Pt Nanoparticles Deposited and Tannic Acid-Reduced Graphene

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Pt Nanoparticles Deposited and Tannic Acid-Reduced Graphene Oxide for Switchable Bioelectronics and Biosensors Based on Direct Electrochemistry Bilge Akkaya, Bekir Çak#ro#lu, and Mahmut Ozacar ACS Sustainable Chem. Eng., Just Accepted Manuscript • Publication Date (Web): 11 Jan 2018 Downloaded from http://pubs.acs.org on January 11, 2018

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ACS Sustainable Chemistry & Engineering

Pt Nanoparticles Deposited and Tannic Acid-Reduced Graphene Oxide for Switchable Bioelectronics and Biosensors Based on Direct Electrochemistry

Bilge Akkayaa, Bekir Cakiroglub and Mahmut Ozacara, b,*

a

b

Sakarya University, Science & Arts Faculty, Department of Chemistry, 54187 Sakarya, Turkey

Sakarya University, Biomedical, Magnetic and Semiconductor Materials Research Center (BIMAS-RC), 54187 Sakarya, Turkey

*Corresponding Author. Tel.:+90 264 295 60 41; fax:+90 264 295 59 50. E-mail address:[email protected](M.Ozacar).

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Abstract

In this study, we reported a novel biosensor based on direct electrochemistry of glucose oxidase (GOx) at Pt nanoparticles deposited and tannic acid-reduced graphene oxide nanocomposite modified glassy carbon electrode. Tannic acid (TA) was utilized for the simultaneous green reduction of Pt4+ and graphene oxide (GO), along with modifying the reduced GO for the GOx immobilization, and thus constructing switchable surface with pH and temperature alterations. Upon the electrochemically oxidation of TA to quinone, enhanced electron transfer was obtained, and a third generation biosensor was fabricated by using π-π interaction between GO and TA, and Schiff-base assisted hydrogen bonds between GOx-TA interactions. The redox peaks were observed at a formal potential of −0.462 V with a peak separation (∆Ep) of 56 mV, which reveals the fast electron transfer. The linear response to glucose oxidation was in the range of 2-10 mM with a limit of detection of 1.21 µM and a sensitivity of 27.51 µAmM−1cm−2. Upon the deposition of poly(Nisopropylacrylamide) (PNIPAAm) onto the constructed biosensor via hydrogen bonds, on-off biosensor was fabricated with a zipper-like interfacial properties upon the formation of shrunken and compact globule PNIPAAm structure, and varying surface charge. Therefore, this study confirmed the versatile aspect of natural TA without using complicated methods.

Keywords: Reduced graphene oxide, Green chemistry, Glucose oxidase, Smart interfaces, pH controlled surface.


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Introduction

The blood glucose measurement in vitro or in vivo, have been paid much attention to produce reliable methods, and to devise electrode materials for the improvement of the response, and enzyme-based electrochemical biosensors since the development of the first glucose biosensor. Graphene and its derivatives, such as graphene oxide (GO), reduced graphene oxide (rGO) have recently emerged as an advanced materials in a various application areas owing to its unique mechanical and electronic properties, which enable us to employ this superior material in biosensor areas1. Graphene based materials display excellent electrical conductivity, high surface area to volume ratio, biocompatibility, obtainability with a lower cost compared with other carbonaceous materials, and strong mechanical strength2, and are employed in various biosensors, such as metal ion sensors and immunosensors3,4. Despite such advanced electrochemical capability of graphene derivatives, there still has been significant interest in the development of new graphene-based nanomaterials to give impetus to the electroanalytical applications. Recently, third generation biosensors (TGB) have been fabricated based on the direct electrochemistry between oxidoreductases (e.g. glucose oxidase (GOx)) and electrode5. Graphene-derivatives have been used as an electrode material in TGB 6

, and this material can be modified to enhance surface area, electrical conductivity, catalytic

activity and biocompatibility, resulting in improved enzyme performance. The noble nanoparticles (NPs), especially platinum NPs, can lead to synergistic effect on electrocatalytic activity by catalyzing hydrogen peroxide, which releases from the oxidoreductase biocatalytic reactions, thus enhancing the sensitivity of the enzyme immobilized electrochemical biosensor. Also, Pt NPs can expand the surface area by allowing the fast diffusion of target analytes7, 8. There are green reduction approaches of GO in the literature, and one of the important reducing agents is natural polyphenols (e.g. tannic acid (TA)), which modify rGO



surface by binding through π-π interactions9,10. Also, polyphenols can bind to protein molecules via mainly hydrogen bonds, and this phenomenon has been used for the enzyme immobilization using TA modified surfaces11. TA can bind to the protein molecules through covalent Schiff base bonds only by basic pretreatment of TA to gain quinone moieties12. Recently, on-off biosensors have been gaining much attention with their interesting switchable surfaces with temperature and pH alteration. The stimuli-responsive polymer poly(N-isopropylacrylamide) (PNIPAAm) has been used extensively for the thermosensitive behavior in the drug delivery and biosensor applications. According to the literature, TA can establish interaction with PNIPAAm and other polyamides via hydrogen and hydrophobic bonds under lower critical solution temperature (LCST) of around 31-32 °C13-14. However, there is no work using this phenomenon in the switchable biosensor surfaces.

