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Construction and Evaluation of a Self-Calibrating Multiresponse and Multifunctional Graphene Biosensor

Recently, many studies have been focused on the development of graphene-based biosensors. However, they rely on one type of signal and need to be calibrated by other techniques. In this study, a nonenzymatic graphenebased biosensor has been designed and constructed. Its ability to detect glucose and Escherichia coli by three different types of signals has been investigated. For its preparation, dopamine-functionalized polyethylene glycol and 2,5-thiophenediylbisboronic acid were conjugated onto the surface of graphene sheets by nitrene [2 + 1] cycloaddition and condensation reactions, respectively. Multivalent interactions between boronic acid segments and biosystems consequently increased the quantifiable fluorescence emission and UV absorption of dopamine segments. Additionally, changing the electrochemical behavior of the functionalized graphene sheets was possible and resulted in a measurable output signal. Conjugation of mannose onto the surface of the biosensor improved its magnitude of signals and specificity for sensing E. coli in a complex medium. The efficiency and accuracy of each signal was monitored by others, which resulted in a real-time self-calibrating biosensor. Taking advantage of the versatility of the three different indicators, including florescence, UV, and electrochemistry, the functionalized graphene sheets have been used as selfregulating biosensors to detect a variety of biosystems with a high accuracy and specificity in a short time.


■ INTRODUCTION
Biosensors are analytical devices with a fast response and ability to directly convert biointeractions to quantifiable and processable physiochemical signals. 1−3 They are used for sensing different analytes ranging from small molecules, e.g., saccharides, 4 to large objects including glycans, 5 nucleic acids, 6 proteins, 7 pathogens, 8 and cancer cells. 9 Usually biosensors consist of interacting functional groups and detectors assembled on a platform. 10 Two-dimensional (2D) nanomaterials are of great interest as sensor platforms because of their unique mechanical and physicochemical properties. 11,12 Graphene derivatives including graphene oxide (GO), reduced graphene oxide (rGO), and graphene quantum dots (GQDs) are the most versatile platforms that have been used to construct biosensors. 13−20 Due to their huge surface area, flexibility, and optoelectronic properties, they are able to improve the sensitivity, response time, and limit of detection of biosensors efficiently. 21−23 In the graphene-based sensors, detectors can be attached to the graphene platform either by covalent or by noncovalent approaches. 24−29 Functionality is one of the key factors that improves the sensibility of graphene-based sensors. 30 A large number of interactive functional groups that are conjugated to the surface of graphene platforms cause strong multivalent interactions with biosystems 31−35 and amplify the intensity of detector signal. (Macro)molecules with a boronic acid functionality have been widely applied for the fabrication of biosensors. 36,37 They are able to bind to cis-1,2-and 1,3-diols of different biosystems such as carbohydrates, proteins, and pathogens at alkaline conditions. Lipopolysaccharides, as the basic components of the external cell wall of Gram-negative bacteria, 38 are able to attach to boronic acids through which these microorganisms can be detected. 39 It has been reported that boronic acid segments noncovalently immobilized on graphene sheets were successfully able to sense glucose molecules. 40,41 However, low stability of the boronic acid functional groups that are attached to the surface of graphene noncovalently hamper application of such systems at physiological and complex media. This could be a drawback, particularly when sensing large biosystems such as pathogens that are able to separate boronic acids from the surface of graphene sheets. Therefore, covalent attachment of boronic acid functionalities to the surface of graphene sheets improves the efficiency of such biosensors.
Catechol derivatives such as dopamine are another class of materials that strongly bind to boronic acids. 42 Dopamine is a catecholamine neurotransmitter that plays several important roles in the brain and body. 43,44 In addition, it shows redox property and can be oxidized by different oxidants to dopaquinone. 45 It shows a fluorescence emission at 330 nm when excited at 266 nm. 46,47 Therefore, both the electrochemical properties and the fluorescence signals of dopamine can be also used as indicators to detect different biosystems. 48,49 As a result, controlled and covalent attachment of boronic acid/ dopamine combinations onto the surface of graphene sheets leads to new biosensors with the ability of multivalent interactions and detecting biosystems. However, in situ calibration of biosensors still is an unsolved problem, which should be overcome to obtain real-time signals with a high accuracy. 50 In order to quantify and standardize a biosensor signal it should be calibrated by other techniques. In most cases, the calibration technique is not compatible with the interacting interfaces and sensing conditions.
In this work, we solved this problem by constructing a multiresponse graphene-based sensor. A telechelic polymer with dopamine and azide functionalities has been synthesized and attached to the surface of graphene by nitrene [2 + 1] cycloaddition reaction. Then functionalized graphene sheets were modified by 2,5-thiophenediylbisboronic acid, and 2D nanomaterials with boronic acid functionalities were obtained. Interactions between boronic acid groups and cis-1,2-or 1,3diols of biosystems changed the fluorescence emission, UV absorption, and electrochemical behavior of the functionalized graphene sheets. Changes in these optoelectronic properties were then used as signals to detect glucose and E. coli. Signal and specificity of this biosensor for detection of E. coli in a complex media were efficiently improved by conjugating mannose onto its surface. The accuracy of each signal was tested by other

