6.0 on a 16-channel SPCE and dry it naturally, which was denoted as GO/SPCE.And use scanning electron microscope (SEM) for characterization.

Preparation of modified SPCE by (ERGO+/ERGO−) n and ERGO nano-films
In this experiment, electrochemical reduction was carried out to prepare (ERGO+/ERGO−) n /SPCE and ERGO/SPCE by immersing (GO+/GO−) n /SPCE and GO/SPCE in 0.1 M PBS (pH 7.0), then cyclic sweeping for 25 cycles was conducted with potential from −1.1 V to 0.7 V and scanning rate of 100 mV s −1 .

DA detection using electrochemical sensing
To examine electrocatalytic performance of 16-channel SPCE coated with electrochemically reduced graphene oxide multilayer nano-films, cyclic voltammetry (CV) was applied to measure electrochemical features of bare SPCE, GO/SPCE, (GO+/GO−) n /SPCE, ERGO/SPCE, and (ERGO+/ERGO−) n /SPCE in 5 mM K 3 [Fe(CN )6 ] 3−/4− and 0.1 M KCl.Making a comparison among pristine carbon electrodes, GO/SPCE, (GO+/GO−) n /SPCE, and ERGO/SPCE, electrochemical properties of (ERGO+/ERGO−) n /SPCE were deeply examined.Then electrocatalytic measurements for dopamine solutions with different concentrations were conducted based on (ERGO+/ERGO−) n /SPCE.The influence of film concentration of GO+ and GO− and number of assembled layers on the electrocatalytic performance of (ERGO+/ERGO−) n /SPCE was investigated.And the influence of pH value of PBS buffer and scanning rate on the detection of DA on the (ERGO+/ERGO−) n multilayer film modified electrode was figured out as well.CV test parameters were determined as −0.3 V initial potential, 0.6 V maximum potential, −0.3 V minimum potential, 100 mV/s scan rate, and 0.005 V sampling interval.

Characterization
The surface topography of all samples was measured by SEM (Regulus 8100).UV-vis spectra were acquired on a microscope slide using a UV-vis spectrophotometer (DR 6000), which can exhibit thickness growth of the composite nano-films.Electrochemical tests were all conducted using a CHIE 760 electrochemical workstation (China) and HSBS16X electrochemical workstation (China).SPCE was composed of an AgCl reference electrode, an auxiliary carbon electrode, and the bare carbon electrode or the working ERGO+/ERGO− multilayer films modified carbon electrode.