In this work, we report a novel Pt NPs deposited and TA-reduced graphene oxide nanocomposite for glucose oxidase (GOx) immobilization and its application for glucose sensing with a decent sensitivity based on direct electrochemistry. To obtain a good selectivity, and catalytic activity, GOx, mostly used enzyme in the glucose biosensors, was chosen owing to its high catalytic activity and specificity to glucose. Also, Pt NPs have synergistic effect on the electrochemical response, and improve the sensitivity. GO and Pt4+ reduction were carried out in a facile and low-cost way by using natural reducing agent, TA. Also the deposited TA on the rGO via π-π interactions provided the biocompatible surface for GOx immobilization through hydrogen bonds. The synthesized nanocomposite was dropcasted on a glassy carbon electrode (GCE). Also, pH and temperature controlled switchable biosensor surface was obtained by using the TA- PNIPAAm interactions. The electrochemical measurements were carried out with cyclic voltammetry (CV) and differential pulse voltammetry (DPV) techniques.


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Materials and Methods

One-pot synthesis of platinum nanoparticles deposited reduced graphene oxide composite (rGO-Pt NPs)

The graphene oxide (GO) was synthesized according to the previously published method 15. Then, 1 mL of GO (0.5 mg mL-1) was dispersed in 2 mL of 5 mg mL-1 TA aqueous solution under vigorous stirring, and, 650 µL of platinum standard solution and 6µL of 0.5 M sodium hydroxide solution were added into the suspension at room temperature. The reduction of GO was enhanced by keeping the resulting mixture at 90 °C for 30 min. Then, the black reduced GO (rGO) was centrifuged and washed twice with deionized water (DW) to eliminate the residual TA and redispersed in 1mL DW for the GCE coating.

Fabrication of the rGO-Pt NPs-GOx modified GCE

Before nanocomposite deposition, the GCE (3 mm in diameter) were polished with alumina powder and subsequently rinsed and sonicated in DW. Subsequently, glassy carbon electrodes were modified with 8 µL of the composite (0.5 mg mL-1) by drop-casting and dried in air. The enzyme immobilization was realized by immersing the nanocomposite-coated electrodes into 250 µL of GOx solution (40 mg mL-1) at 4 °C for 4 h. Finally, the modified GCE was rinsed with PBS to remove the loosely bound GOx and was stored at 4 °C in a refrigerator under dry conditions when not in use.

PNIPAAm covering on rGO-Pt NPs-GOx/GCE



The rGO-Pt NPs-GOx nanocomposite on the GCE was covered with PNIPAAm to investigate the temperature, pH sensitive behavior. 50 µL of PNIPAAm (20 mg mL-1) solution in pH 7.4 PBS was drop-casted on the nanocomposite modified GCE, and the obtained sensitive bioconjugate (PNIPAAm- rGO-Pt NPs-GOx) was left to dry at room temperature for 30 min.

Results and discussion

Preparation and characterization of electrodes, and electron transfer through TA

Graphene oxide (GO) was synthesized according to modified Hummers’ Method15 (Fig. S1), followed by chemically reduction, and the electrochemical and on-off biosensor were fabricated as depicted in Scheme 1. Simultaneous green reduction of GO and Pt4+ was carried out with green reducer TA. Also, TA was deposited on the rGO surface via π-π interactions between the phenolic groups of TA, and benzene rings of rGO. GOx was immobilized on TA coated rGO via covalent assisted hydrogen bonds by immersing the rGO-Pt NPs-GOx/GCE into the enzyme solution.

The effects of different parameters on electrochemical biosensor fabrication were investigated. The optima of GOx immobilization time, platinum amount and nanocomposite volume for the deposition were determined for the electrochemical biosensor fabrication (Fig. S2, Fig. S3, Fig. S4), and their optimum values were found to be 4 h, 650 µL of 1000 ppm platinum standard solution, and 8 µL of composite solution, respectively. The current response was diminished with the increasing immobilization time. Although, 850 µL platinum solution led to a bit more current response, 650 µL was chosen owing to the cost care, and


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upon the deposition of different volume of nanocomposites, the redox currents remained constant after 8 µL of nanocomposite deposition.

