Silver nanoparticles on graphene quantum dots as nanozyme for efficient H2O2 reduction in a glucose biosenso

Silver nanoparticles on graphene quantum dots as nanozyme for efficient H2O2 reduction in a glucose biosensor

Silver nanoparticles on graphene quantum dots as nanozyme for

Hoang Vinh Tran1

, Tuan Anh Le2 , 3

, Bach Long Giang4

, Benoit Piro5

and Lam Dai Tran6 , 7

1 Department of Inorganic Chemistry, School of Chemical Engineering, Hanoi University of Science and Technology (HUST), 1st Dai Co Viet Road, Hanoi, Vietnam

2 Phenikaa University Nano Institute (PHENA), Phenikaa University, Hanoi 12116, Vietnam

3 Faculty of Materials Science and Engineering, Phenikaa University, Hanoi 12116, Vietnam

4 Nguyen Tat Thanh University, 300 A Nguyen Tat Thanh, Ho Chi Minh City, Vietnam

5 Université Paris Diderot, Sorbonne Paris Cité, ITODYS, UMR 7086 CNRS, 15 rue J-A de Bạf, 75205 Paris Cedex 13, France

6 Institute for Tropical Technology, VAST, 18 Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam

7 Graduate University of Science and Technology, VAST, 18 Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam E-mail: hoang.tranvinh@hust.edu.vn

Keywords: hydrogen peroxide reduction, silver nanoparticles (AgNPs), graphene quantum dots (GQDs), electrochemical sensor, glucose oxidase (GOx)

Abstract

In this work, we developed graphene quantum dot-supported silver nanoparticles (AgNPs@GQDs) as Nanozyme for efficient electrocatalytic reduction of H2O2 We applied this composite material in a glucose oxidase-based glucose sensor, by drop casting a mixture of AgNPs@GQDs and chitosan (CS)

on glassy carbon electrodes (GCE) Various conditions such as thickness of the AgNPs@GQDs/CS film, pH and temperatures were optimized The proposed sensor presented excellent selectivity and sensitivity, a linear dependence on glucose concentration in the range 0.1–10 mM and a limit of detection of ca 0.01 mM.

1 Introduction Glucose is the most important simple sugar in human metabolism Our body naturally regulates blood glucose level as a part of metabolic homeostasis and itsfluctuation in blood is the trigger alarm for various diseases among which the most common are diabetes, characterized by too high blood sugar levels over a prolonged period, The general range of blood glucose concentration in healthy persons is about 3–8 mM, whereas it is about 9–40 mM for diabetic subjects For this reason, glucose sensors have always been in high demand [1–4] Since Clark and Lyons[5], a huge amount of studies regarding enzymatic glucose sensors have been reported due

to their high selectivity and good sensitivity The use of enzymes induces high costs and their activity is highly dependent on environmental conditions(pH, temperature, immobilization procedures); however, glucose oxidase is incomparably specific and no synthetic equivalent has been found to date Most enzyme-based glucose sensors were developed following two detection methods: colorimetric[6,7] or electrochemical [8,9]

Colorimetric methods are often applied in hospitals or analytical centres, while commercial electrochemical glucose sensors are now widely available for individual glucose test at home The recent democratisation of the latter brings back electrochemical glucose sensors in front of the scene

In traditional electrochemical glucose biosensors, glucose oxidase(GOx) is used as specific enzyme for oxidation of glucose to gluconic acid Under aerobic conditions, it produces hydrogen peroxide(H2O2) as a side product, which is electrochemically reduced onto a catalytic electrode to provide the sensor’s signal This approach has some limitations, however, such as laborious enzyme immobilization processes and the use of noble metals as catalytic electrodes

In this work, we proposed a novel strategy for the fabrication of an electrochemical glucose biosensor without noble metal nor immobilized GOx Compared to traditional electrochemical glucose sensors, horseradish peroxidase(HRP) has been replaced by silver nanoparticles supported on graphene quantum dots

RECEIVED

28 May 2019

REVISED

27 August 2019

ACCEPTED FOR PUBLICATION

23 September 2019

PUBLISHED

2 October 2019

© 2019 IOP Publishing Ltd

(AgNPs@GQDs), which are much more stable upon use and storage and less expensive AgNPs@GQDs were immobilized onto glassy carbon electrodes(GCE) using chitosan (CS) as binder AgNPs@GQDs/CS-modified GCEs were used for H2O2reduction at a potential of−0.5 V versus SCE In these proof-of-concept experiments, GOx was simply introduced in solution

