Functionalized Graphene Oxide Bridging Between Enzyme and Au

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Functionalized Graphene Oxide Bridging Between Enzyme and Au Sputtered Screen Printed Interface for Glucose Detection Muhammad Asim Akhtar, Razia Batool, Akhtar Hayat, DongXue Han, Sara Riaz, Shifa Ullah Khan, Muhammad Nasir, Mian Hasnain Nawaz, and Li Niu ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.9b00041 • Publication Date (Web): 05 Mar 2019 Downloaded from http://pubs.acs.org on March 6, 2019

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ACS Applied Nano Materials

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Functionalized Graphene Oxide Bridging Between Enzyme and Au Sputtered Screen Printed Interface for Glucose Detection

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Muhammad Asim Akhtar†, Razia Batool†, Akhtar Hayat†, Dongxue Han‡,§, Sara Riaz⊥, Shifa Ullah Khan∇, Muhammad Nasir†, Mian Hasnain Nawaz*†,‡ and Li Niu* ‡,§.

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Abstract: This work reports the fabrication of an electrochemical glucose biosensor based on functionalized graphene, assembled onto gold sputtered screen printed electrode (Au-SPE). With the aim of simplicity, versatility and low-cost biosensors dually functionalized graphene oxide (GO-SH) containing excess of carboxylic acids and thiol functionality, was synthesized and characterized by fourier transform infrared spectroscopy (FTIR), Raman and UV-Vis spectroscopy. The increased carboxylic groups of graphene backbone provided more active sites for the remote functional groups of glucose oxidase (GOx) during 1-Ethyl-3-(3dimethylaminopropyl) carbodiimide (EDC) assisted immobilization. The modified electrode interface was characterized by scanning electron microscopy (SEM), atomic force microscopy (AFM) and cyclic voltammetry (CV). This hybrid electrode interface showed higher sensitivity for glucose (3.1732 μA mM-1 cm-2) with detection limit of 0.3194 mM (S/N=3). Furthermore, the fabricated biosensor demonstrated a linear response in 3 mM to 9 mM glucose concentration with the correlation coefficient of 0.94693, a performance well beyond the similarly fabricated electrode interfaces in terms of selectivity and efficiency. The enhanced electrochemical performance is assumed to originate simultaneously from sputtered morphology of Au and the bifunctionality of graphene backbone. This work highlights the significant potential for Au sputtered electrode interfaces coupled with bifunctional graphene backbone towards several biomedical applications.

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Introduction

Interdisciplinary Research Centre in Biomedical Materials (IRCBM), COMSATS University Islamabad, Lahore Campus 54000, Pakistan ‡ State

Key Laboratory of Electroanalytical Chemistry, c/o Engineering Laboratory for Modern Analytical Techniques, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, P.R. China § Center

for Advanced Analytical Science, c/o School of Chemistry and Chemical Engineering, Guangzhou University, Guangzhou 510006, P.R. China ⊥

Department of Chemistry, COMSATS University Islamabad, Lahore Campus 54000, Pakistan

Key Laboratory of Polyoxometalate Science of Ministry of Education, Faculty of Chemistry, Northeast China normal University, Changchun 130024, China ∇

*(M.H.N.) [email protected]; *(L.N.) [email protected]

KEYWORDS: screen printed electrode, Au sputtered electrodes, thiol functionalized graphene, glucose oxidase, glucose detection

In recent era inexpensive, accurate, sensitive and selective sample analysis is one of the major challenges 1-2. In the quest of introducing newer and disposable electrode systems screen printed interfaces are getting more attention since their discovery 3-4. These electrode systems have advantage of ease of engineering the substrate surface towards specific and desired properties. In general, screen printed electrodes have carbon or gold based working electrodes while silver as reference electrode which can be printed on an economical plastic or paper sheets. The conductivity and functionality of these printed working interfaces have widely been canvassed for developing miniatured, simple and disposable analytical platforms. On similar lines, metal nanoparticles on flat surfaces show distinct analytical properties as compared to bulk material 5. The unique characteristic properties and biocompatibility of nanoparticles, erupts the scope of their applications in different areas

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including electrochemical based miniatured devices 6-9. Likewise, numerous methods have been employed for the preparation of nanoparticles based simple and portable electrochemical interfaces including simple deposition, electrochemical and chemical grafting, probe assisted adsorption, screen printing and plasma assisted sputtering on working interfaces. Among these, sputtering of conducting materials (especially gold and carbon) onto the working electrode provides advantages in heightening the electron transfer ability as well as provision of wide spectrum of active cites for further modifications. Moreover, sputtered gold nanoparticles provide such environment so that biomolecules retain its biological activity when immobilized and assists electron transfer between biomolecule and electrode surface.

