Accepted Manuscript Title: Direct electron transfer of glucose oxidase and dual hydrogen peroxide and glucose detection based on water-dispersible carbon nanotubes derivative Author: Hsiao-Chien Chen Yi-Ming Tu Chung-Che Hou Yu-Chen Lin Ching-Hsiang Chen Kuang-Hsuan Yang PII: DOI: Reference:

S0003-2670(15)00081-1 http://dx.doi.org/doi:10.1016/j.aca.2015.01.027 ACA 233688

To appear in:

Analytica Chimica Acta

Received date: Revised date: Accepted date:

20-10-2014 15-1-2015 19-1-2015

Please cite this article as: Hsiao-Chien Chen, Yi-Ming Tu, Chung-Che Hou, Yu-Chen Lin, Ching-Hsiang Chen, Kuang-Hsuan Yang, Direct electron transfer of glucose oxidase and dual hydrogen peroxide and glucose detection based on water-dispersible carbon nanotubes derivative, Analytica Chimica Acta http://dx.doi.org/10.1016/j.aca.2015.01.027 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Direct electron transfer of glucose oxidase and dual hydrogen peroxide and glucose detection based on water-dispersible carbon nanotubes derivative

Hsiao-Chien Chen a, Yi-Ming Tu b, Chung-Che Hou b, Yu-Chen Lin c, Ching-Hsiang Chen d, Kuang-Hsuan Yang e,*

a

Department of Biochemistry and Molecular Cell Biology, School of Medicine, College of

Medicine, Taipei Medical University, 250, Wuxing St., Taipei 11031, Taiwan. b

Department of Chemical and Materials Engineering, Chang Gung University, 259 Wen-Hwa

1st Rd., Tao-Yuan 33302, Taiwan. c

Wah Hong industrial Co. Ltd., 6 Lixing St., Guantian Dist., Tainan City,72046,Taiwan.

d

Graduate Institute of Applied Science and Technology, National Taiwan University of Science

and Technology, 43 Keelung Rd., Sec. 4, Taipei 10607, Taiwan e

Department of Food and Beverage Management, Vanung University, 1, Van Nung Rd.,

Shuei-Wei Li, Chung-Li City 32061, Taiwan.

* Corresponding author: Tel: + 886-3-4515811 ext. 84609; Fax: + 886-3-4628015 E-mail addresses: [email protected] (K.-H. Yang)

Highlights

► 1. Dual hydrogen peroxide and glucose sensor ► 2. Direct electrochemistry of glucose oxidase used MWCNT-Py/GC electrode ► 3. Change sensing function by adjusting pH value.

Graphical abstract Abstract A

water-dispersible

multi-walled

carbon

nanotubes

(MWCNTs)

derivative,

MWCNTs-1-one-dihydroxypyridine (MWCNTs-Py) was synthesis via Friedel–Crafts chemical acylation. Raman spectra demonstrated the conjugated level of MWCNTs-Py was retained after this chemical modification. MWCNTs-Py showed dual hydrogen peroxide (H2O2) and glucose detections without mutual interference by adjusting pH value. It was sensitive to H2O2 in acidic solution and displayed the high performances of sensitivity, linear range, response time and stability; meanwhile it did not respond to H2O2 in neutral solution. In addition, this positively charged MWCNTs-Py could adsorb glucose oxidase (GOD) by electrostatic attraction. MWCNTs-Py-GOD/GC electrode showed the direct electron transfer (DET) of GOD with a pair of well-defined redox peaks, attesting the bioactivity of GOD was retained due to the non-destroyed immobilization. The high surface coverage of active GOD (3.5 ×10−9 mol cm−2) resulted in exhibiting a good electrocatalytic activity toward glucose. This glucose sensor showed high sensitivity (68.1 µA mM–1 cm–2) in a linear range from 3 μM to 7 mM in neutral buffer solution. The proposed sensor could distinguish H2O2 and glucose, thus owning high selectivity and reliability.

Keywords: carbon nanotubes; glucose; hydrogen peroxide; direct electron transfer; electrostatic attraction; Friedel–Crafts chemical acylation.