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Article signals, which led to a self-calibrating biosensor with high versatility to detect different biosystems.

■ EXPERIMENTAL SECTION
Full information including materials, instruments, and synthetic protocols is available in the experimental section of the Supporting Information.

■ RESULTS AND DISCUSSION
In this work, a new type of graphene-based biosensor using our recently published procedure for the controlled functionalization of graphene sheets 51−53 has been reported.  A telechelic poly(ethylene glycol) with dopamine and azide functional groups was synthesized and conjugated to the surface of graphene oxide by nitrene [2 + 1] cycloaddition reaction. In order to synthesize the telechelic polymer, cyanuric chloride was conjugated to the end functional group of poly(ethylene glycol) monomethyl ether and the product was reacted with dopamine. Nucleophilic substitution of the chloride group of dopaminefunctionalized poly(ethylene glycol) by sodium azide resulted in the final telechelic polymer. After conjugation of telechelic polymer to the surface of graphene sheets, 2,5-thiophenediylbisboronic acid was attached to their catechol groups and 2D nanomaterials with boronic acid functional groups were obtained (Scheme 1).
Telechelic polymer and functionalized graphene sheets were characterized by different spectroscopy and microscopy methods. Figure 1A(a−f) displays IR spectra of PT, PTD, PTDN 3 , PTDN-GO, 2,5-thiophenediylbisboronic acid, PTDN-GO-DBA, and PTDN-GO-DBA-Mann. In the IR spectrum of PT absorbance bands at 2895, 1611, and 1580 cm −1 were assigned to C−H bonds of poly(ethylene glycol) and CN bonds of triazine ring. The appearance of absorbance bands at 3045−3656 and 2129 cm −1 in the IR spectra of PTD and PTDN 3 confirmed the successful synthesis of telechelic polymer with catechol and azide functionalities. After conjugation of PTDN 3 to nGO, the absorbance band of the azide group disappeared, which is an indicator for the nitrene [2 + 1] cycloaddition reaction. Absorbance bands at 1518 and 652 cm −1 and amplified absorbance band of hydroxyl groups at 3050− 3560 cm −1 in the IR spectra of PTDN-GO-DBA indicated conjugation of 2,5-thiophenediylbis-boronic acid to PTDN-GO. In the IR spectra of PTDN-GO-DBA and PTDN-GO-DBA-Mann, absorbance bands at 620 and 640 cm −1 , which belong to the boronate esters, as well as an absorbance band at 980 cm −1 , corresponding to the C−S bond, represent conjugation of boronic acid segments to the functionalized graphene sheets.
The structure of PT and PTD was evaluated by 1 H-and 13 C NMR spectroscopy. Signals of different protons and carbons are depicted in the NMR spectra of these compounds ( Figure 1B). Based on these data, a telechelic polymer was successfully synthesized with the desired functionalities.
Synthesized materials were further characterized by elemental analysis (Table 1). Nitrogen, carbon, and hydrogen contents of PTDN 3 were in agreement with the calculated amounts (ESI ,  Table S1, ESI). The low carbon content of nGO showed that it was highly oxidized. The destructed π-conjugated system of nGO resulted in low fluorescence quenching, which was crucial for biosensing by fluorescence signal. According to elemental analysis, the density of functional groups (conjugated polymer) was high and it was one functional group per 6 carbon atoms of nGO (ESI, p 2). After conjugation of 2,5-thiophenediylbisboronic acid to PTDN-GO, the nitrogen content decreased to 1.27%. This nitrogen content indicated that one boronic acid functional group had been attached to each catechol group of PTDN-GO (ESI, p 2). The sulfur content of PTDN-GO-DBA decreased to 1.1%, which indicated the conjugation of 2,5thiophenediylbis-boronic acid to PTDN-GO. Conjugation of mannose eventually decreased the nitrogen content of PTDN-GO-DBA to 1.24%. Figure 2a and 2b represents the tapping mode SFM height images of nGO and PTDN-GO-DBA on a freshly cleaved and atomically flat mica surface. The nGO sample had structures with (longest) lateral dimensions 26 ± 10 nm (sheets up to 100 nm also were observed) and heights mostly of 0.8 ± 0.05 nm (error is standard deviation). We attributed those thin sheets to