The immobilized GOx was determined according to the Bradford Method16, the immobilized GOx amount was found to be 3.2 ± 0.6 mg (enzyme) mg-1(nanocomposite). Also, DNSA glucose determination test demonstrated that immobilized GOx increased the enzyme activity by 140 % compared to that of free GOx. According to the literature, enzyme activity can enhance upon immobilization owing to the formation of much favorable 3D structure for the enzymatic activity17. According to the inductively coupled plasma measurements, it was confirmed that about 5 % of the nanocomposite consists of platinum nanoparticles. The morphologies of rGO and rGO-Pt NPs were investigated by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and high-resolution TEM (HRTEM). Fig. 1A and 1B display the SEM and TEM images of rGO with a wrinkled and a high surface area for the enhanced enzyme immobilization. Fig. 1C and 1D show TEM images of rGO-Pt NPs, and the Pt NPs demonstrated a good size distribution in the range of 2.0 nm to 4.5 nm (inset of Fig 1C), and uniformly distributed on the rGO, which enhance the reproducibility of the biosensor. The HRTEM image of the rGO-Pt NPs (Fig. 1E) demonstrated that the synthesized Pt NPs possess a particle size of about 3.4 nm. The d-spacings of adjacent fringe was found to be 2.2 Å, which corresponds to the {111} plane of face-centered cubic (fcc) Pt, and confirms the formation of high crystalline Pt NPs. Energy dispersive X-ray spectroscopy (EDS) analysis confirmed the Pt NPs presence by yielding a peak of Pt M around 2 keV, as shown in Fig. S5.

The information about TGA, Zeta potential analysis, and UV-Vis measurements of the nanocomposites were given in Fig. S6, Fig. S7, and Fig. S8, respectively. UV-Vis spectra of



the nanocomposites were given in Fig. S8 to investigate the time dependent reduction process of GO. Accordingly to Fig. S8, TA can attached to the reduced graphene oxide surface via π-π interactions along with modifying the surface for enzyme immobilization using hydrogen bonding between nitrogen containing moieties of enzyme and hydroxyls of TA, which was confirmed by FTIR. In Fig. S8 (b), GO displays two characteristic absorption peaks. An intense absorption band at 230 nm attributed to π–π⁎ transition and the second band at 305 nm corresponding to the n–π* transition of the C=O bond were observed. Upon the reduction with TA for 30 min, and 60 min, n–π* transition band disappeared, which confirms the reduction of GO. According to the UV-Vis spectra, the π–π* transition and n–π* transition of GO was blue shifted to lower wavelength of 212 nm and ca. 275 nm, respectively, in the spectra of rGO for 30 min, and 60 min (Fig. S8 c and d). This finding confirms the π–π stacking interaction between the hydrophobic aromatic regions of GO and that of TA along with hydrogen bond formation (H-type π-π stacking molecular model)18. Thus, TA can attached to rGO surface, leading to modification of surface for enzyme immobilization.

In order to confirm the synthesis of graphene oxide, reduction and modification of its surface with TA, and GOx immobilization, FTIR analyses were performed. FTIR spectra of samples measured in the range of 400–4000 cm−1 are demonstrated in Fig. 1F. In the GO spectrum, the peak at 1726 cm−1 is assigned to the C=O stretching vibration of COOH groups, while the broad bands centered at about 1380 cm−1 and 3170 cm−1 are associated to the deformed and stretching mode of OH, respectively19,20, and the peaks at1039 cm−1 and 1620 cm−1 are attributed to the C-O, and C=C stretching vibrations. In the rGO spectrum, the peaks in the GO diminished or nearly disappeared, and peaks attributed to TA appeared, which confirm the reduction and TA deposition on rGO. In the spectrum of rGO, the adsorption peaks at 1712 cm−1, and 1613 cm−1 correspond to the C=O stretching, aromatic C-O stretching,


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respectively21. Also, the peaks at 1329 cm−1 originated from in plane -OH bending, at 1060 cm−1 associated to aromatic C-H deformation vibrations, at 1444 cm-1 originates from symmetric stretching vibration of C-O, and at 1512 cm-1 assigned to C-C stretching in rings were also observed11. The peak at 1581 cm-1 can be attributed to the presence of quinone moieties, which result from the oxidation of hydroxyls of TA upon GO and Pt NPs reduction. This peak was also observed in our previous study, in which quinones formed with the increment in the medium pH. However, this peak totally disappeared in the rGO-Pt NPs-GOx spectrum, which verifies the Schiff base bond formation22. The presence of amide II at 1540 cm−1 and amide I at 1646 cm−1 confirmed the immobilization of GOx and the preservation of secondary structure after the immobilization23. The carbonyl group (C=O) stretching vibration in rGO spectrum was shifted from 1697 cm−1 to 1713 cm−1 in the rGO-Pt NPs-GOx, indicating the hydrogen bonds between TA and GOx24,25. Therefore, improved enzyme immobilization was carried out to construct the enzyme based biosensor.