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Experimental

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Materials and characterizations

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Functionalization of graphene (GO-SH)

On the other hands, graphene a single layered carbon atom honeycomb close packed structure gained much attention in recent era in exploring fascinating properties of graphene including excellent electrical conductivity 10. Furthermore, incorporation of these single carbon 2D sheets into complex assemblies and composites may induce unique electronic and mechanical characteristics. Chemical functionalization is yet another interface to play vital role towards tuning up singular properties of these embodied materials 8. Introduction of thiol functionality on graphene surface could induce Au-S assisted self-assembly of graphene onto Au surfaces even at room temperature 6, 11-12. Furthermore, due to intrinsic carboxylic groups of graphene oxide, the carbon surface gets negatively charged when dispersed in water 13. It facilitates immobilization of biomolecules, especially enzymes, via strong hydrogen bonding and electrostatic interactions with their amino groups. Moreover, electron transfer between enzyme and the electrode surface is the key feature that determines the analytical performance of enzymatic biosensors. Based on these features, such bifunctional graphene oxide sheets simultaneously, decorated with thiol and carboxylic groups can facilitate efficient enzyme immobilization on Au sputtered electrode surface towards fabrication of enzyme based sensitive biosensors 14-15. In this work we functionalized graphene oxide by one-pot monothiolation. In this technique epoxide and hydroxyl groups were targeted for thiolation. This was performed by hydrobromic acid which reduced graphene oxide, followed by thiourea addition and hydrolysis with sodium hydroxide which eventually resulted in GO-SH. That has enhanced electrical conductivity due to regeneration of sp2 carbon network and lower electronegativity of sulfur 16. On the other hand, Au nanoparticles were deposited on the working interface of the screen printed electrode that imprinted nanoisland like structures of gold on the underlying carbon layers. Finally, thiol functionalized graphene was used for the modification of gold sputtered screen printed electrodes for glucose detection.

Graphite powder (-20 +60) was purchased from Sigma Aldrich. Potassium ferro/ ferri cyanide were purchased from Uni-Chem chemicals and reagents. Glucose, glucose oxidase and 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) were purchased from Sigma-Aldrich. Phosphate buffer saline (PBS) tablets were purchased from bio-WORLD (bioplus fine research chemicals) and were used to prepare PBS solution (pH 7.4) as the supporting electrolyte. Distilled water was used in all experiments, all the chemicals were of analytical grade, and were used as received unless otherwise stated. Cyclic voltammetry measurements were carried out with Palmsens electrochemical work station. Three electrode system of screen printed electrodes (SPE) consisting of silver as reference, and carbon as counter and working electrodes was used. Modification of working electrode with gold nanoparticles was done by gold sputtering for 180 seconds by using sputter coater model Quorum SC7620. All electrochemical measurements were carried out under ambient conditions. Fourier transform infrared spectroscopy (FTIR) was carried out on a Thermo Nicolet 6700. Raman spectroscopy was performed by using InVia Raman Microscope Renishaw UK at wavelength 514 nm and UV-Vis spectra were recorded on Perkin Elmer spectrophotometer Lambda. To study the surface morphologies of modified electrodes, the working interface of SPE was manually cut and Au sputtered for scanning electron microscopy (SEM) by using TESCAN VEGA 3 whereas atomic force microscopy (AFM) was carried out by using Park system AFM XE7 in non-contact mode.

Graphene oxide (GO) was prepared by a modified Hummer’s method 17 using natural graphite powder (-20 +60) as starting material. For thiol functionalization, 50 mL of GO aqueous suspension (1 mg/mL) was prepared in deionized water followed by ultrasonication for 1 hr. HBr (3 mL) was then added to the suspension at 30 oC followed by 2 hrs. vigorous stirring. Subsequently, 3 g of thiourea was added and the suspension was allowed to stir for further for 24 hrs. at elevated temperature (80 oC) 16. The solution was then cooled to room temperature followed by addition of 4M NaOH (30 mL). The reaction mixture was then filtered after stirring


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for 30 min at room temperature and washed with ether/water. The black material was then collected and dried in vacuum oven for 72 hrs. at 30 oC to obtain GO-SH.