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Article typical height between 2 and 8 nm. The variation of the heights of different PTDN-GO-DBA sheets was attributed to the polydispersity index (PDI) of PEG chains within these structures and the possibility of attachment of the functionalities to thicker (few layered) nGO crystals. The lateral dimensions of the PTDN-GO-DBA sheets were found to be within 63 ± 10 nm. We attributed the larger lateral sizes of these sheets to the disordered arrangement of the PEG chains within the 2D nGO sheets and lying on the mica surface along the sheet edges ( Figure S1, ESI). Therefore, it was possible to assign the increase of the height and lateral dimension of PTDN-GO-DBA sheets to their functionalization.
Since the goal of this work was to construct a biosensor based on the optoelectronic properties of the functionalized graphene oxide sheets, the UV absorption, fluorescence emission, and electrochemical characteristics of the synthesized graphene derivatives were investigated.

Article
Fluorescence resonance energy transfer (FRET) depends on the electron state 54 of fluorophore and its distance from the platform. 55−58 These properties have been abundantly used for construction of the graphene-based biosensors. 59 Changes in the fluorescence resonance energy transfer, after interactions between analytes and fluorophore, can be used to quantitatively probe various biosystems. 60,61 Yum et al. have shown that interactions between aromatic boronic acids, which are attached to the surface of carbon nanotubes and glucose, change the electronic state of these fluorophores and affect their energy transfer with carbon nanotube. 54 The change in the fluorescence of the carbon nanotube upon this energy transfer has been used to detect glucose.
On the basis of these studies, interactions between biosystems and boronic acid groups are supposed to change the fluorescence of dopamine segments (Figure 3a). PTDN-GO-DBA showed a strong UV absorption at 248 nm, which was assigned to the dopamine and boronic acid segments ( Figure  3b). As nGO was highly oxidized, the FRET was not too strong, and a considerable emission for the dopamine segment at 605 nm was observed (Figure 3c). This characteristic is the basis for sensing biosystems by fluorescence signal.
Interactions between targeted biosystems and PTDN-GO-DBA improved the dispersibility and consequently UV absorption of graphene sheets due to the decreased light scattering. 62,63 Accordingly, an increase in the UV absorption of PTDN-GO-DBA at 270 nm, upon addition of glucose and E. coli, was used as another signal for detection these biosystems (Figure 3d and 3e, respectively).
To test the reliability of fluorescence and UV signals, an aqueous solution of glucose (50 μM) and a solution of E. coli (5000 CFU/mL) in PBS were added to a constant concentration of PTDN-GO and PTDN-GO-DBA in PBS, and their fluorescence and UV spectra were recorded. While addition of glucose and E. coli (Figure 3f and 3g, respectively) did not significantly change the fluorescence spectra of PTDN-GO, it did increase the fluorescence intensity of PTDN-GO-DBA at 605 nm dramatically (Figure 3f and 3g).
In addition to fluorescence and UV signals, an electrochemical property of PTDN-GO-DBA was used to detect glucose and E. coli. Sulfur atoms of PTDN-GO-DBA are able to improve interactions between the graphene sheets and the gold surfaces. Accordingly, bare gold electrodes (GE) were modified by PTDN-GO-DBA (ME) ( Figure S2, ESI), which were then incubated with an aqueous solution of targeted biosystems. The intensity of the current peak in the cyclic voltammograms (CV) of PTDN-GO-DBA was decreased after adding a PBS solution (pH 8.0) of glucose (15 μM) or E. coli (5000 CFU/mL) containing 5 mM [Fe(CN) 6 ] 3−/4− . The reason for such an observation is the attachment of glucose or E. coli to the surface of modified electrode through boronic acid functionalities. This process hinders the diffusion of the [Fe(CN) 6 ] 3−/4− toward the electrode surface, diminishes the electron transfer between analyte and electrode, and causes a decrease in the intensity of the current peak. Such an electrochemical signal is used as an indicator to detect glucose and E. coli (Figure 4a and 4b). Figure 5A and 5B shows raising the intensity of UV absorption and fluorescence emission of PTDN-GO-DBA upon adding different concentrations of glucose and E. coli. By extrapolation of these plots to zero, different concentrations of glucose and E. coli can be detected. The lowest concentrations of glucose and E.