Direct electrochemistry of GOx on rGO-Pt NPs/GCE

The nanocomposite modified electrodes were scanned at the potential range of 0.1-0.9 V right before the experiments to oxidize phenolic hydroxyls of TA to obtain quinone moieties26, which enable electron transfer (Scheme 1). During the electron transfer between the GOx and electrode, formed quinones groups are reduced to hydroquinones at the direct electron transfer (DET) potential. This phenomenon confirms that electrochemically tailored TA can continuously transfer the electrons from the GOx to the electrode. Thus, an electrochemical biosensor, which possess fast electron transfer was fabricated. Additionally, PNIPAAm was coated on the prepared electrochemical biosensor, to impart the switchable interface to the



biosensor based on hydrogen bonds, and the pH and thermosensitive surface was comprehensively explained in the Section 3.4. Direct electrochemistry of GOx on rGO-Pt NPs has been investigated by cyclic voltammetry (CV). Fig. 2A demonstrates the CV curves of GOx/GCE, GO-GOx/GCE, rGO-GOx/GCE, and rGO-Pt NPs-GOx/GCE in deaerated 0.1 M PBS. GOx/GCE (Fig. 2A, line a), and GOGOx/GCE (Fig. 2A, line b) demonstrated no redox peak, indicating the insufficient and inappropriate GOx immobilization. On the other hand, the rGO-GOx/GCE (Fig. 2A, line c) yielded a pair of redox peaks, which is associated to the electrochemical reaction of FAD. This finding can confirm that TA deposition and GO reduction allow the appropriate enzyme immobilization for the direct electron transfer. However, rGO-Pt NPs-GOx/GCE (Fig. 2A, line d) gave a more enhanced and quasi-reversible redox peaks with the anodic peak potential (Epa) at -0.434 V and the cathodic peak potential (Epc) at -0.490 V. The peak to peak separation (∆Ep) is found to be 56 mV. The formal potential (E°) calculated from the average of cathodic and anodic peak potential is -0.462 V. The improved redox peak of rGO-Pt NPsGOx/GCE can be associated to the enhanced surface area and increased electrical conductivity resulting from the Pt NPs deposition. This value is close to the standard electrode potential of −0.459 V (vs. Ag/AgCl) for FAD/FADH227, suggesting that the DET between GOx and the nanocomposite was accomplished. This finding confirms that as a protein binding agent, TA provided a short distance between GOx and rGO for the DET, and the Pt NPs reinforced the redox peaks. According to the literature, DET can be achieved by using adsorption based immobilization, or covalent immobilization with the short crosslinking agent to reduce the active site-surface distance27,28. Enzyme-free electrodes demonstrated no redox peak in the potential region of experiments (data not shown). The effect of pH on the DET of GOx on rGO-Pt NPs/GCE was studied at a scan rate of 100 mV.s-1 using CV at GOx working range of pH 5.0-8.6, which is good agreement with the


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effective GOx working pH range and a strong dependence on medium pH was observed. A decrement in pH leaded to a positive shift in both Epa and Epc, indicating that hydrogen ions involve in the electrochemical reaction of GOx, and DET potential decreases with decreasing pH. As shown in Fig. 2B, the formal potential of GOx has a linear dependence on the pH in the range from 5.0 to 8.6. The slope value for pH vs. formal potential is found to be ˗(56.4 ± 0.4) mV pH-1 (R2 = 0.988)( Fig. 2B, inset). The observed slope value (56.4 mV pH-1) is very close to the theoretical value (58.6 mV/pH) for two-proton couple with two-electron transfer of GOx as shown Eq. (1)29. The observed slope value is comparable to the studies in the literature6,27 . The small decrement in the value compared to the literature can be attributed to the acidic nature of TA, and there is no substantial effect on the efficiency of biosensor. According to the Fig. 1B, the maximum current appeared between pH 7.0 and 8.6, and physiologic pH 7.4 was chosen as pH of experiment media for being appropriate for body fluids.

FAD 2H 2e

FADH

(1)

Fig. 2C illustrates the effect of scan rate on CV characteristics of the DET of rGO-Pt NPsGOx/GCE. As the scan rate increased from 0.02 to 0.2 V s-1, both anodic (Ipa) and cathodic (Ipc) peak currents increased linearly with scan rate, indicating that the redox reaction is a surface controlled process. Nevertheless, the value of ∆Ep was independent on the scan rates, and remained constant. The electron transfer rate (ks) between GOx and rGO-Pt NPsGOx/GCE was calculated using Laviron equation (n∆Ep < 0.2 V):

log ks α log1 α 1 αlogα logRT⁄nFυ α1 αnFΔEp⁄2.303RT (2)



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where α is the charge transfer coefficient (~0.5) and υ (Vs-1) is the scan rate. The constants R, T and F represent their usual meanings (R = 8.314 J K-1 mol-1, T = 298 K, F = 96,485 C mol1