2 Experimental

2.1 Chemicals

Citric acid(C6H8O7,H2O), urea ((NH2)2CO), ammonia (NH3) solution 28%wt, acetic acid (CH3COOH) solution 99%wt, silver nitrate(>99%), 3,3′,5,5′-tetramethylbenzidine ((C6H2(CH3)2-4-NH2)2) (…99%,

Mw=240.34 g.mol−1), glucose oxidase (GOx) from Aspergillus Niger (Type VII, lyophilized powder,

…100,000 units.g−1solid), chitosan (from shrimp shells, …75% deacetylated, Mv110 000–150 000) and

phosphate buffered saline(PBS) tablets were purchased from Sigma Aldrich (PBS solutions were prepared by dissolving a PBS tablet into 200 ml distilled water to obtain a solution at pH 7.4) Glassy carbon working

electrodes(GC, 3 mm diameter, S=0.07 cm2) were purchased from BAS Inc Chitosan solutions were

prepared by dissolving 1 g chitosan in 100 ml of 1% v/v acetic acid/water then stirring overnight at room temperature Alumina slurry used for GC electrode polishing was from ESCIL, Chassieu, France All other reagents(H2SO4, acetic acid) and solvents (acetonitrile -ACN-, ethanol -EtOH-)-, were PA grade

2.2 Synthesis of GQDs and AgNPs@GQDs

Graphene quantum dots were synthesized from citric acid and urea by hydrothermal method in an autoclave at

160°C for 4 h [10] Silver nanoparticles were synthesized by a green method using GQDs as reducing reagent:

1 ml of 0.1 M AgNO3was added into the GQD solution then the mixture was heated to 90°C for 3 h until completed reduction of silver ion into silver nanoparticles[10,11] The precipitate was centrifuged at

30 000 rpm for 1 h to collect AgNP@GQDs as a black solid, which was dried at 80°C under vacuum for 24 h Finally, 1 mg of AgNPs@GQDs was dispersed into 10 ml of chitosan solution to obtain AgNPs@GQDs/CS

2.3 Electrode preparation

GCEs were polished with 1μm alumina slurry on a polishing cloth then washed with water, ethanol and ACN in ultrasonic bath for 2 min each AgNPs@GQDs/CS was drop-casted on freshly polished GCE by adding 20 μl of the corresponding solution and let it dry at RT This procedure gives AgNPs@GQDs/CS-modified electrodes (noted AgNPs@GQDs/CS/GCE) for which the surface density of AgNP can be controlled by adding various AgNPs@GQDs amount into the CS solution

2.4 Characterization

The AgNPs@GQDs/CS/GCEs were electrochemically characterized in a 10 mM PBS solution by cyclic

voltammetry(CV) between −0.1 V and −0.9 V versus SCE, at a scan rate of 50 mV.s−1 The three-electrodes cell consisted of a GC working electrode(3 mm in diameter), a platinum (Pt) grid counter electrode and a

commercial calomel electrode(SCE, supplied from Radiometer Analytical) Experiments were run with a potentiostat Autolab PGSTAT30 Solutions were systematically deaerated with argon before and during

experiments UV–vis spectra were measured using an Agilent 8453 UV–vis spectrophotometer for wavelengths between 200 and 1200 nm The morphologies and crystal structure of the nanoparticles were characterized using

a Field Emission Scanning Electron Microscope(FE-SEM) using a FE-SEM JEOL JSM-7600F Particles size distribution was analysed by Dynamic Light Scattering(DLS) on a Zetasizer nano ZS-100 (Malvern, UK)

2.5 Glucose detection

5 ml PBS were added into the electrochemical cell with 20μl GOx solution (10 mg.ml−1) then degassed under Ar for 10 min to remove oxygen At this stage, cyclic voltammetry was performed to record the background signal For glucose detection, a mixture of 0.5 ml glucose solution in PBS was added in the electrochemical cell, stirred

at 200 rpm for 30 min at RT, then a CV was recorded The reduction peak at a potential of−0.5 V versus SCE was used as signal, after subtraction of the background current Scheme1described the fabrication strategy of the AgNPs@GQDs/CS/GCEs and glucose detection

3 Results and discussion

3.1 Characterization of the AgNPs@GQDs/CS layer

Figure1(a) presents a TEM image and UV–vis spectra (inset) of AgNPs@QGD with mean particle size of ca