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Article coli (detection limit) that was detected by UV and fluorescence signals are shown in Table 2.
In order to investigate the electrochemical sensing in detail, the electrochemical behavior was evaluated for GE before and after modification with PTDN-GO-DBA in PBS (pH8) containing [Fe(CN) 6 ] 3−/4− (5 mM) and KCl (0.1 M). Figure  6a demonstrates two well-defined redox peaks with 85 mV potential separations for GE. The CV of ME showed a shift of the oxidation/reduction peak to more negative/positive potentials in comparison to GE, respectively. Also, the observed current response and the shape of the peak of ME were lower than and different from GE, respectively (Figure 6a). Changes in the current and potential peaks were due to the slower electron transfer through the ME.    Figure  6b). The resistance of charge transfer (R ct ) for ME (9.250 kΩ) was significantly higher than GE (0.552 kΩ) due to the immobilized PTDN-GO-DBA. While GE showed an almost straight line, indicating a diffusion-limited electrochemical behavior, the insulating layer of PTDN-GO-DBA on the electrode surface acted as a barrier to the interfacial electron transfer and led to a semicircular EIS spectrum. The CV curves of this compound were investigated at various scan rates (10− 200 mV/s). It was found that the peak current increased along with the rising scan rate. The oxidation and reduction potentials also shifted to more negative and positive values with the increased scan rates, respectively (Figure 6c). At lower scan rates, the cathodic peak current increased linearly with increasing square root of scan rates (Figure 6d). This result indicated that the redox reaction was a surface process and that the ME had good stability and reproducibility. In agreement with EIS results, CVs of ME at different scan rates showed a direct correlation between scan rate and both anodic and cathodic current peaks. This result indicated a diffusioncontrolled redox process.
After electrochemical characterization of ME it was applied for the sensing of glucose and E. coli ( Figure 7A). The modified electrode was exposed to glucose solutions with different concentrations, and CV and EIS were recorded. Clearly, the peak current decreased and peak-to-peak separation increased upon increasing the glucose concentration. Because boronic acid functional groups of PTDN-GO-DBA were immobilized on the electrode and bound to glucose, the effective surface area of the electrode consequently decreased. This process limited diffusion of the redox couple toward the electrode surface ( Figure 7B). Also, changes in CV and EIS after applying certain concentrations of E. coli proved successful detection of this pathogen by ME ( Figure 7C). The current peaks exhibited a linear dependence on the glucose (0−40 μM) and E. coli (0− 1.25 × 10 4 CFU/mL) concentrations. Different concentrations of glucose and E. coli can be detected by modified electrode using calibration curves in Figure 7D and 7E. The detection of limit (LOD) of the modified electrode for sensing glucose and E. coli are shown in Table 2.
One of the main advantages of our biosensor is the simultaneous determination of targets by three different signals. This advantage provided a possibility to test each signal by two other signals and achieving a self-calibrating biosensor. After determination of the concentration of glucose and E. coli by electrochemical signal, the same sample was tested by UV and fluorescence signals. Results of self-calibration experiments are shown in Figure 8. As it can be seen, there is a high agreement between the results obtained by different signals. Any error could be detected by deviation from such linear relationship between UV, fluorescence, and electrochemical signals.