). At the scan rate of 0.1 V s-1, the ks value for rGO-Pt NPs-GOx/GCE has been estimated to

be 3.05 s-1. Although, there are studies, which possess superior rate constant such as, for GOx immobilized on rGO-ZnO (7.55 s-1)30, for immobilized GOx on rGO-magnetic nanoparticles (13.78 s−1)31, the current study is better or comparable to the values observed for GOx immobilized on ERGO-MWCNTs (3.02 s-1)28, on rGO-Ag NPs nanoparticles (2.0 s-1)32, on graphene/gold nanoparticles/poly neutral red (1.73 s-1)33, and on graphene and cobaltphthalocyanine composite (3.57 s-1)34. When compared to poly neutral red, and cobaltphthalocyanine, it can concluded that TA is both appropriate modifying agent for enzyme immobilization, and a decent alternative electrically active linker, which makes the GO a decent conductive material with faster electron kinetics owing to the Pt NPs deposition. The surface amount (Γ, molcm-2) of electroactive GOx immobilized on the surface of composite can be estimated according to the formula below35,

Q nFAΓ

(3)

where F is the Faraday constant, Q is the charge consumed in the redox reaction, which can be found from the integration of cathodic peak, Γ is the electroactive glucose oxidase amount, n and A stand for the number of transferred electrons, which corresponds to 2 in this reaction, and the area of the GC electrode (0.071 cm2), respectively. The amount of electroactive glucose oxidase at the rGO-Pt NPs-GOx/GCE was estimated to be 2.72 ± 0.4×10−11 mol cm−2, which is comparable or larger than that observed for the saturated monolayer surface coverage of GOx on the electrode (1.7x10-12 mol.cm-2)36. The amount of immobilized GOx was significantly larger than that of monolayer amount, and that of rGO-GOx/GCE (4.14±0.2x10-


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12

). This finding confirms that enhanced electroactive GOx amount was achieved with Pt NPs

deposition8, and the surface was efficiently employed in terms of GOx binding. Also, literature confirms the efficient electroactive GOx immobilization, and improved electrochemical signals with the preserved enzyme structure, upon the utilization of electroactive molecules and metal NPs resulting in expanded surface area and conductivity6,34. In this context, Liu et al confirmed that Au NPs deposited rGO facilitates the electron transfer on electrode surface, and provides a large surface area for the immobilization of antibody. Therefore, metal nanoparticles deposited graphene derivatives have beneficial properties for the improved sensitivity in biosensor applications4.

Performance of rGO-Pt NPs-GOx/GCE based glucose biosensor

DPV and CV measurements were carried out in O2-saturated PBS (0.1 M, pH 7.4) using rGOPt NPs-GOx/GCE in the absence and presence of 1, 2, 4, 6, 8, 10, 12 mM of glucose with the scan rate of 100 mV.s−1. As shown in Fig. 2D and Fig. 3A, the reduction peaks were diminished with the increasing glucose concentration. The addition of glucose solutions into O2-saturated PBS (0.1 M, pH 7.4) led to an increase in the Ipa intensity and a decrease in the Ipc intensity (Fig. 3A). These findings confirmed the bioelectrocatalytic activity of rGO-Pt NPs-GOx/GCE towards glucose through DET mechanism as shown in Eqs. (4) and (5):

GOD FADH O → GOD FAD H O

(4)

GOD FAD glucose → gluconolactone GOD FADH

(5)



In the O2-saturated medium, nearly all GOx molecules are oxidized biocatalytically according to the Eq. (4). Therefore, the electrochemically reducing current required for the oxidized GOx, has the maximum value. Upon the addition of glucose, the reduction current for FAD of GOx, diminished gradually. This is owing to the fact that with the addition of glucose, more GOx (FAD) was converted to GOx (FADH2) according to the biocatalytic reaction between GOx (FAD) and glucose (Eq. (5)). Therefore, the cathodic current for GOx (FAD) decreased linearly with the increasing of glucose concentration a certain concentration range (Fig. 3B). The fabricated biosensor was oxygen sensitive and the glucose measurements were carried out by considering the glucose concentration in merely O2 saturated PBS as 0. The inset of Fig. 3B shows that the reduction peak current intensity of the fabricated electrode decreases linearly at the range of 2-10 mM. Thus, a mediator-free TGB was fabricated. The linear regression equation can be expressed as Ip(µA) = 74.289 ± 0.646 (µA) - 1.953 ± 0.101 (glucose concentration) (µA mM-1), R2 = 0.989. The linear measurement range observed in this biosensor is comparable or broader compared to those reported for biosensors constructed based on TGB given in Table 1. The calibration plot over the linear concentration range exhibited a sensitivity of 27.51 mA mM−1cm−2, with a correlation coefficient of 0.9893. The limit of detection (LOD) was determined according to the formula, LOD = 3 Sb/S (Sb: standard deviation of ten blank measurements and S: sensitivity), and was found to be as low as 1.21 µM (S/N=3). The sensitivity, linear range and detection limit of developed biosensor were enhanced and are comparable with recent reports summarized in Table 1. When compared to the literature, decent sensitivity can be attributed to three main reason. First is the substantial prevention of enzyme conformational changes upon immobilization owing to the biocompatible nature of TA, and improved enzyme activity was achieved. Second is that Pt NPs can reduce the releasing H2O2 by leading to synergistic effect on the reduction current for the FAD at the DET potential. Third reason is that metal nanoparticles containing composites