Modification of screen printed electrode Screen printed working electrode was gold sputtered using sputter coater for 180 secs at 2 mbar chamber pressure and 10 mA process current, keeping the other electrodes intact. A handmade plastic cover was used to cover the electrode with 4 mm hole (the size of working electrode) to expose the working electrode to the plasma. Followed by Au sputtering, 5 μL homogeneous dispersion of thiol functionalized graphene solution (0.2 mg/mL) was drop casted onto the working electrode to get surface coverage of 2.5×10-4 mg/ mm of working interface, which is calculated by 5 μL of 0.2 mg/mL of suspension, dropped on 4 mm of working electrode. The modified electrode was dried at room temperature (3 hr.) at room temperature. For carboxylic group activation of functionalized graphene, 5 μL EDC hydrochloride solution (100 mM) was dropped onto the surface of working electrode and incubated for 45 min at room temperature followed by rinsing with plenty of water 18. Subsequently, 5 μL of glucose oxidase (GOx) solution (600 U/mL) was dropped on activated functionalized graphene modified gold sputtered electrode and incubated for 30 min followed by water rinsing to remove left over GOx molecules. The fabricated electrode was denoted as GOx decorated thiolated graphene modified gold sputtered screen printed electrode (GOx-GO-SH-Au-SPE). This whole procedure of modification of screen printed electrode is shown in scheme 1. Scheme 1. Schematic representation (illustrated by Adobe Illustrator CS5) of GO-SH synthetic route towards fabrication of GOx-GO-SH-Au-SPE interface for glucose detection.

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Results and discussion The synthesis of GO-SH was monitored via FTIR, Raman and UV-vis spectral changes. FTIR was performed at attenuated total reflectance (ATR) mode for graphene oxide and GO-SH ranging from 4000 cm-1 to 645 cm1 to evaluate functionalization, as shown in figure 1A. The spectrum of GO-SH showed appearance of small band in the region 2416-2650 cm-1 which was attributed to the stretching vibration of S-H group and it confirms the thiol functionalization of graphene oxide. 16, 19 The stretching band of C=O of carboxylic group showed enhanced absorption in the region of 1615-1733 cm-1 indicating the increased number of carboxylic groups in GO-SH. Absorption peaks at 1541 cm-1,1230 cm-1 and 1050 cm-1 were assigned to the stretching of C=C, C-O of carboxylic groups and C-O-C of epoxy groups respectively. Similarly, the Raman spectra of GO and GO-SH showed obvious D and G bands as shown in figure 1B. The ratio of peak intensities of D and G bands (ID/IG) suggests the number of defects in graphene oxide. ID/IG for graphene oxide and GO-SH were recorded to be 0.8758 and 1.0054, respectively.



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Figure 1. FTIR (A), Raman (B) and UV-Vis spectra (C) of graphene oxide (a) and GO-SH (b). UV-vis spectra were recorded in corresponding aqueous suspensions of GO and GO-SH.

The higher degree of defects for GO-SH spectra indicating the functionalization of graphene oxide 20. This is also in accordance with the crystalline size of graphene oxide and GO-SH which is calculated by using the Cancado formula 21: La (nm) = 560/ E4 (ID / IG)-1 i.e. 18.955 nm and 16.511 nm, respectively. The smaller value of full width at half maxima (FDHM) corresponding to D band of GO-SH (87.8283) as compared to graphene oxide (159.3873) also suggests the low degree of structural disorder that contains sp2 and sp3 hybridized carbon moieties 22. Furthermore, the characteristic peak of GO at 231 nm in UV-vis spectra of GO-SH was red shifted to 202 nm, as shown in figure 1C. It is well-known that the reduction of graphene oxide results in red shift of UV-vis absorption peak 23 and higher the reduction state, greater will be the red shift.

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Figure 2. AFM images and corresponding roughness of bare screen printed (A) and Au-SPE (B). The working interface was manually cut in desired dimensions for topographic analysis.