Running header: Dual hydrogen peroxide and glucose sensor

1. Introduction

Glucose is a primary source of energy for the cells. It is transported from intestines or liver to cells via the bloodstream. Human body can regulate glucose concentration naturally. The normal blood glucose concentration is within 4.4-6.6 mM. As the patient suffers diabetes, the metabolic disorder results the glucose level higher or lower than normal level. Diabetic patients are one of the leading causes of death and disability [1]. Therefore, the regular blood glucose monitoring is critical. Glucose biosensor based on electrochemical technique has received much attention due to easy to use and real-time detection with high sensitivity [2,3]. In addition, developing glucose biosensor based on glucose oxidase (GOD) has been widely used because of its high specificity, relatively low cost and good stability [4]. Also, GOD is one of the well-known redox electroactive enzymes that can be used for the electrocatalysis of glucose [5,6]. However, direct electron transfer (DET) between GOD and general electrode is difficult because the redox electroactive site is embedded deeply in the enzyme matrix [7]. In addition, according to the GOD biocatalysis, glucose transforms into gluconolactone accompanying with producing H2O2 and consuming oxygen [8]. Hence, many efforts in developing glucose biosensors have been focused on detecting H2O2 and oxygen [9,10]. The suitable working potential for sensing H2O2 is

considered at negative potential which can avoid the interference of some species in human blood such as uric acid, ascorbic acid and dopamine [11]. However, in the most oxygen sensitive electrodes, the potential are often applied at negative potential [9]. The very close potential of H2O2 and oxygen means the mutual effect occurs in the determination of glucose, resulting in the inaccurate data. So far, this issue has not been widely investigated and resolved. Besides, the quantity and bioactivity of GOD involving in the amount of producing H2O2 or consuming oxygen majorly dominate sensor’s performances. Carbon nanotube (CNT) has aroused attention as a carrier in the application of electrochemical biosensor due to its unique structural, mechanical and electronic properties [12]. The high specific surface area is conducive to load large amount of GOD. Furthermore, the excellent conductivity contributes to the realization of DET between GOD and electrode [13,14]. The retained bioactivity majorly depends on the method of immobilization. Numerous studies have investigated in immobilizing methods including covalent bonding [15], entrapment [16,17], mixture [18], assembly, [19,20] and electrostatic attraction [11,21]. Among these methods, electrostatic attraction is recognized as an ideal technique due to the non-destroyed process to GOD that can avoid enzyme deactivation. The previous literatures demonstrate polybenzimidazole and its derivatives are sensitive to H2O2 in the presence of carboxylic acid. The reaction of H2O2 and carboxylic acid forms peroxyacid that can oxidize electroactive imine structure further to form N-oxide. The N-oxide structure is restored by electrochemical reduction, thus generating the current signal [22,23]. However, these insulated polymers hinder electron transfer, resulting in decreasing the sensor’s performance.

In this study, the small molecule, 2,6-dihydroxypyridine-4-carboxylic acid (DHPCA) is used for modifying multi-walled carbon nanotubes (MWCNTs) via Friedel–Crafts chemical acylation to synthesize MWCNTs-1-one-dihydroxypyridine (MWCNTs-Py). The directly bonding small molecule (–Py) on MWCNTs can avoid the consumption of electron during electron transfer process. The MWCNTs-Py/GC electrode shows high performance of detecting H2O2 in the presence of acetic acid. Also, negatively charged GOD can be immobilized directly on positively charged MWCNTs-Py by electrostatic interaction. The highly conjugated composite realizes the DET within GOD and electrode. The most important is the MWCNTs-Py-GOD/GC electrode is sensitive to glucose in the neutral solution without interfering by H2O2.

2. Materials and methods

2.1. Materials

2,6-dihydroxypyridine-4-carboxylic acid (97%), MWCNTs, phosphorus pentoxide (P2O5) acetic acid (99.7%), glucose assay kit and glucose oxidase (EC 232-601-0) were from Sigma-Aldrich. Polyphosphoric acid (PPA), glucose and H2O2 (30%) were from Showa. The supporting electrolyte consisted 0.2 M phosphate buffer solution (PBS). Deionized (DI) water was used in all experiments.

2.2. Synthesis of MWCNTs-Py

The synthesis method of MWCNTs-Py was according to Friedel–Crafts chemical acylation [24]. 100 mg of MWCNTs, 100 g of PPA, 5 g of P2O5 and 5 g of DHPCA were placed in 250 ml flask and stirred at 100 oC under nitrogen for 2 h. Then the temperature increased to 130 oC for 24 h. After reaction, the solution was cooled down to the room temperature and was diluted by DI water. The precipitate was obtained by high-speed centrifugation (12000 rpm). The purification of washing by DI water, methanol, and high-speed centrifugation was repeated for five times. Finally, the precipitated was collected and dried at 50 oC in the vacuum oven for 24 h.

2.3. Preparation of the MWCNTs-Py/GC electrode 50 mg of MWCNTs-Py was dispersed in 100 ml of DI water. In addition, 0.1 % of chitosan was prepared in the above solution. 2 L of the dispersed MWCNT-Py solution was dropped onto a GC electrode (0.071 cm2) and dried in a vacuum aspirator at 50 °C for 2 h.