40 nm The UV–vis spectrum of the AgNPs@GQD solution (figure1(a), inserted, curve 2) shows a strong adsorption band at 420 nm which is attributed to the characteristic surface plasmon absorption of AgNPs, while this absorption is not observed in the case of GQDs(figure1(a), inset, curve 1) The shoulder at 357 nm is attributed to the presence of residual GQDs The x-ray diffraction diagram of GQDs(figure1(b), curve i) presents a weak broad peak(002) centered at 2θ∼27.7° which indicates the disordered stacking structure of graphene layers This peak is weak because it strongly depends on the degree of oxidation of GQDs(the attached hydroxyl, epoxy/ether, carbonyl and carboxylic acid groups can increase the interlayer spacing [12]) In

contrast, the XRD pattern of the AgNPs@GQDs(figure1(b), curve ii) shows three main characteristic peaks at 2θ=37.5°, 43.1° and 64.8° which match very well with those of the standard AgNPs (PCPDF card number

40783) [13] with Miller indices (111), (200) and (220) Line broadening in the pattern can be quantitatively evaluated using the Debye–Scherrer equation (equation (1)), which gives a relationship between peak

broadening in XRD and particle size:

l

b q

D k.

where D is the thickness of the crystal, k is the Debye–Scherrer constant (0.9), λ is the x-ray wavelength

(0.154 06 nm), β is the line broadening in radian, obtained from the full width at half maximum, and θ is the Bragg angle According to this equation, particle sizes are estimated to be 40±5 nm These results are consistent with those obtained by TEM AgNPs@GQDs’s size distribution were analysed by DLS (figure1(c)) It can be seen that AgNPs@GQDs have a narrow size distribution from 8 nm to 100 nm and a main size around 30–40 nm, which is in agreement with TEM results SEM images of AgNPs@GQDs/CS/GCE are shown in figure1(c) It can

be seen that AgNPs@GQDs appear as white spots distributed over the whole GCE’s surface with low aggregation due to the protect by GQDs(TEM in figure1(a)) [10,11]

The electroactivity of AgNPs@GQDs/CS/GCE was studied by cyclic voltammetry (CVs) in Ar-saturated PBS and results are shown infigure1(d) CV of bare GCE (curve i) has a reduction peak at −0.6 V versus SCE due

to reduction of oxygen; however the current is very low(lower than 2 μA) CV of CS/GCE (curve ii) shows passivation by chitosan A similar behaviour is obtained with AgNPs@GQDs/CS for a low AgNPs@GQDs concentration(figure1(d), curve iii) However, at higher concentrations (figure1(d), curve iv and v), CVs show a significantly higher capacitance

Optimization of the AgNPs@GQDs layer was done by measuring the oxygen reduction current after H2O2

adding, for several AgNPs@GQDs densities Figure2shows CVs of GCE(figure2(a)), CS/GCE (figure2(b)) and AgNPs@GQDs/CS/GCE with various AgNPs@GQDs concentrations (figures2(c)–(f)), in Ar-saturated PBS (black curve) and in Ar-saturated PBS containing 0.5 mM H2O2(red curve) Very low reduction currents were obtained on bare GCE(figure2(a)) and CS/GCE (figure2(b)) H2O2reduction started to appear for a

AgNPs@GQD concentration of 0.125 mM(figure2(c)) and is maximum for 0.5 mM (figure2(e)) A

Scheme 1 Schematic view of the proposed strategy for design and fabrication of electrochemical glucose sensors based on

AgNPs@GQDs /CS/GCEs.

concentration of 1 mM lead to a too large capacitive current compared to the faradic one, and the peak shifted to more positive potentials(figures2(d)–(f))

The H2O2reduction peak potential(Epon AgNPs@GQDs/CS/GCE (−0.5 V versus SCE) (figure2(e)) is higher than that of AgNPs/CS/GCE (−0.7 V versus SCE) [14], which indicates that GQDs facilitate the electron transfer between AgNPs@GQDs/CS and the electrode Compared to other modified GCEs for H2O2reduction, the potential is less negative than for CS/2D ZnAl layered double hydroxide (Ep=−0.6 V) [15] or Pt/RGO/ CS/HRP [16] suggesting that AgNPs@GQDs/CS/GCEs possess excellent electrocatalytic reduction activity However it is slight more negative than Mn3O4-MnO2nanorods/graphene nanocomposite (MM-HNRS/GS) (Ep=−0.4 V) [17] or carbon black/bimetallic PdCu modified GCE (PdCu/CB/GCE) (Ep=−0.243 V) [18] The intensity of the reduction peak versus scan rate was measured(figure3(a)) and plotted versus the square root of scan rate(figure3(b)); the result shows that the reduction process is diffusion-limited

