The Electrochemical Properties of 1-Pyrenebutyric acid/Graphene The Electrochemical Properties of 1-Pyrenebutyric acid/Graphene Composites and Their Application in Glucose Biosensors Composites and Their Application in Glucose Biosensors

: The electrochemical properties of 1-pyrenebutyric acid/graphene composites (PBA/G) obtained by one-step synthesis via 仔 - 仔 stacking was investigated. The electrochemical impedance titration curve shows the surface charge changes as function of solution pH by using ferricyanide/ferrocyanide redox couple as the probe. An apparent pK a value is estimated as 6.2 according to the impedance titration curve. In addition, a glucose biosensor was constructed by immobilizing glucose oxidase (GOD) on the surface of PBA/G via covalent interaction. This biosensor shows a linear response to glucose within the concentration up to 5 mmol 窑 L -1 with a detection limit of 0.085 mmol 窑 L -1 . A small apparent Michaelis-Menten constant (5.40 mmol 窑 L -1 ) of the immobilized GOD suggests that the immobilized GOD retains its bioactivity and shows high catalytic activity to glucose.


Introduction
Recently, two-dimensional graphene nanosheets have attracted extensive interest due to its extraordinary mechanical, thermal and electronic properties.
They have the potential for a wide range of applications such as ultracapacitors [1] , chemical sensors and biosensors [2][3] , hydrogen storage [4] and field-effect transistors [5] .Graphene sheets have been synthesized by different methods such as chemical vapor deposition, epitaxial growth, reduction of graphene oxide sheets and carbon nanotube cutting [6][7] .Two key challenges in the synthesis of graphene sheets are the availability in large quantities and the prevention of irreversible aggregation.Nowadays, large-scale production of graphene sheets could be accomplished by the reduction of graphene oxide sheets.
However, most resultant graphene sheets tend to form agglomerates or even restack to form graphite via van der Waals interaction, which significantly limits the applications of graphene in many fields.In order to resolve this problem, graphene nanocomposites were prepared by combining graphene sheets with biocompatible molecules including surfactants, polymers, DNA and proteins [8][9][10][11] .This approach holds great promise for the bioassay applications because of their good water-solubility and versatile functional groups [12][13][14][15] .1-pyrenebutyric acid (PBA), an organic fluorescent probe, provides potential applications in labeling macromolecules [16] and synthesizing biosensor materials [17] due to its outstanding optical and chemical stability.With the strong affinity to the basal plane of graphite via 仔-仔 stacking, 1-pyrenebutyric acid has been immobilized on graphene sheets to obtain 1-pyrenebutyric acid/graphene composites (PBA/G) [18] .The resulting composites have good wa-ter-solubility and could be widely used for graphene-based biosensors.Gao et al. constructed an enzyme-based signal-on DNA sensor for the specific detection of target DNA down to attomolar level with PBA/G as a nanoprobe substrate [19] .Liu et al. demonstrated a novel electrochemical biosensor for pathogenic virus detection based on the pyrene derivatives/graphene composites [20] .Obviously, it is important to understand the electrochemical properties of PBA/G and explore their applications further in the biosensors area. In

Instruments
UV-vis absorpti on spectra were recorded on a UV 3600 spectrophotometer (Shimazu, Japan).Raman scattering measurements were performed on a Renishaw InVia micro-Raman system (Renishaw, England) with an excitation wavelength of 514 nm.
Electrochemical impedance spectroscopy (EIS) measurements were carried out with an Autolab PG-STAT 302 (Metrohm, Switzerland).Other electro-chemical measurements were performed on a CHI 660D electrochemical working station (CH instruments, USA) using a three-electrode system with a modified glassy carbon (GC) electrode (3 mm in diameter) as the working electrode.A platinum wire and an Ag/AgCl reference electrode (saturated KCl) were used as the counter and reference electrodes, respectively.

Preparation of PBA/G Composites
Graphene oxide (GO) was synthesized from spectral graphite according to a modified Hummer爷s method [21] .PBA/G was fabricated by the reduction of GO in the presence of PBA according to the reported method [18] .In detail, 29 mg PBA was dissolved by  Fig. 1A shows the UV-vis absorption spectra of PBA, PBA/G and the centrifugal supernatants.Consistent with literature [22] , the main absorption peaks of PBA appear at 242 nm, 276 nm and 342 nm.
Here increases in comparison with that of GO, which is consistent with that report previously [18] .

Electrochemical Properties of PBA/G
The surface properties of graphene and PBA/G modified GC electrodes were characterized in K 3 Fe(CN) 6 solution by cyclic voltammograms (CVs).
As shown in Fig. 2  probe [20] .This result demonstrates that the surface of PBA/G is negatively charged under these conditions.(stage I in Fig. 3).With the increase of pH (5 ~7), the carboxyl groups gradually deprotonate to yield a negatively charged carboxylate ion on the surface of modified electrode, which gradually increases the electrostatic repulsion between the probe and the negatively charged carb oxylate groups and inhibits interfacial electron transfer.Therefore, the R ct values increase with solution pH (stage II in Fig. 3).At higher solution pH (>7), the carboxyl groups are fully deprotonated and the electrostatic repulsion between the probe and the electrode reaches maximum.Thus another plateau is observed (stage III in Fig. 3).The resultant electrochemical impedance titration curve represents the change in surface charge with solution pH.According to the responding pH value of the midpoint of the titration curve [23] ,

Electrochemic al impedance titration is useful
an apparent pK a value of PBA/G could be estimated to be 6.2 which is larger than that of PBA in the so-lution (ca.4.8 [24] ).This may be due to the coexistence of the graphene sheets which delocalizes the charges from PBA.