2.4. Preparation of the MWCNTs-Py-GOD/GC electrode 5 ml of MWCNTs-Py solution was mixed with 50 mg of GOD at 4 oC for 3 h. The free GOD was removed by high-speed centrifugation (12000 rpm) for three times. The MWCNTs-Py-GOD was re-dispersed in 5 ml DI water. Also, 0.1 % of chitosan was prepared in the solution. 2 L of the dispersed MWCNT-Py-GOD solution was dropped onto a GC electrode and dried at 4 °C for 2 h before measurement.

2.5. Equipment and measurements Isoelectric point (IEP) and particle size were analyzed on Zeta potential analyzer from Malcern Zetasizer Nano ZS. Field-emission scanning electron microscopy (FE-SEM) was

performed using a JSM-6330 F. Absorption spectra were recorded on a UV2450 spectrophotometer (Shimadzu). FTIR spectra were obtained using a Bruker-Tensor 27 spectrometer at a spectral resolution of 8 cm–1. X-Ray photoelectron spectrometry (XPS) measurements were performed using a VG Scientific ESCALAB 250 system. Raman spectra were measured from 1000 to 3000 cm–1 at room temperature by using UniRAM system as integrated by CL Technology Co., Ltd.. Electrochemical measurements were performed on a CHI 660A electrochemical workstation (CH Instruments, USA). A conventional three-electrode system was employed. An Ag/AgCl (saturated KCl) electrode and a platinum plate electrode were used as a reference and counter electrode.

3. Results and discussion

3.1. Characteristic of MWCNTs-Py

MWCNTs-Py was synthesized from MWCNTs and DHPCA via Friedel–Crafts chemical acylation using PPA and P2O5 as catalysts for reacting 48 hr at 140 oC (Fig. 1A). Based on this electrophilic substitution reaction, carboxylic group (–COOH) of DHPAC transformed into acylium ion (–C+=O) which was an active species to replace the sp 2 C–H of MWCNTs. Fig. 1B showed the absorption spectra of MWCNTs, DHPCA and MWCNTs-Py in aqueous solution. No absorption peak was observed to MWCNTs within the range of 200–900 nm due to the van der Waals and π-π interactions resulting in aggregation and precipitation in DI water (Fig 1B inset). DHPCA showed a π-π* transitions peak at 331 nm. However, MWCNTs-Py showed an obvious peak at 250 nm with a shoulder band at 350 nm. The former peak was attributed to the π-π*

transition of MWCNTs and the latter shoulder band was contributed from DHPCA. Besides, the absorption of MWCNTs mixing with DHPCA showed that the π-π* transition at 250 nm contributing from MWCNTs was not observed (Fig. 1B(d)) due to the precipitation of MWCNTs. Therefore, the absorption spectrum of mixed solutes was similar to the pure DHPCA in which its π-π* transition at 331 nm did not change and was totally different to that of MWCNTs-Py. The 19 nm of red shift indicated the conjugated level of grafting Py on MWCNTs was better than DHPCA. In addition, the peak of DHPCA at 331 nm disappeared after modification, meaning no residual unreacted DHPCA was present. The well-dispersion of MWCNTs-Py in aqueous solution could be explained by the presence of hydrophilic group, hydroxyl group (–OH) (Fig. 2B inset). In addition, the imine structure of –Py was protonated to form =N+H– in the acidic solution during Friedel–Crafts chemical acylation. The charge of MWCNTs-Py was measured by zeta potential analyzer and was 49.7 mV. The positively charged MWCNTs-Py generated charge repulsion within MWCNTs-Py, thus preventing the formation of aggregation in aqueous solution. The structure of MWCNTs-Py was further characterized by FT-IR and XPS. Fig. 1C showed the FT-IR spectra of MWCNTs, DHPCA and MWCNTs-Py. The characteristic peaks of MWCNTs had been widely surveyed, including stretching vibration of C=C (νC=C ) at 1533 cm–1 and νC–O at 1189 cm–1 which was attributed to the atmospheric oxidation or residual oxides during purification process of MWCNTs (Fig. 1C(a)) [25]. The majorly characteristic peaks of DHPCA included νC=O at 1699 cm–1, νCOO– (carboxylic salt) overlapping νC=N+ (protonated imine) at 1606 cm–1, νC=C at 1533 cm–1, combination of O–H deformation vibration with νC–N at 1313 cm–1 and νC–O at 1208 cm–1 (Fig. 1C(b)). Broad bands in the 2900–3300 cm–1 range were assigned to the νC–H unsat. at 3109 cm–1 and hydrogen bonded νO–H overlapping νN+–H at 2907 cm–1,