Results and discussion
3.1.Preparation of (ERGO+/ERGO−) n /SPCE for biosensing As illustrated in figure 1, driven by an intermolecular electrostatic interaction between GO+ with positive amine groups and GO− with negative hydroxy groups, modified layer-by-layer multilayers can be formed on a SPCE substrate though an alternating deposition process of simple drop casting.After a number of absorption cycles, unreduced (GO+/GO−)n nano-films as a functional coating with designed thickness can be obtained for the further electroreduction treatment, thereby making (GO+/GO−)n to be (ERGO+/ERGO−)n with high electrocatalytic properties for sensitive dopamine detection.
Surface morphologies of bare electrodes, ERGO films modified SPCE, and (ERGO+/ERGO−) n multilayer films modified SPCE were displayed using SEM images.In figure 2(a), it is obviously found that bare SPCE surfaces are highly rough.After modification of self-assembled films composed of ERGO+ and ERGO−, the modified electrode surfaces were covered with a great many raised wrinkles as proved in figure 2(b), which indicated that ERGO multilayer films have been successfully fabricated and modified onto the SPCE surfaces.The unique interfaces can effectively guarantee large area for electrocatalytic activity and electron transfer.On the contrary, considering thickness of the ERGO film based on the simple drop coating method is too thick, the electrode surface was covered to be a flat surface (figure 2(c)), which was not favorable for an electron transfer process, which was corresponding to the result of the following CV measurements.Except the top view, crosssectional SEM images have been a useful tool to analyze as-prepared films, such as thickness and inner structures.Although cross-sectional SEM images of (ERGO+/ERGO−) 12 multilayers were not readily acquired on a flexible SPCE substrate, it can demonstrate successful formation of GO-based film on a rigid silicon wafer [33].
In general, absorbance variation obtained from UV-vis spectra can be regarded as a powerful tool to reflect the thickness growth trend of multilayered nano-films, considering stronger absorbance as increased film thickness under a specific light wavelength.Therefore, measuring UV-vis was selected to firmly demonstrate LBL self-assembly of (GO+/GO−) n multilayers.In figure 3(a), UV-vis absorbance of (GO+/GO−) n films increasingly got stronger as an incremental number of deposition cycles under an interval of four bilayers.Herein, n was determined as 0, 4, 8, 12, 16, and 20, respectively.Moreover, the data in figure 3(b) demonstrated that absorbance at wavelength of 310 nm performed a linear relationship with number of stacking layers (R 2 = 0.989), indicating uniformly growing multilayers through a LBL process, offering a promising manner for creating biosensing interfaces.
3.2.Electrochemical properties of (ERGO+/ERGO−) 12 /SPCE electrodes Typically, 5 mM [Fe(CN) 6 ] 3−/4− mixed with 0.1 M KCl was selected as an electrochemical probe, considering it is sensitive to interfacial states and surface chemistry, especially for C sp 2 edge plane defects.In order to explore whether the CV can successfully electro-reduce graphene oxide multilayer films and whether the LBL selfassembly method can further improve electrochemical characteristics of ERGO film-based electrodes, CV experiments were carried out to deeply examine electrochemical curves of bare SPCE, GO/SPCE, (GO+/GO−) 12 /SPCE, ERGO/SPCE, and (ERGO+/ERGO−) 12 /SPCE.Figure 4(a) compared the voltametric responses of as-prepared electrodes in 5 mM [Fe(CN) 6 ] 3−/4− .In figure 4(a), after electro-reduction, the values of anodic peak current (I pa ) and cathodic peak current (I pc ) are significantly enlarger, compared to that of bare electrodes.And (ERGO+/ERGO−) 12 /SPCE showed smaller oxidation peak potential difference and an increase of about 30% for response current compared to ERGO/SPCE, implying that the CV process can  successfully reduce GO.This is probably caused by the resulting ERGO for significantly enlarged edge plane defects, electroactive sites, and surface area.Furthermore, that indicates that electrochemical sensors with deposited LBL multilayered films exhibit better characteristics than the sensors prepared by a conventional drop casting approach.Figure 4(b) showed electrochemical impedance spectroscopy (EIS) of bare SPCE, GO/SPCE, (GO+/GO−) 12 /SPCE, ERGO/SPCE, and (ERGO+/ERGO−) 12 /SPCE.EIS was selected for deeply testing the interfacial or surface properties of as-prepared electrodes.For specific electrode interfaces, electron-transfer resistance (R ct ) is directly equal to semicircle diameter of acquired Nyquist plots.In figure 4(b), the R ct of (ERGO+/ERGO−) 12 /SPCE prominently decreased.The results revealed that (ERGO+/ERGO−) 12 multilayer film formed better electron conduction pathways and higher efficiency between electrodes and multilayers, significantly improving ferricyanide diffusion toward electrode interfaces, corresponding to CV results.12 /SPCE Dopamine (DA), as a neurotransmitter, has been a key biomolecule that particularly functions in central nervous systems.Meanwhile, neurological diseases or disorders represented by Schizophrenia and Parkinson can be probably resulted from DA deficiency.Therefore, it is highly desired to develop simple, rapid, accurate DA detecting techniques exhibiting excellent sensitivity and selectivity, especially using electrochemical detection based on electroactive DA and oxidation-reduction reactions.

Electrochemical behaviors of DA based on (ERGO+/ERGO−)
In the present work, the electrochemical biosensor was developed based on (ERGO+/ERGO−) n multilayer film for the detection of DA. Figure 5(a) records CV curves of a solution containing 1 mM DA in PBS buffer for both bare SPCE and (ERGO+/ERGO−) 12 /SPCE electrodes (100 mV/s scan rate).Cathodic/anodic peaks

Parameter optimization of multilayers
GO concentration and the number of assembled layers can well control GO film thickness.Meanwhile, the property to electrochemical response can be regulated as well.Therefore, to obtain the best electrocatalytic or sensing performance for DA detection, typical factors like GO+ or GO-concentration and deposited layers were further optimized.
First, GO+ and GO− concentration with a great impact on thickness and structures of multilayers was studied to survey variation of anodic peak currents using cyclic voltametric.In figure 6(a), anodic peak currents value markedly went up as raised GO+ or GO− concentration and reached the peak at 1 mg mL −1 .Electron transfer efficiency was distinctly hindered while GO+ and GO− concentration exceeded 1 mg mL −1 due to the higher thickness/density of GO-based films and insufficient reduction.However, compared to 0.5 mg mL −1 building materials, there was no clear difference for the anodic peak current value.Therefore, in order to fully make use of materials with low cost, 0.5 mg mL −1 G −1 O −1 + or GO− was determined as a key parameter for preparing multilayers.
Second, the number of self-assembled layers under same preparation conditions was also studied to examine the electrocatalysis (figure 6(b)).It can be seen that I pa calculated from CV curves got higher as incremental bilayers, and reached the highest value at 12 layers, meaning that LBL self-assembly was controllable.As deposited layers increased at early stage, ERGO content in resulting multilayers raised accordingly, leading to enlarged surface area and intensive electrocatalytic sites.However, when the number of assembled layers exceeded 12, ERGO multilayer films were thick enough, giving rise to inadequate reduction even under same treatment conditions.Therefore, (ERGO+/ERGO−) 12 /SPCE with 12 layers was determined as the optimal assembled sensors.It should be noted that all the above experimental data were obtained after electro-reduction of graphene oxide-based multilayer films.