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can lead to the electrochemical signal amplification3. Therefore more improved electrochemical response was observed with the Pt NPs deposition. Li et al fabricated PtPd nanoparticles/reduced graphene oxide based glucose biosensor, and owing to the fact that operation potential was high compared to that of this biosensor and selectivity was low without enzyme, the effect of interfering agents was observed high. Also the obtained sensitivity value of the fabricated biosensor surpasses the value of the nonenzymatic biosensor (1.47 µAmM-1cm-2)44. When the concentration of glucose was higher than 12 mM, the cathodic peak currents deviated from linearity and levelled off, showing the characteristics of the Michaelis-Menten kinetic mechanism. The apparent Michaelis-Menten constant (Km) is an important parameter for explaining enzyme–substrate reaction kinetics which can be obtained by using the electrochemical version the Lineweaver-Burk equation as follows45:

1⁄Iss = 1⁄Imax + Km ⁄Imax .1/Cglucose

(6)

where Iss is the steady-state response current after the addition of substrate, Imax is the maximum current under saturated substrate conditions, and Cglucose is the bulk concentration of glucose. The enzymatic reaction velocity increases with the substrate concentration, and it deviates from linearity at higher concentration. Km is an indicator of enzyme catalyzed reaction kinetics, and the smaller Km value demonstrates that immobilized enzyme has a higher affinity to its substrate, and decent enzymatic activity is observed. Km value of the GOx-rGO-Pt NPs/GCE was calculated as 3.43 mM, which was compared to those of other GOx immobilized biosensors in Table 1. The lower Km value confirms the preservation of GOx native structure in rGO-Pt NPs/GCE, resulting in higher affinity toward the glucose in enzymatic reaction.



On/off-switchable bioelectrocatalysis with a zipper-like biointerface

Upon the deposition of PNIPAAm on the as-fabricated biosensor, the on-off biosensor was obtained, and the characterization and biosensing in the on state were carried out. UV-Vis, and FTIR spectra of the obtained PNIPAAm containing bioconjugates demonstrated a pH sensitive behavior of the switchable interfaces. According to the Fig 4A, Fig. S9, and Fig. S10 A and B, the UV bands and FTIR peaks switched substantially with the pH alteration rather than temperature change. In the Fig. 4A, the UV- Vis spectra of the PNIPAAm- rGO-Pt NPsGOx demonstrate clearly the TA- PNIPAAm interactions. At pH 4, π-π and n-π transitions of rGO were revealed the blue-shifted behavior confirming the hydrophobic bonds promoted hydrogen bonding. However, these bands were disappeared at pH 7, indicating the diminishing interactions between PNIPAAm and TA. Also, in the Fig. S10, FTIR measurements demonstrated the mainly pH responsive behavior of the PNIPAAm- rGO-Pt NPs-GOx46. EIS can give information on the impedance changes of the electrode surface during the modification process. The Nyquist plots of the modified electrodes consist of a semicircle part at higher frequencies corresponding to the electron transfer limited process and a linear part at lower frequencies corresponding to the diffusion process. The electron transfer resistance (Rct) at the PNIPAAm-rGO-Pt NPs-GOx modified electrodes can be obtained from the diameter of the semicircle. Fig. 4B displays the EIS curves of the electrodes. The Rct value of the sensitive PNIPAAm- rGO-Pt NPs-GOx at 40 °C pH 4 (1290 Ω) is larger than that of the bare GCE (675 Ω) indicating that PNIPAAm containing nanocomposite reduced the conductivity. When the temperature was decreased to 20 °C at the same pH, the Rct value increased to 2875 Ω, indicating that conductivity of biosensor surface was diminished with the decreasing temperature towards redox couple of [Fe(CN)6]3−/4−. Upon the pH was increased to 7, Rct values were even more increased to 5060 Ω and 9240 Ω


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at 40 °C and 20 °C, respectively. This can be explained that at pH 7, ionization of hydroxyl groups of TA can occur much more compared to pH 4, and negatively charged TA on the surface can repel the [Fe(CN)6]3−/4− redox couple by leading to increment in the Rct values. In addition, more small Rct values observed at the 40 °C compared to at 20 °C, at pH 4 and 7 were attributed to the shrunk structure of the polymer with broken inter- and intra-hydrogen bonds upon the phase transition above 32 °C, which allows the easy [Fe(CN)6]3−/4− transition47. Mishra et al. also observed the highest redox current of Fc(COOH)2 on the poly(N-isopropylacrylamide-co-diethylaminoethylmethylacrylate)

containing

switchable

biointerface at 40 °C, and at pH 548. It was concluded that, the redox couple conductivity of the thermosensitive PNIPAAm-rGO-Pt NPs-GOx depends mostly on the pH of the medium owing to the neutral-negative transition of the surface charge upon pH alteration, which alters the permeability of the [Fe (CN)6]3−/4− to the electrode surface. This finding was also confirmed with CV curves, FTIR and UV-Vis spectra of the nanocomposites.