respectively. After chemical modification, the peak at 1699 cm–1 shifted to 1650 cm–1, indicating that the carboxylic acid had transformed into acylium ion and had grafted on MWCNTs to form ketone structure. Besides, the other peaks showed the small shift, meaning the MWCNTs-Py had been synthesized successfully via Friedel–Crafts chemical acylation. Furthermore, the broad band at 2893 cm–1 characterizing as the positively charged N+–H was agreement with the result of zeta potential. The mixture composite was also characterized by FT-IR. Although the characteristic peak of MWCNTs was hardly observed, the positions of all peaks did not shift comparing with DHPCA (Fig. 1C(d)). In addition, the peak at 1650 cm–1 assigning the ketone group which formed after chemical modification was not found. The results of absorption and FT-IR spectra demonstrated that DHPCA was chemically bonded onto MWCNTs via Friedel–Crafts chemical acylation to form MWCNTs-Py and was not obtained by adsorption or mixing. Additionally, C1s XPS of MWCNTs could be deconvoluted into two peaks, including sp2-hybridised carbon atoms of the graphite in the MWCNTs (C=C/C–C) at 284.5 eV and the atmospheric oxidation or residual oxides of MWCNTs (C–O–C) at 285.9 eV which was characterized as the defective structures (Fig. 1D(a)) [26,27]. The defective level of MWCNTs was estimated by the ratio of the area of defective structure to total area and was approximate to 11.8 %. In contrast, the C1s spectrum of MWCNTs-Py was deconvoluted into four peaks. In addition to the two peaks at the same position to MWCNTs, the two new peaks at 286.1 eV and 287.6 eV were assigned to the –Py species. The close electronegativity of carbon atom and hydrogen atom resulted in the close binding energy where C–O–C at 285.9 eV and C–O–H at 286.1 eV. Besides, the ketone group (C=O) bonded on the MWCNTs accepting abundant electrons showed lower binding energy (287.6 eV) than that of carboxylic group reported in the

literature [27]. Furthermore, the area ratio of C=O to C–O–H was 2.05 which was consistent with the theoretical data of 2.00, demonstrating that the expected Friedel–Crafts chemical acylation was successfully achieved. Raman spectrum of MWCNTs was often characterized the G band and D band (Fig. 2A(a)). The peak at 1575 cm–1 was assigned to vibrations of sp2-bonded carbon atoms in the two dimensional, hexagonal graphite layers of the nanotubes [28,29]. The peak at 1346 cm–1 (D band) accompanying with the shoulder peak at 1606 cm–1 (D′ band) was corresponded to sp3-hybridised carbon atoms at defect and disordered sites within the nanotubes [28,29]. The higher Raman shift at 2688 cm–1 (2D band) was characterized the overtone of the D band. Comparing with MWCNTs, the blue shift of G band and D were observed to MWCNTs-Py, demonstrating the covalent modification on MWCNTs was occurred via Friedel–Crafts chemical acylation (Fig. 2A(b)) [30]. Furthermore, the intensity ratio of G band to D band (ID/IG ) was from 0.99 of MWCNTs to 1.01 of MWCNTs-Py. The very close values indicated the modification based on Friedel–Crafts chemical acylation almost did not destroy the conjugated structure of MWCNTs. Also, TEM image showed the clear hollow structure to MWCNTs and MWCNTs-Py. This result revealed the coaxially tubular structure was retained after chemical modification.

Fig. 1 and Fig. 2 are here.

3.2. Electrochemical behavior of MWCNTs-Py/GC electrode

The electrochemical behavior of MWCNTs-Py was investigated by cyclic voltammetry (CV) at a scan rate of 0.05 Vs−1 in PBS (pH 7.0) (Fig. 3A). Comparing to MWCNTs/GC electrode, the

MWCNTs-Py/GC electrode showed a well-defined redox peaks between 0.4 V and –0.6 V. The anodic (Epa1) and cathodic (Epc1) peak potentials were 0.03 V and –0.20 V with formal potential (the average of Epa and Epc; E0′) of 0.09 V. This pair of peaks was attributed to electroactive protonated imine [31]. Additionally, one pair of weak peaks was observed at 0.23 V (Epa2) and 0.17 V (Epc2) corresponding to the small amount of imine structure [11]. As the scan rate increased from 0.05 to 0.5 V s–1, the anodic and cathodic peak currents of MWCNTs-Py increased simultaneously and were directly proportional to the square root of the scan rates, indicating that the electrochemical response of the MWCNTs-Py/GC electrode was a typical diffusion-controlled process; i.e., the electron transfer involved in this redox reactions occurred much faster than the diffusion of counter-ions into the electrode surface (Fig. 3B and 3C) [32]. The fast electron transfer contributed the unclear shift of peak potential while the scan rate increased. As the literature reported, the anodic and cathodic peak potential of insulated polymer containing imine structure showed clear positive and negative shift, respectively while the scan rate increased due to the slow electron transfer rate [23,32]. In this study, the unclear shift of peak potential was attributed to the monomer –Py bonding on MWCNTs directly, which was favorable to directly electron transfer. Also, retaining conjugated structure of MWCNTs during chemical modification enhanced the electrochemical behavior of MWCNTs-Py. Furthermore, MWCNTs also provided high surface area for grafting –Py. The surface coverage concentration (Γ) of –Py on MWCNTs-Py/GC electrode was calculated according to Eq (1) [33]:

(1)

where A is the electrode area (0.071 cm2), υ is scan rate (V s–1), F is Faraday’s constant, n is the electron transfer number (n = 1) and T is the temperature. The Γ of electroactive –Py on the MWCNTs-Py/GC electrode was estimated to 2.55 ×10−8 mol cm−2. This value was higher about 100 times than the previous literatures [23,32]. The high Γ of –Py demonstrated the high degree of chemical modification was achieved and the electroactivity of –Py could be maintained.

Fig. 3 is here.

3.3. MWCNTs-Py/GC electrode-based H2O2 sensor

It had been reported that the imine structure could be chemically oxidized by peracetic acid which was formed from the mixture solution of H2O2 and acetic acid [11, 23]. The oxidized imine structure, N-oxide could be reverted to imine structure by accepting two electrons via electrochemical reduction, thus increasing the reduction current. Based on this mechanism, the enzyme-free hydrogen peroxide sensor can be developed. In this study, DHPCA with pyridine ring is possessed of imine structure which is a sensitive probe to H2O2 in the presence of acetic acid. Besides, this small molecule is directly bonded on MWCNT by covalent bond. This modified MWCNT was favorable for electron transfer within –Py and MWCNT, thus improving the performance of electrochemical sensor. In order to meet the above conditions, acetic acid was added for providing carboxylic acid in this study. Fig. 4A(a) showed the CV of MWCNTs-Py/GC electrode in 0.2 PBS with acetic acid at pH 6.4. Similar to the CV of MWCNTs-Py/GC electrode at pH 7.0, it also showed a pair well-defined redox peaks. The anodic (Epa1) and cathodic (Epc1) peak potentials positively shifted to 0.04 V and –0.18 V.

According to the reaction EAg|AgCl|Cl‐ = E0

Ag|AgCl|Cl‐–[RT/F]

lnaCl‐, where a is the chemical

activity for the relevant species. It disclosed the standard potential corresponding to the reference electrode would shift positively due to the lower chemical activity in the acidic solution. As in the presence of H2O2 (0.5 mM), the reduction current increased apparently from 0.13 V to –0.6 V accompanying with the decrease of oxidation current (Fig. 4A(b)). The decreased oxidation current indicated that the parcel of imine structure was chemically oxidized by peracetic acid to form pyridine N-oxide, thus decreasing the coverage concentration of –Py for electrochemical oxidation. Comparing to the electrochemical reduction of imine structure in one electron transfer, pyridine N-oxide was involved in two electrons transfer, resulting in the increase of reduction current. This change became more pronounced as the H2O2 concentration increased to 1.0 mM, indicating the more peracetic acid was formed to oxidize MWCNTs-Py. Amperometric measurement was performed to evaluate the performance of the MWCNTs-Py/GC electrode toward increasing H2O2 concentrations at an applied potential of –0.5 V in PBS (pH 6.4) (Fig. 4B). Successive additions of H2O2 at a stirrer rotation speed of 400 rpm showed that the current responded rapidly with time. The response time achieving 95% of the steady-state current was 1.8 s. The rapid response was attributed to H2O2 could diffuse freely through the porous surface constructing by MWCNTs. Also, the presence of MWCNTs with retaining its originally conjugated structure enhanced the electron transfer rate. The reduction current increased with the increase of H2O2 concentrations. However, it was hardly to observe this response while the MWCNTs-Py/GC electrode was at pH 7.0, meaning that the MWCNTs-Py could not be oxidized to form MWCNTs-Py N-oxide in the absence of acetic acid. The corresponding calibration plot for the MWCNTs-Py/GC electrode was presented in the inset of Fig. 4B. The MWCNTs-Py/GC electrode was sensitive to H2O2 in a linear range from 1 µM to

12.5 mM with the sensitivity of 911.4 µA mM–1 cm–2 (R2 = 0.996) for five measurements (n = 5). The detection limit was 0.77 µM at a signal-to-noise ratio of 3. The sensitivity based on fabricated MWCNTs-Py/GC electrode was comparable to the previous literatures [11,22]. Moreover, the tolerable H2O2 concentration within detecting range was higher than other imine structure-based sensors due to the abundant of –Py on MWCNTs preventing the oxidation reaction achieving saturation.