The surface morphologies of bare and gold sputtered screen printed electrodes were characterized by atomic force microscope (AFM), as shown in figure 2. The topographical height of bare electrode was found to be 0.60 μm, figure 2A. However, with the gold sputtering the topographical height was reduced to 0.34 μm which is attributed to the fact that gold nanoparticles homogeneously cover the whole surface of the electrode, figure 2B. The corresponding surface profile and along the diagonal root mean square roughness (Rq) indicated that the bare screen printed electrode bear large sized particles with rougher surface i.e. 36 nm. On the other hand, gold sputtered screen printed electrode has small sized and equally distributed particles that attribute to Rq value of 13.8 nm which is lower than the bare electrode. The uniform distribution of gold nanoparticles leads to the high electroactive surface area of the electrode, promote the electron transfer between the electrode surface and the GOx and also enhance the enzyme loading on to the electrode surface 24-25.


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Figure 3. SEM micrograph of bare electrode (A), GO-SH-Au-SPE (B), and after electrochemical studies (C). For SEM analysis the working interface of SPE were manually cut into the dimensions suitable for SEM sample holder and pasted on the holder via conducting adhesive tape followed by Au sputtering.

The surface morphology of GO-SH-Au-SPE electrodes was then examined by scanning electron microscope, as shown in Figure 3. The results are consistent with the previously discussed topographic results. The surface of bare electrode clearly illustrates large sized particles that correspond to the carbon paste of SPE, figure 3A. After modification of electrode with the gold sputtering and GO-SH immobilization, the SEM image of electrode surface clearly showing the gold nanoparticles and GO-SH nanoparticles dispersed homogeneously on to the electrode surface, figure 3B. The stability has been an important question in the quest of electrode modifications and fabrications of different sensing and biosensing devices. The stability of the electrode is owing to the covalent binding of electrode surface and the biomolecule. Which is essential for the reproducibility and repeatability of the electrode 25-26. Hence, to evaluate the stability of our fabricated glucose biosensor, the surface morphology of GOx-GO-SH-Au-SPE after electrochemical studies was also analyzed. The electrode surface was found identically same, both before and after the electrochemical studies (figure 3 B & C) indicating the best stability of the modified SPE interfaces.

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Figure 4. Cyclic voltammetric response (A) and electrochemical impedance spectroscopy (B) of bare SPE (a), Au-SPE (b), GO-SH-Au-SPE (c), EDC activated GO-SH-Au-SPE (d), GOx-GO-SH-Au-SPE (e) in 5 mM [Fe(CN)6]4-/3- (1:1), Scan rate 50 mVs-1.

For electrochemical characterization of Au-SPE and GO-SH-Au-SPE, cyclic voltammetry (CV) and electrochemical impedance spectroscopy were performed in 5 mM potassium ferro/ferri cyanide solution, figure 4. In cyclic voltammetry a pair of well-defined quasi reversible redox peaks were observed on Au-SPE, figure 4C. The electrode modification by thiol functionalized graphene (GO-SH-Au-SPE) resulted in peak broadening. The formal potential of Au-SPE and GO-SH-Au-SPE was calculated by averaging the cathodic and anodic peak potentials as 0.1465 V and 0.1445 V. while the peak to peak separation was estimated to be 0.921 V and 1.233 V, respectively. Which implies that GO-SH adsorbed on the surface of Au-SPE and it hinders electron transfer from surface to electrolyte. However, with GO-SH modification via EDC the peak to peak separation dramatically decreased to 0.523 V, whereas the formal potential remained same (0.1455 V). It confirmed the GO-SH modification resulting the electron transfer properties enhancement 18. According to the carbodiimide



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crosslinker chemistry EDC reacts with carboxylic group of GO-SH that in results intermediate in form of ester formation. Primary amino groups have properties to replace this intermediate ester by nucleophilic attack and ultimately bonding with the original carboxyl group through amide bond, consequently a by-product of EDC in the form of soluble urea derivative is released 27. Hence, EDC can provide an efficient way to attach glucose oxidase (GOx) with the GO-SH back bone. These effects can easily be observed in electrochemical impedance measurements, Au-SPE show lower resistance for electron transfer as compared to the GO-SH-Au-SPE, figure 3B. However, with the modification of graphene via EDC the semi-circle diameter of Nyquist plot decreased, which implies that electron transfer rate dramatically increased. It is important to note that deprotonated COO of functionalized graphene oxide is negatively charged and provide higher electron transfer resistance, however on activation of terminal COOH groups with EDC/NHS and subsequent attachment of GOx resulted in the increase of electron transfer resistance. The same can be envisioned in the CV and EIS spectra presented in figure 4. The electrochemically active surface area of the EDC activated GO-SH-Au-SPE was calculated from the peak current obtained by the cyclic voltammograms in ferro/ferri cyanide [Fe(CN)6]3-/4- (redox probe) using the following Randels–Sevcik equation. ∗ 1/2 𝑖𝑝 = 2.69 × 105𝐴𝑛3/2𝐷1/2 𝑟𝑒𝑑 𝐶 𝑣