According to the CV curves of the PNIPAAm containing nanocomposites, it was revealed that pH and temperature responsive on-off biosensor could be formed using the TAPNIPAAm interactions, and surface charge alteration. Fig. 4C shows the CV curves of the polymer coated biosensors. According to the figure, the most enhanced redox peak of the [Fe(CN)6]3−/4− was observed at 40 °C and pH 4. This finding confirms that at higher temperature, the polymer takes the shrunken structure above the lower critical solution temperature (LCST) of 32 °C, which facilitate the transfer of the redox couple to the electrode surface by uncovering the surface with the hydrogen bonds cleavage upon conformational changes48,49. Therefore, the highest electrochemical response was observed at pH 4 with temperature increment. However, at 20 °C and pH 4, the redox peaks of the redox couple decayed owing to the fact that TA can bind to the polymer via hydrogen bonds, by covering



the electrode surface and reducing the peak currents. Nevertheless, non-ionized hydroxyls of TA at pH 4 avoid the possible repellence between the redox couple and TA coated surface, and a pair of redox peaks were observed. In the case of pH 7at 40 °C and 20 °C, the redox peaks totally disappeared because of the fact that TA is present in its negatively charged form at neutral pH, and a repellence can occur between the TA and negatively charged redox couple by hindering the electron transfer through to the electrode surface and the electrochemical signal disappeared50. Therefore, 40 °C and pH 4 was on state, and 20 °C and pH 7 was off state conditions of the biosensor. Consequently, multi stimuli-responsive smart biointerface was fabricated and this phenomenon can be beneficial for the development of versatile materials for electrochemical systems. Repeatability tests of polymer modified electrodes in the on and off states were demonstrated in Fig. 4D. A reversible interaction between TA and PNIPAAm led to the decent repeatability of the biosensor. The glucose measurements by using the on-off biosensor was carried out at 40 °C, and pH 4 (on state), as shown in Fig. 3C. From the Fig. 3D, the on-off biosensor exhibited a sensitivity of 43.06 mAmM−1cm−2 with a R2 of 0.9893, LOD of 1.02 µM, a linear measurement range of 2-10 mM. Parlak et al51 have constructed a switchable biosensor using poly(4-vinyl pyridine) conjugated graphene oxide for glucose sensing, and at pH 4, the linear measurement range was 0.01-6.0 mM and the sensitivity was 127.57 mA mM-1 cm-2. When compared to this study, a decent sensitivity and linear range were obtained with a facile and straightforward fabrication process, and the biosensor can switch the measurement response with medium pH and temperature.

Conclusion


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In conclusion, simultaneous GO and Pt4+ reduction were carried out in a facile and low-cost way by using natural reducing agent, TA. We have synthesized a novel rGO-Pt NPs nanocomposite in a one-pot manner, which combined the catalysis of releasing H2O2 by Pt NPs, biocompatibility and modifying role of TA, excellent electron transfer properties of TA with the electrochemically formed quinones groups, and enhanced surface area of rGO. The obtained nanocomposite enabled the DET between FAD and modified electrode. By using DET, the electrochemical detection of glucose was carried out based on TGB system, and wide linear measurement range was achieved with high sensitivity and stability. Also, the fabricated biosensor yielded high selectivity as the operation potential was set to a low potential, with a decent accuracy for the glucose measurement in serum samples, which offers a new approach for developing novel type of electrochemical biosensors. The chemically and electrochemically oxidized TA provided the electron transfer between GOx and electrode. It is confirmed that TA is suitable natural product for the green reduction processes, enzyme immobilization, and electron transfer in bioelectronics related areas. Also, by using the interactions between TA and PNIPAAm, smart surface coating of biosensor was carried out to fabricate on-off biosensor. At 40 °C and pH 4, upon the hydrogen bonds cleavage, and appropriate surface charge formation for redox couple diffusion, switchable biosensor determined the glucose concentration with a decent sensitivity. In this study, we achieved a novel nanocomposite by using the electrochemical behavior of TA, and we hope that this paper will be a pioneer work for the electrochemical systems that use polyphenols, in the future. Rapid, environment-friendly, low-costly synthesized rGO-Pt NPs nanocomposite is suitable for the electrochemical glucose biosensor, and this biosensor can be extended to the on-off biosensor, and other enzyme based TGBs.

Acknowledgment



This investigation has been supported by the Scientific Research Projects Commission of Sakarya University (Project number: 2017-02-04-024). M.O. thanks Turkish Academy of Sciences (TUBA) for partial support. We also thank to Dr. Soner Çakar for TGA study, Prof. Dr. Necmettin Tozlu, Dr. Engin Şahin, Dr. Đbrahim Hakkı Karataş, Mr. Süleyman Tekmen for TEM study, Dr. Atıf Koca, and Miss. Duygu Akyüz for the chronoamperometric measurements, and Mr. Celal Caner for ICP study.