3.2 Detection of glucose

We applied the H2O2reduction property of the AgNPs@GQDs/CSGCEs to an electrochemical glucose sensor

In an electrochemical cell containing 50μl of 20 mg.ml−1GOx, afirst CV was acquired then 50 μl of glucose at

Figure 1 (a) TEM (inset: UV–vis spectra of (1)GQDs and (2)AgNPs@GQDs); (b) XRD of (a) GQDs and (b) AgNPs@GQDs; (c)

AgNPs@GQDs size distribution by DLS (inserted: digital photo of AgNPs/QGQDs : (1) stock solution and (2) diluted solution); (d) SEM of AgNPs@GQDs /CS-modified GCE; (E) Cyclic voltammograms of (i) bare GCE; (ii) chitosan-modified GCE (CS/GCE); (iii-v) AgNPs@GQDs /CS-modified GCE with various concentrations of AgNPs@GQDs: (iii) low, (iv) medium and (i) high AgNPs@GQDs concentrations, respectively CVs were recorded in Ar-saturated PBS solution Scan rate 50 mV.s−1.

various concentrations were added into the electrochemical cell under continuous stirring at 100 rpm for

20 min to let the enzymatic reaction take place(equation (2)):

Glucose O2GOx Gluconic acid H O2 2 2

To investigate the specificity for glucose, we used various saccharides and other molecules as substrate, namely galactose, glycine and fructose, at a concentration of 10 mM CVs before and after addition of these molecules are shown onfigure4 The reduction current at−0.5 V only slightly increased from −2.5 μA to −4.5

μA, while for the same glucose concentration a strong reduction current of ca −18 μA was obtained

To see real time response, chronoamperometry(CA) was used instead of CV, at a constant potential of

−0.5 V versus SCE) The response time was ca 50 s after injection; as shown, under similar conditions, the current change is ca 0.1μA for lactose and 1 μA for glucose (figures4(c), (d)), which demonstrates a sufficient specificity

A calibration curve was built by recording peak currents for various glucose concentrations up to 10 mM; CVs and the corresponding calibration curve are shown infigures5(a) and (b), respectively In the range

0.1–2 mM, an acceptable linear relationship was found, with I

(μA)=(3.7403±0.3859)+(3.2274±0.3763)×Cgluocse(mM) (R2=0.956) The limit of detection (LOD) was 0.01 mM(S/N=3) The coefficient of variation was 4.8% (n=10)

Figure 2 Cyclic voltammograms of (a) bare GCE; (b) CS/GCE and (c)–(f) increasing surface concentration of AgNPs@GQDs on CS/ GCE in PBS solution in absence (i, black) and presence (ii, red) of 0.5 mM H 2 O2.

The reproducibility of this strategy for ultrasensitive detection of glucose based on AgNPs@GQDs/CS/GCE was evaluated by intra- and inter-assays Eight repetitive assays were carried out at a glucose concentration of 0.5 mM, and the coefficients of variation of intra-assays and inter-assays were 4.5% and 4.7%, respectively, which shows a good reproducibility Moreover, AgNPs@GQDs/CS/GCEs were kept in a moisture-saturated atmosphere at 4°C for 3 weeks, and 95.1% of the current was retained, indicating an excellent stability

Figure 3 (a) Cyclic voltammograms of AgNPs@GQDs/CS/GCE at various scan rates from 10 mV.s −1 to 500 mV.s−1in Ar-saturated PBS containing 0.5 mM H2O; (b) Plot of I p versus v1/2.

Figure 4 (a) CVs of AgNPs@GQDs/CS/GCE after injection of 10 mM of different interferents+GOx solution; (b) Maximum peak currents extracted from (a); and amperometric i-t curves of for different concentrations of (c) glucose and (d) lactose.

4 Conclusions

In this work, a simple and convenient detection for glucose based on AgNPs@GQDs/CS-modified glassy carbon electrodes was reported for thefirst time in the field of biosensing analysis AgNPs@GQDs acts as a good catalyst for electroreduction of H2O2generated from the reaction of glucose with glucose oxidase and O2

AgNPs@GQDs/CS/GCE show low LOD, good stability and acceptable selectivity It also shows simple operation and high sensitivity, which therefore makes this modified electrode very promising for analytical applications involving H2O2such as glucose sensors

Acknowledgments

The authors are grateful to Vietnam’s National Foundation for Science and Technology Development

(NAFOSTED) with Project code 104.99-2016.23 for providing financial support H V Tran thanks University Paris Diderot, France, for an internship grant

ORCID iDs

Hoang Vinh Tran https://orcid.org/0000-0003-1777-5526

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