Investigation of scan rate and pH for DA detection
To firmly prove electrochemical behaviors of DA on (ERGO+/ERGGO−) 12 /SPCE surfaces or interfaces, CV responses of detecting DA (1 mM) using a series of scan rates were displayed in figures 7(a) and (b).In figure 7(a), I pc evenly increased as elevated scanning rates ranged from 20 to 200 mV s −1 .As a result, I pa presented a linear trend with scan rates.Meanwhile, the derived linear equation is described as I pa = 0.3965 ν + 16.8017 (I pa : μA, ν: mV/s) (R 2 = 0.987).The I pc is also linear as the scan rates, thus I pc = −0.3039ν+ 1.6639 (I pc : μA, ν: mV/s) (R 2 = 0.997).
Additionally, considering changed charge density of building materials under various pH conditions, the measured I pc and I pa using (ERGO+/ERGGO−) 12 /SPCE sensors for detecting DA was deeply examined by CVs  conducted under pH from 6.0 to 8.0 (figures 7(c) and (d)).The negatively shifted anodic peak potential (E pa ) as raised pH value proved that perhaps protons participated in interfacial reaction.The linear trend between E pa and pH was formulated by E pa = 0.6226-0.06245pH (E pa : V) with a slope of −62.45 mV pH −1 (R 2 = 0.997).The slope value of DA was approximately equal to 59 mV pH −1 (Nernst theoretical value), which proves that the quantity of protons and electrons involving oxidation was nearly identical.Moreover, anodic peak currents were sharply reduced once pH exceeded 7.5.Considering the physiological pH environment, detection sensitivity, and selectivity, neutral pH 7.0 PBS was selected for electrochemically detecting DA in tests.

The DA detection based on electrochemical measurements
In figure 8(a), the typical amperometry responses of the (ERGO+/ERGO−) 12 multilayer film-coated electrodes to different DA concentration were measured using +0.38 V working potential.It can be seen that remarkably enhanced oxidation currents were acquired as increased DA concentration.Figure 8(b) proved the linear trend of DA concentration and current response derived from the data in figure 8(a).Figure 8(b) indicates that the response current was linearly proportional to DA concentration especially from 1.0 × 10 −6 M to 1 × 10 −3 M, while the calculated regression equation is expressed as I = 0.00819 C DA + 0.03475 (I: μA, C DA : μM) (R 2 = 0.9996).Meanwhile, the resulting detection limit is 0.39 μM with a signal-to-noise ratio (S/N) of 3, which is low enough compared to literatures [17,[34][35][36][37][38][39], as summarized in table 1.Moreover, the linearity range as a key sensing parameter performed wider than most of reported graphene-based dopamine biosensors (table 1).

Stability and reproducibility for (ERGO+/ERGO−) 12 /SPCE
The interference of UA and AA was a critical issue in dopamine detection by means of electrochemical biosensing, resulting from oxidized molecules under similar potentials, leading to overlapping voltametric responses.To examine selectivity of (ERGO+/ERGO−) 12 /SPCE-based electrochemical biosensors to detect dopamine, the (ERGO+/ERGO−) 12 /SPCE electrode was assembled and applied to sense electrochemical behaviors making use of mixing molecules.In figure 9, the oxidation peak potential of AA is about 90 mV while that of DA is around 195 mV based on (ERGO+/ERGO−) 12 /SPCE electrodes.The oxidation peak potential for UA was located at 305 mV.Peak separation was successfully achieved and the mutual interference was eliminated.
The stability and reproducibility of the (ERGO+/ERGO−) 12 /SPCE were also thoroughly researched by repeatedly recording the sensing current responses in 1 mM DA solution.The fabrication reproducibility for five experimental electrodes was carried out under the same conditions, thereby obtaining RSD of 3.36%.When modified electrodes were employed intermittently and stored in PBS solution (pH 7.0) at ambient temperature lasting for up to 14 d, the resulting current signal slightly decreased by less than 2.8% relative to the initial current response.Thus, the LbL multilayered film modified electrode performed high reproducibility and stability.

Conclusion
In summary, the functional (ERGO+/ERGO−) n multilayer nano-films were successfully assembled for the first time through simply LBL self-assembly and electrochemical reduction processes and used to modify SPCE surfaces.Compared with bare GCE and ERGO/SPCE, (ERGO+/ERGO−) n /SPCE exhibited much higher electrocatalytic activities and sensing performances with significantly increased peak current signals and decreased charge transfer resistance.It was remarkably derived that the detection limit is at a very low level of 0.39 μM (S/N = 3).In addition, the (ERGO+/ERGO−) n multilayer films-based electrochemical sensors can be applied for detecting DA with higher sensitivity, causing higher stability and reproducibility stability than ERGO/SPCE electrodes.This was not only because of unique structural features of ERGO with electrochemical sites, but also because of the controlled building materials and post-treatments.The novel sensing behaviors and simple realization of (ERGO+/ERGO−) n multilayer films can be a promising pathway for detecting biomolecules represented by DA.

Figure 6 .
Figure 6.(a) Variation of I pa as raised GO+ and GO− concentration.(b) Variation of I pa as the raised number of deposited layers.The testing condition is 1 mM DA, 0.1 M PBS, and 100 mV/s scan rate.

Table 1 .
Sensing performances of various biosensors for detecting DA.