Supporting Information

Apparatus and reagents, TGA and zeta potential characterizations of biosensor, electrochemically active surface area of Pt NPs, repeatability, reproducibility, stability of biosensor, chronoamperometric measurements, interference study and glucose analysis in serum were given in Supporting Information file.

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‘For Table of Contents Use Only’ Figure Captions Scheme 1. Schematic illustration of the TGB and on-off biosensor for the biosensing of glucose. Figure 1. (A) TEM image of rGO, (B) rGO-Pt NPs (inset shows the size distributions of Pt NPs), (C) HRTEM image of rGO-Pt NPs, (D) FTIR spectra of GO (a), rGO (b), and rGO- Pt NPs-GOx (c). Figure 2. (A) CV curves of GOx/GCE (a), GO-GOx/GCE (b), rGO-GOx/GCE(c), and rGO-Pt NPs-GOx/GCE (d) in deoxygenated 0.1 M PBS (pH 7.4) solution at scan rate 100 mV s-1(All CV measurements in this study were carried out in the anodic sweep), (B) The pH effect of the solution on CV responses, (C) The scan rate effect of the experiment on CV responses, (D) Glucose measurement using DPV technique. Figure 3. (A) CV curves of rGO-Pt NPs-GOx/GCE in O2 -saturated 0.1 M PBS at a scan rate of 100 mV s-1 in the presence of different concentration of glucose (0, 1, 2, 4, 6, 8, 10, 12 mM) (B) The cathodic peak current versus glucose concentration plot (inset: linear part of the curve). (C) CV curves of PNIPAAm-rGO-Pt NPs-GOx /GCE in 5 mM Fe [CN] 3-/4- and 0.1 M



ABS at a scan rate of 100 mV s-1 in the presence of different concentration of glucose (0, 1, 2, 4, 6, 8, 10 mM) (D) The cathodic peak current versus glucose concentration plot for the on-off biosensor (inset: linear part of the curve). Figure 4. (A) UV-Vis spectra of rGO-Pt NPs-PNIPAAm at two different temperature and pH values, (B) Nyquist plot of the bare GCE, and PNIPAAm-rGO-Pt NPs-GOx at two different temperature values, (C) CV curves of PNIPAAm-rGO-Pt NPs-GOx at different temperature and pH values, (D) Dependence of amperometric currents vs. number of cycles at -0.1 V switched between two different temperature values.

Table Captions Table 1. The comparison of the rGO-Pt NPs-GOx/GCE and other direct electron transfer based biosensors reported in the literature for glucose determination


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Scheme 1.



A

B

C

D

E

Figure 1.


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Figure 2.



Figure 3.


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Figure 4.



Page 34 of 35

Table 1.

Modified electrode

Linear range (mM)

Sensitivity (µAmM-1cm-2)

LOD (µM)

Km (mM)

Ref.

rGO-Pt Np-GOx/GCE

2-10

27.51

1.21

3.43

This study

ERGO-MWCNT-GOx-Nf/GCE a

0.01-6.5

7.95

4.7

-

28

GO-DDAB-GOx/GCE b

Up to 0.6

0.22

20

1.5

37

rGO-CD-GOx/GCE c

0.05-3

59.74

12

1.78

38

GNS-PEI-Au NPs-GOx/GCEd

0.001-0.1

93

0.32

-

6

GO-CNT-GOx/GCE

2-8

19.31

-

-

27

Cs-PGA-GOx/GCE e

0.5-5.5

2.13

0.12

-

39

rGO-PPy-Au NPs-GOx/GCE f

0.2-1.2

123.8

-

-

40

G-MWCNTs-AuNPs-GOx/GCE

0.005-0.175

29.72

4.8

2.09

41

Up to 2

7.29x10-5

50

-

42

0.001-4.7

28.4

0.1

6.77

43

rGO-MNPs/GCEi

0.05-1

5.9

0.1

0.16

31

rGO-ZnOj

0.2-6.6

13.7

0.2

2.2

30

GN-Py-GOx/GCE g GN-PDA-GOx/Au Electrodeh

a

ERGO: electrochemically reduced graphene oxide; MWCNT: multi-walled carbon nanotubes; Nf: nafion

b

DDAB: didodecyldimethylammonium bromide

c

CD: β-cyclodextrin

d

GNS-PEI: graphene-polyethyleneimine

e

Cs-PGA: chitosan- poly(glutamic acid)

f

PPy: polypyrrole

g

GN-Py: graphite nanoparticle- pyrene

h

GN-PDA:graphene-polydopamine

i

MNPs: magnetic nanoparticles

j

ZnO: zinc oxide


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Graphical Abstract

Tannic acid is abundant in nature, and its usage in biosensors and switchable bioelectronics was firstly described in this study.


Pt Nanoparticles Deposited and Tannic Acid-Reduced Graphene

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