(1)

Where, C* is the concentration of ferrocyanide (5×10-3 M), was the Dred is the diffusion coefﬁcient of potassium ferrocyanide (6.3 × 10-6 cm2 s-1) and 𝑣 is the scan rate (50×10-3 V). The calculated electrochemically active surface area of the EDC activated GO-SH-Au-SPE was found to be 0.148 cm2. Furthermore, after GOx incubation the redox peak of 5 mM potassium ferro/ferri cyanide solution was shifted to higher potential, indicated the conjugation of GOx with GO-SH modified surface. This potential shift could be attributed to the blockage of electron transfer from electrode surface to the electrolyte. The formal potential of GOx-GO-SHAu-SPE was estimated as 0.162 V and peak to peak separation was about 0.824 V. This increase in the formal potential also confirms the above-mentioned argument. The well-defined and quasi reversible redox peaks suggest favorable direct electron transfer between the electrode and the redox centers of GOx molecules. The electrochemical response of GOx is mainly due to the FAD which is the part of GOx molecule. Based upon this electrochemical response of GOx and by direct electron transfer between substrate and GOx, used to prepare bioelectrocatalytic sensing devices. 28-29

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Figure 5. Cyclic voltammograms of the GOx-GO-SH-Au-SPE in 5 mM [Fe(CN)6]4-/3- at different scan rates: 25, 50, 75, 100, 125, 150, 175 mVs-1 (A), peak potential (Ep) vs. the logarithm of scan rate (log v), the linear ﬁtting at scan rates from 25 mVs-1 to 175 mVs−1 (B).

Figure 5 shows the effect of scan rate in 5 mM [Fe(CN)6]4-/3-. It was found that the redox potentials Epa and Epc of GOx shift and anodic and cathodic peak currents also increased with the increase in scan rate. The peak currents are linearly corelated to the scan rate (figure 5A); linear regression equations: Ipa = 0.4426v + 65.8414 , r = 0.96076 ; Ipa = -0.4125v - 66.9571 , r = 0.9477), according to the Laviron’s equation. 30 Ip = n2F2AΓv/4RT

(2)

Where n is the number of electrons transfer, which in this case corresponds to 2, F is the Faraday constant (F= 96485 C mol-1), Γ is the electroactive glucose oxidase amount, A is the area of the screen printed working


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electrode (0.502 cm2), R and T represent their usual meanings (R= 8.314 J K-1 mol-1, T= 298 K). The amount of electroactive glucose oxidase at the surface of GOx-GO-SH-Au-SPE was estimated 2.34 × 10-7 mol cm-2. Moreover, the relationships of Epa and Epc with logv were constructed with the linear regression equations Epa(V) = Epa= 0.3896 Logv + 1.1822, (r = 0.9743) and Epc(V) = -0.3605 Logv - 0.8258, (r = 0.9899), figure 5B.

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c 20

I (µA)

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E (V) vs Ref

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Figure 6. Cyclic voltammograms of bare (a), Au-SPE (b) and GOx-GO-SH-Au-SPE (c) electrodes in the presence of 5 mM glucose solution in pH 7.4 PBS, Scan rate 50 mVs-1.

Figure 6 shows cyclic voltammogram of bare, gold sputtered electrode and GOx-GO-SH-Au-SPE in 5 mM glucose solution which was prepare in air-saturated PBS (pH 7.4), the shape of cyclic voltammogram show no redox peak in case of bare and gold sputtered electrodes however GOx-GO-SH-Au-SPE shows reduction peak at -0.47 V. Oxygen is natural mediator of oxidase so in the presence of dissolved oxygen O2, glucose is oxidized to gluconic acid while O2 is reduced to H2O2 by GOx, as has been indicated in reaction (1). Moreover, O2 can electrochemically be reduced on electrode surface as shown in reaction (2), forming a cathodic current which can be envisioned in the cyclic voltammogram.