The self-calibration feature of the constructed biosensor to detect E. coli was investigated by intentional deviation of one signal and consequently correction by two other signals. The fluorescence signal of biosensor, for example, was deviated by adding a certain amount of another fluorescence probe such as Rhodamine B. The fluorescence signal of biosensor was quenched partially by a fluorescence probe, and a deviation from the initial response was induced (Figure 8a and 8b). The deviation of the fluorescence signal was then calculated by UV and electrochemical diagrams and then applied to correct the fluorescence signal. The fluorescence signal corrected by both UV and electrochemical signals (Figure 8c) was in a good agreement with the initial fluorescence signal (Figure 8a), indicating the self-calibration ability of this biosensor. The selfcalibration concept was also proven for the electrochemical signal of an old electrode. The signal of the old electrode was    Boronic acid-based sensors are generally unselective or show limited selectivity, 66,67 which is also a drawback for our system. To solve this problem mannose was immobilized to PTDN-GO-DBA via cis-diol attachment. Since mannose showed specific interactions with E. coli, 68−70 the modified biosensor was applied to detect E. coli in a complex media.
UV, fluorescence measurements, and differential pulse voltammetry (DPV) were used to investigate the specificity of biosensor to detect E. coli in human blood serum (HBS). E. coli solutions with known concentrations were mixed with the human blood serum. Then these mixtures were investigated by UV and fluorescence spectroscopy (Figure 9). It was found that the intensity of UV absorption and fluorescence emission of graphene sheets increased upon interactions with E. coli. However, these parameters did not considerably change after interaction with glucose. This result proved specific interactions between PTDN-GO-DBA-Mann and E. coli. The detection limits of PTDN-GO-DBA-Mann are shown in Table 2.
Differential pulse voltammetry (DPV) was also used for investigating the specificity of the biosensor to detect E. coli in human blood serum. E. coli samples with known concentrations were mixed with the human blood serum, whereupon the mixture was added to the gold electrode modified by PTDN-GO-DBA-Mann (ME-Mann). Tbe DPV was recorded as well ( Figure 10a). DPV of ME-Mann exhibited an oxidation peak at 0.31 V in the absence of E. coli. However, a gradual decrease in DPV measurements was observed upon addition of different concentrations of E. coli (1.25 × 10 3 −10 4 CFU/mL) ( Figure  10b). This is due to the electron-transfer hindering effect caused by E. coli immobilized on GE. The linear relationship between the peak current and the E. coli concentration with an acceptable correlation coefficient (0.988) showed that this biosensor was able to selectively detect E. coli in a complex media with a high accuracy. However, addition of different concentrations of glucose to human blood serums did not significantly decrease the DPV curves of ME-Mann (Figure 10c). This was due to occupation of boronic acids by mannose and therefore weak interaction with glucose. A detection limit as low as 1.3 × 10 3 CFU/mL was found for sensing E. coli in human blood serum by this biosensor.

■ CONCLUSIONS
The aim of the present study was to prepare a nonenzymatic, self-calibrating, multiresponse, and multifunction biosensor based on functionalized graphene sheets. The biosensor was able to detect biosystems by three different types of signals at the same time. The selectivity of the biosensor toward E. coli in a complex medium was improved by attaching mannose to its surface. Taking advantage of the versatility and accuracy of each signal as well as multivalent interactions between the graphene platform and biosystems, functionalized graphene sheets could be used as a powerful, fast-response, and self-examining biosensor.