Fig. 4 is here.

3.5. MWCNTs-Py-GOD/GC electrode-based glucose biosensor

H2O2 is one of by-products of glucose reaction catalyzed by GOD in biological systems [8]. Therefore, numbers of researches in developing glucose biosensor were based on the detection of produced H2O2 [10,11]. However, H2O2 and glucose sometimes coexist in some samples, resulting in the harsh challenge to distinguish their respective signal. It had been demonstrated that MWCNTs-Py/GC electrode was sensitive to H2O2 in acidic solution, but was not in neutral solution. This property provided a good opportunity for preparing dual H2O2 (at acidic solution) and glucose (at neutral solution) sensor. In the fabrication of glucose biosensor, the bioactivity of GOD majorly dominated the sensor’s performance. Using electrostatic technique to immobilize GOD is widely recognized as an ideal candidate because the bioactivity of the immobilized GOD is well preserved. Based on the result of zeta potential, the charge of MWCNTs-Py is 49.7 mV. This positive charge is attributed to the protonated –Py (=N+H–) by PPA. In addition, the isoelectric point (IEP) of GOD

is about 4.2, meaning that the charge of GOD at pH 7.0 is negative. By the positively charged MWCNTs-Py and negatively charged GOD, GOD can be immobilized directly via electrostatic attraction on the –Py of MWCNTs-Py. The zeta potential disclosed the charge of MWCNT-Py changed from 49.7 mV to –23.5 mV after adsorbing GOD, meaning that abundant GOD was attracted to form MWCNT-Py-GOD. The amount of immobilized GOD was quantified by the absorption spectra of residual GOD. GOD aqueous solution showed an obvious peak at 277 nm and its absorption intensity was proportional to its concentration (Fig. 5A and inset). Hence, the quantity of loading GOD on MWCNTs-Py could be estimated by the freely un-adsorbed GOD and was 1326.4 U (62.5 mg) per mg of MWCNTs-Py. The adsorbed quantity was much higher than PBBIns-Gs and wet anion exchange resin due to the high surface area of MWCNTs and strong positive charge MWCNTs-Py [11,34]. In addition, the bioactivity of MWCNT-Py-GOD was characterized by H2O2 assay kit using fresh free GOD as a reference. The relative bioactivity of immobilized GOD on MWCNTs-Py was 93.2 %, demonstrating that the immobilization of GOD based on electrostatic attraction could mostly maintain its natural structure. The electrochemical behavior of MWCNTs-Py-GOD/GC electrode was performed within the scanning range of 0 to –0.7 V at pH 7.0 (Fig. 5B(b)). Comparing to the MWCNTs-Py/GC electrode, a pair of well-defined redox peaks was observed at –0.39 V (Epa) and –0.49 V (Epc) with a E0′ of –0.44 V (Fig. 5B(a)). It meant the bioactive GOD had been successfully immobilized on the modified electrode and the obvious direct electron transfer occurred within the redox active center (FAD/FADH2) as Eq. (2) shown.

GOD (FAD) + 2e– + 2H+ ↔ GOD (FADH2)

(2)

Also, the surface coverage of GOD concentration could be calculated by Eq. (1) and was 3.5 ×10−9 mol cm−2. The coverage density of GOD was larger about 10 times than literatures, indicating MWCNTs-Py composite had good biocompatibility and could maintain the bioelectrocatalytic activity of GOD [11, 35,36]. As increased the glucose concentration, the reduction peak current decreased significantly accompanying with the decrease of oxidation peak current (Fig 5B(c,d)). It disclosed the oxygen in the solution was consumed during the oxidation of glucose based on the following mechanism.