Glucose Biosensors Based on PBA/G Composites
Glucose oxidase (GOD) could catalyze the oxidation of glucose to produce H 2 O 2 in the presence of O 2 and has been widely used in construction of glucose biosensors [25][26] .In the present work, GOD, as a model enzyme, was immobilized on the surface of PBA/G by covalent modification to construct a glucose biosensor.Fig. 4 shows the impedance spectra (Nyquist plots) corresponding to the stepwise modification processes.The R ct (42.22 k赘, Fig. 4b) of the PBA/G modified GC electrode was much larger than that of the bare GCE (151.4 赘, Fig. 4a), suggesting that the successfully formation of PBA/G membrane which can hinder the electron transfer from the redox probe of [Fe(CN) 6 ] 3-/4-.When GOD was immobilized via covalent interaction, the R ct of the resultant GOD/PBA/G film (67.62 k赘, Fig. 4c) increases largely, indicating that a glucose biosensor based on the PBA/G composites was successfully constructed.The performance of the resulting biosensor was tested by recording the amperometric response of the oxidation of hydrogen peroxide produced from the enzyme reaction (the detection potential: 0.4 V).
As shown in Fig. 5, the positive current at the GOD/PBA/G modified GC electrode increases with the injected glucose (each addition of 1 mmol 窑L -1 ).
The calibrated steady-current responses with glucose concentration are given in the inset of Fig. 5.
The relationship is approximately linear up to 5 mmol 窑L -1 with a detection limit of 0.085 mmol 窑L -1 .
The high affinity of GOD was found by the determination of the apparent Michaelis-Menten constant ( ) which can be estimated in terms of the Lineweaver-Burk equation [26] .
where, i ss and i max are the steady-state current and the maximum current, respectively.C is the glucoseconcentration. Fig. 6 shows the Lineweaver-Burk plots based on the data from the inset of Fig. 5.The i max and determined from the slope and the intercept of the straight line in Fig. 6 are 0.18 滋A and 5.40 mmol窑L -1 , respectively.The value is smaller than that of GOD immobilized by the other reported methods, which demonstrates that the immobilized GOD using our method possesses high catalytic activity with respect to glucose.In addition, the stability of the immobilized GOD was investigated in 0.1 mol窑L -1 phosphate buffer (pH 7.4) containing 1 mmol窑L -1 glucose with a detection potential of 0.4 V.The result shows the activity of the enzyme retains approximately 71% of its original value after a storage time of 16 h.

Conclusions
In summary, the electrochemical properties of water-soluble PBA/G composites were investigated by electrochemical techniques.The surface charge of PBA/G varied with the pH values of bulk solution and the apparent pK a of PBA/G determined from the electrochemical impedance titration curve is 6.2.A glucose biosensor based on PBA/G was constructed by attaching GOD on the surface of PBA/G via covalent interaction.This biosensor shows a linear re-

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mL NaOH (83.3 mmol 窑L -1 ) solution and then 0.1 mg窑mL -1 GO suspension was added to the above solution.The resulting mixture was reduced with hydrazine solution at 80 o C under vigorous agitation for 24 h.The product PBA/G was centrifuged, thoroughly washed with dilute NaOH solution and ultrapure water respectively and then dried under vacuum at room temperature.As a comparison, graphene was fabricated by the same procedure without addition of PBA.2.4 Preparation of GOD/PBA/G ModifiedGC ElectrodesGlassy carbon (GC) electrode was well polished with 0.05 滋m alumina slurry, and then cleaned ultrasonically in ethanol and water for 3 min, respectively.The PBA/G and graphene modified GC electrodes were prepared by casting 10 滋L PBA/G or graphene suspension onto the pretreated bare GC electrode using a micropipette tip and dried in air.The PBA/G modified GC electrode was immersed in 0.1 mol 窑L -1 phosphate buffer (pH 7.4) containing 0.15 mol窑L -1 EDC and 0.3 mol窑L -1 NHS for 1 h to activate the 要COOH groups in PBA/G.GOD was covalently attached to PBA/G by incubating the activated PBA/G modified GC electrode in 10 mg窑 mL -1 GOD solution for 1 h.

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Results and Discussion 3.1 Characterization of the Graphene and PBA/G Fig. 1A, these characteristic peaks of PBA observed in the spectrum of PBA/G are red-shifted (250 nm, 286 nm and 354 nm), which is attributed to the 仔-仔 stacking interaction between PBA and graphene.The Raman spectra of PBA and PBA/G are shown in Fig. 1B.Compared to the spectra of GO and graphene, the shoulder band around 1610 cm -1 and the weak band at 1235 cm -1 in the spectrum of PBA/G indicate the formation of PBA/G nanocomposites.As shown in Tab. 1, the D/G ratio of PBA/G (curve a), the graphene modified GC electrode shows a couple of well-defined redox wave (驻E p = 108 mV, i pc /i pa 抑1) due to the excellent conductivity of graphene.However, no obvious reversible redox wave is observed for the PBA/G modified GC electrode (Fig. 2, curve b), which is attributed to the electrostatic repulsion between the carboxylate groups of PBA/G and the Fe (CN) 6 3-

Fig. 5
Fig. 5 Typical current time response curves of GOD/PBA/G modified GC electrode at 0.4 V in 0.1 mol 窑L -1 phosphate buffer (pH 7.4) upon successive addition of 1 mmol 窑L -1 glucose.Inset: relationship between response current and glucose concentration.

Fig. 6
Fig. 6 Lineweaver Burk plots based on the data in the inset of Fig. 5.