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Figure 7. Cyclic voltammograms of GOx-GO-SH-Au-SPE in pH 7.4 PBS with different concentrations of glucose. Scan rate 50 mVs-1 (A), linear calibration curve peak current vs glucose concentration (3, 4, 5, 6, 7, 8 and 9 mM) (B)

With the different concentrations of glucose in to air-saturated PBS cyclic voltammetry was performed, as shown in figure 7. It has been seen that the reduction current signal of GOx-GO-SH-Au-SPE decreased with the increase in glucose concentration. Presence of glucose triggered an enzyme-catalyzed reaction, in which



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glucose oxidation occurred with the consumption of dissolve oxygen that resulted in decrease of O2 reduction current reaction. We can say that the restraining of electrocatalytic reaction is due to the presence of glucose as a consequence of the enzyme-catalyzed reaction between the glucose and oxidized form of GOx. This shows that GOx immobilized on the electrode surface maintain its enzyme activity in the presence of O2. 𝐺𝑂𝑥

𝐺𝑙𝑢𝑐𝑜𝑠𝑒 + 𝑂2 𝑂2 +4𝐻

+

―

Gluconic acid + 𝐻2𝑂2

+4𝑒 →2𝐻2𝑂

(2)

(3)

Figure 7B showing the calibration plot obtained from reduction peak current vs glucose concentrations. This shows the linear range of glucose concentration with the reduction current was from 3 mM to 9 mM with the correlation coefficient of 0.94693 and detection limit of 0.3194 mM at a signal to noise ratio of 3. The sensitivity of GOx-GO-SH-Au-SPE to D glucose was found to be 3.1732 μA mM-1 cm-2 well beyond the physical limits of human body 31. As the Glucose is naturally present in human blood and serum in level of about 5 mmol/L, hence the selected concentration ranges were tested. As analytical performances of any sensory device depend on the sensitivity and linear range, so the practicality of our fabricated biosensor can clearly be envisioned from the selected range of analyte concentrations. Moreover, the sensitivity of the present study is also much superior to the only reported example of such nanoconstructs (46 μA) for the glucose detection32.

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Figure 8. Cyclic voltammetric response of GOx-GO-SH-Au-SPE towards interfering molecules including ascorbic acid, L-cystine, hydrogen peroxide, and uric acid at 5 mM in PBS (pH 7.4) with scan rate 50 mVs-1.

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Conclusion

To further access the selectivity of the GOx-GO-SH-Au-SPE biosensor for its use in complex environment containing interferents, the cyclic voltammetric responses of ascorbic acid, L-cystine, H2O2, and uric acid at same concentration as that of glucose (5 mM), were obtained, as shown in figure 8. It was found to be quantitatively negligible reduction peaks were present. It confirms the selectivity of the GOx-GO-SH-Au-SPE towards glucose detection. The results were reproducible as no apparent changes were observed for different electrodes prepared via same protocol for these experiments. Likewise, the modified electrodes were subjected to detect glucose directly or were stored for one month at room temperature to work as ready to use detection device. During these experimentations it was also observed that no apparent changes in the response were observed even after storing the modified electrodes for one month at ambient conditions.

In summary, Au sputtered working interface of SPE successfully anchored GO-SH backbone as conductive support for effective immobilization of GOx. The fabricated biosensor denoted as GOx-GO-SH-Au-SPE efficiently electro catalyzed the reduction of dissolved oxygen in pH 7.4 PBS solution. The reduction current decreased with the increase in glucose concentration, hence based on the decrease in electrocatalytic response a glucose biosensor has been developed. The GOx-GO-SH-Au-SPE biosensor displayed higher sensitivity (3.1732 μA mM-1 cm-2) and a linear range from 3 mM to 9 mM for glucose biosensing. To the best of our knowledge, these performance properties are the best among the sputtered electrodes used so far for the glucose biosensors. Because of the easy preparation and good properties of GOx-GO-SH-Au-SPE the asprepared biosensor could be a promising tool for electrochemical sensing of glucose. Moreover, the present


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work was focused on a proof of concept to demonstrate the applicability of the designed novel transducer construct towards detection of glucose. Future work will be extended to design specific and selective transducer surfaces based on this nanoconstruct for analysis of various biomolecules such as cholesterol, D-alanine etc where applicability of method with real samples is anticipated.

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Acknowledgment This work was supported by national research program for universities and startup research grants of Higher Education Commission of Pakistan (20-4993/R&D/HEC/14/614 and 21-329/SRGP/R&D/HEC/2014).

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Functionalized Graphene Oxide Bridging Between Enzyme and Au

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