GOD (FAD) + glucose → gluconolactone + GOD (FADH2)

(3)

GOD (FADH2) + O2 → GOD (FAD) + H2O2

(4)

Amperometric response toward glucose recording for the MWCNTs-Py-GOD/GC electrode at the applied potential of –0.55 V under saturated oxygen was shown in Fig. 5C. With successive addition of glucose, the reduction current decreased due to the enzymatic oxidation reaction of glucose consuming oxygen. However, the current of glucose based on the modified electrode of mixing GOD with MWCNTs did not response after successive addition of glucose (Fig. 5C inset (bottom)). This result might be attributed to the process of fabricating electrode. The immobilization of GOD onto MWCNTs-Py was based on the electrostatic interaction. This MWCNT-Py-GOD was further washed by PBS and used high-speed centrifugation (12000 rpm) to remove the free GOD. The immobilization of GOD onto MWCNTs without containing DHPCA was also prepared according to the above purification process. However, the GOD could be removed easily due to the non/weakly adsorbing to MWCNTs, resulting in no response

after adding glucose. It reveals that GOD can not be directly immobilized onto MWCNTs without modifying by DHPAC. The response time achieving 95% of the steady-state current based on MWCNTs-Py-GOD/GC electrode was 2.7 s. The modified electrode responded linearly to the glucose concentration over the range of 3 μM to 7 mM with a sensitivity of 68.1 µA mM–1 cm–2 for five measurements (n = 5). The glucose biosensor performances based on MWCNTs-Py-GOD/GC electrode showed high performance comparing to literatures (Table 1).

Fig. 5 and Table 1 are here.

3.4. Reproducibility, stability and selectivity of H2O2 and glucose sensors

The reproducibility of the H2O2 sensor was investigated by repeating five times of loading MWCNTs-Py on GC electrode, measuring 1 mM H2O2 and polishing MWCNTs-Py/GC electrode. The relative standard deviation (R.S.D.) was 4.4%. Similarly, the proposed glucose biosensor was tested by 0.5 mM of glucose; meanwhile the MWCNTs-Py was replaced by MWCNT-Py-GOD. The obtained value of R.S.D. was 5.2%. These results indicated that the process for preparing H2O2 and glucose sensors were stable. In addition, the storage stabilities of sensors were carried out by measuring 1 mM of H2O2 and 0.5 mM of glucose within 2 weeks (Fig. S1). Because the MWCNTs-Py composite was considered as a stable material, MWCNTs-Py/GC electrode was exposed at 25 oC under atmosphere. For 2 weeks storage, this modified electrode corresponding to 1 mM of H2O2 did not change clearly. The glucose sensors based on MWCNTs-Py-GOD/GC electrode retained 81.2% of its initial bioactivity at 4 oC after 2 weeks storage. The good stability could be attributed to the firm and highly concentrated GOD

on MWCNTs-Py, thus preventing the drastically conformational change of GOD. Ascorbic acid (AA) and uric acid (UA) that occur naturally in the human body are often used as the interfering species to test the selectivity of glucose biosensor. Fig. S2 showed the selectivity of proposed glucose biosensor. The current did not response as the addition of AA (1mM) and UA (1mM), indicating that AA and UA did not influence the obtained value. This result could be explained by the electrochemical properties of AA and UA. It had been reported that AA and UA could be directly electrocatalyzed at potentials of 0.25 and 0.54 V, respectively [43]. The applied potential of detecting glucose was negative in this study, thus avoiding the effects of such interfering compounds. Furthermore, the used of bovine serum albumin (100 nM) as the protein in human blood to exam the glucose biosensor is performed. The current also did not response unless adding glucose (1 mM). Therefore, it can be demonstrated the MWCNTs-Py-GOD/GC electrode is possessed of highly selective to glucose.

3.5. Human serum and whole blood sample detections in glucose

To evaluate the accuracy of proposed glucose biosensor in real sample detection, the MWCNTs-Py-GOD/GC electrode was used for detecting the glucose present in human serum. The concentrations of five real samples were also quantified by glucose assay kit. The results were summarized in Table 2. It could be seen that the obtained values from proposed biosensor were all smaller than those from assay kit. This phenomenon was attributed to the produced H2O2 in biological catalysis process reacted with carboxylic acid group existing in some organic component in human serum. This reaction would generate peroxyacid that oxidized –Py to form –Py N-oxide that we had described in H2O2 sensor. Even so, the values measured by

proposed glucose biosensor were very close to those from assay kit, indicating that using MWCNTs-Py-GOD/GC electrode for detecting glucose was accurate and reliable. Furthermore, the detection of glucose in whole blood was also examined. Comparing with serum, the direct detection of glucose in whole blood should be concerned the blood cell and platelet that would affect the diffusion of glucose to the probe. So far, most of the literatures about the developments of glucose biosensor are based on the detection of serum samples. In this study, the detection of glucose concentration in human serum was injecting 0.5 ml of serum into an electrochemical cell which contained 20 ml of PBS. The dilute process and stirring state eliminate the effect of diffusion. Therefore, the obtained values are closely agreed with those measures in hospital (Table 2). These results reveal the proposed biosensor is suitable for monitoring glucose in serum as well as whole blood.

Table 2 is here.

4. Conclusion

A MWCNTs derivative, MWCNT-Py was synthesized via Friedel–Crafts chemical acylation. The retained highly conjugated MWCNTs resulted in the rapid electron transfer. The electroactive –Py provided dual H2O2 (acidic PBS) and glucose (neutral PBS) sensor. The proposed H2O2 sensor based on MWCNTs-Py/GC electrode showed performances, including high sensitivity, wide linear range, rapid response time and good stability. The positively charged MWCNTs-Py also provided advantageous environment for loading negatively charged GOD via

electrostatic attraction. The reproducibility, stability and reliable of MWCNTs-Py-GOD/GC electrode was acceptable to monitoring glucose in the clinical detection of glucose.

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Figure Captions

Fig. 1. (A) Synthesis of MWCNTs-Py. (B) Absorption spectra (a) MWCNTs, (b) DHPCA, (c) MWCNTs-Py and (d) mixing of DHPCA and MWCNTs. Inset: photo images of MWCNTs (left) and MWCNTs-Py in aqueous solution. (C) FT-IR spectra of (a) MWCNTs, (b) DHPCA, (c) MWCNTs-Py and (d) mixing of DHPCA and MWCNTs. (D) C 1s XPS spectra of (a) MWCNTs and (b) MWCNTs-Py.

Fig. 2. (A) Raman spectra of (a) MWCNTs and (b) MWCNTs-Py. TEM images of (B) MWCNTs and (C) MWCNTs-Py.

Fig. 3. (A) CVs of (a) MWCNTs/GC and MWCNTs-Py/GC electrodes in 0.2 M PBS at pH 7.0. (B) CVs of MWCNTs-Py/GC electrode in 0.2 M PBS at various scan rates (0.05~0.5 V s–1). (C) Plots of peak currents versus square root of scan rate.

Fig. 4. (A) CVs of MWCNTs-Py/GC electrode in the presence of (a) 0 mM, (b) 0.5 mM and (c) 1.0 mM H 2O2 at pH 6. (B) Current–time plots of MWCNTs-Py/GC electrodes with the additions of different [H2O2] at (a) pH 6.5 and (b) pH 7.0. Inset: Linear dependences of the response currents vs. [H2O2] (n = 5).

Fig. 5. (A) Absorption spectra of different [GOD]. Inset: Linear dependences of the absorbance intensity vs. [GOD]. (B) CVs of (a) MWCNTs-Py/GC electrode without glucose and (b) MWCNTs-Py-GOD/GC electrode in the presence of (b) 0 mM, (c) 1 mM and (d) 2 mM glucose at pH 7. (C) Current–time plot of MWCNTs-Py-GOD/GC electrode with the additions of glucose at pH 7.0 under saturated oxygen. Inset: current-time plot of MWCNTs-GOD/GC electrode with the additions of glucose (bottom) and surface morphology of MWCNTs-Py-GOD/GC electrode (top). (D) Linear dependences of the response currents vs. [glucose] (n = 5).

Table 1. Analytical properties of different glucose biosensor

GO–PAMAM–Ag/GOD/CS/GC

Sensitivity (A mM–1cm–2) 75.72

Linear range (mM) 0.032–1.89

Response time (s) 10

Nafion/01/GOD/GC

3.54

0.001–0.06

-

38

{GOD/PEI} 3/CNT/GCE

106.57

up to 0.53

-

39

GOx/TCS-TiO2/chitosan /GC

23.20

0.05–1.32

-

40

Gs-nanoAu-GOD/GC

56.93

0.2–2

-

41

AuNPs/G/MWCNTs/GC

29.72

0.005-0.175

5

42

MWCNTs-Py-GOD/GC

68.1

0.003–7.0

2.7

this work

Ref. 37

Table 2. Human serum and whole blood detection based on MWCNTs-Py-GOD/GC electrode. Sample

Standard method (mM)

a

1 2a 3a 4a 5a 6b 7b 8b 9b

c

4.6 6.7 c 5.3 c 5.9 c 6.2 c 7.8d 10.5 d 8.2d 6.5d

a

serum sample

b

whole blood

c

glucose assay kit

d

hospital biochemistry laboratory

Figures

Fig. 1

MWCNTs-Py-GOD/GC (mM)

Relative error (%)

4.41± 0.14 6.45 ± 0.17 5.14 ± 0.10 5.67 ± 0.16 6.01 ± 0.13 7.51 ± 0.18 9.97 ± 0.22 8.31 ± 0.20 6.32 ± 0.14

‐4.1 ‐3.7 ‐3.0 ‐3.9 ‐3.1 ‐3.7 ‐5.0 1.3 ‐2.8

Fig. 2

Fig. 3

Fig. 4

Fig. 5