A novel electrochemical hydrogen peroxide biosensor based on hemoglobin capped gold nanoclusters–chitosan composite

Abstract

A biocompatible nanocomposite containing hemoglobin capped gold nanocluster (Hb-AuNCs) and chitosan was prepared and applied to the modification of a glassy carbon electrode (GCE) for the preparation of a highly sensitive hydrogen peroxide biosensor. The electrochemical behavior of Hb-AuNCs in the composite film was studied in phosphate buffer solution of pH 7.4 and a pair of quasi-reversible redox peak attributed to the electrode reaction of Hb's Fe(III)/Fe(II) redox couple was observed. The FTIR and UV-vis spectroscopy indicated that Hb entrapped in the composite film possesses substantial changes in its secondary structure so that it provided a high peroxidase-like enzyme activity attributing to the excellent electrical conductivity of the encapsulated gold nanoclusters. The AuNCs found to play a critical role as conductive holder and accumulator of redox active centers at the surface of GCEs. The fabricated biosensor showed fast response, acceptable stability, excellent sensitivity, and high electrocatalytic activity toward the reduction of hydrogen peroxide. The oxidation peak current was found to be linearly proportional to H₂O₂ concentration in the range of 55–700 nM with a limit of detection of 16.5 nM (at S/N = 3).

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Introduction

Recently, there is an increasing interest in the development of an electrochemical basis for the investigation of protein structure, mechanisms of redox transformation of protein molecules and their metabolic processes involving redox transformations. Understanding of these reactions fundamentally can provide an insight into physiological electron transfer process as well as an impetus to the further development of biosensors and bio-electrocatalytic systems.^1–3 In this line, there has been much effort for the development of H₂O₂ electrochemical biosensors which can widely improve the understanding of the redox processes of enzymes and proteins, and their transport and bioavailability.⁴ H₂O₂ is not only known for its cytotoxic effects and associated tissue injury, but also plays a key role in physiological and biomedical studies and in monitoring of biological processes. H₂O₂ is also a side product of many oxidative biological reactions catalyzed by enzymes such as glucose oxidase (GOx), lactate oxidase (LOx), cholesterol oxidase (ChoOx), etc.⁵ Therefore, sensitive and accurate determination of H₂O₂ is of vital importance in many biological applications.

Hemoglobin (Hb), a typical multi-cofactor protein that possesses two heme-containing dimers, is considered to be an ideal model protein for the study of electron transfer of heme containing molecules, because of its commercial availability, moderate cost and well-known structure.⁶ Moreover, due to its intrinsic peroxidase activity, Hb can be used in the design of H₂O₂ biosensors.^5,7 However, it has been documented that, on conventional solid electrodes, the fast electron transfer between Hb and the electrode is not possible because the redox center of proteins is embedded in polypeptide chain structures and the proteins are easily absorbed on the electrode surface.⁸ Such slow electron exchange may be due to unfavorable orientation of Hb molecules on electrode surfaces, which increases the distance between its heme center and electrode surface, and also to the adsorption of impurities and denatured proteins onto electrode surface, which can block the electron communication between heme and electrode.⁹ Therefore, great efforts have been devoted to explore new immobilization methods and supporting materials that accelerate the electron transfer of Hb while maintaining its enzymatic activity. In this respect, the selection of appropriate nanomaterials (NMs), including nanoparticles (NPs),^10,11 quantum dots (QDs)^12,13 and carbon nanomaterials^14,15 is an important issue for fabrication of an effective electrochemical biosensor based on Hb as a cheap mimetic enzyme.

Recently, the protein-supported nanoclusters of noble metals with excellent conductivity have been successfully applied as electrochemical interfaces for sensitive detection of H₂O₂,¹⁶ glucose,¹⁷ retinal-binding protein¹⁸ and KB cells.¹⁹ In recent years, we have been involved in one-pot ‘green’ synthesis of fluorescent Hb-AuNCs with reactive functional groups, low-toxicity, good biocompatibility and stability.²⁰ We also found that Hb remains active in the Hb-AuNCs as an excellent sensing platform for hydrogen peroxide, which makes the nanocluster attractive for further investigation in different analytical fields including electrochemical applications.²¹

On the basis of the above findings, herein, we employed Hb-AuNCs as a non-enzymatic electrochemical probe for a very simple and convenient preparation of a biosensor for H₂O₂ with enhanced electrocatalytic activity. In one hand, the AuNCs can induce substantial conformational changes of Hb while keeping its bioactivity on electrode and, in the other, the encapsulated AuNCs possess very important role in enhancing the rate of electron transfer in Hb, which results in improved electrochemical sensing ability. To the best of our knowledge, this is the first time that the direct electrochemistry of Hb-AuNCs and its electrocatalysis towards H₂O₂ has been investigated in a sensing system.

Experimental

Chemicals

Human hemoglobin (Hb, MW 66 000) was purchased from Sigma and used without further purification. Hydrogen tetrachloroaurate (HAuCl₄) was obtained from Alfa Aesar and trisodium citrate, sodium hydroxide and ammonia were purchased from Fluka. The experimental procedures for preparation of hemoglobin were carried out based on the protocol of Williams and Tsay²² and its concentration was monitored by the method of Antonini and Brunori (ε_415nm = 125 mM⁻¹ cm⁻¹ or ε_541nm = 13.8 mM⁻¹ cm⁻¹ per heme),²³ as reported before.^20,21 5 mg mL⁻¹ hemoglobin solutions of pH 7.4 were stored at 4 °C. 0.1 M phosphate buffers of various pH values were prepared by mixing the stock solutions of Na₂HPO₄ and NaH₂PO₄ and adjusted by 0.1 mM NaOH and 0.1 mM H₃PO₄ solutions. All other chemicals were of analytical grade from Merck and used without further purification. All solutions were made up with doubly distilled water.

Apparatus

The electrochemical experiments were performed using an Autolab modular electrochemical system (ECO Chemie, Utrecht, The Netherlands) equipped with a PGSTAT 101 module and driven by NOVA software (ECO Chemie) in conjunction with a personal computer for data storage and processing. The electrochemical cell used was consisted of a three-electrode system using the modified glassy carbon electrode (GCE, d = 3.0 mm) as the working electrode, a platinum wire as a counter electrode and an Ag/AgCl electrode as the reference electrode. Air was injected into deoxygenated phosphate buffer solution by syringe under nitrogen atmosphere. Amperometric experiments were carried out in a stirred system by applying an analytical potential to the working electrode. Aliquots of H₂O₂ standard solution were added successively to the solution. Current–time data were recorded after a steady state current had been achieved. Faradaic impedance measurements were performed in the presence of a 1 : 1 mixture of 5 mMK₃Fe(CN)₆/K₄Fe(CN)₆ as a suitable redox-probe, using an alternating current voltage of 10 mV and frequency range of 0.01 to 10 000 Hz. The UV-vis and IR spectra were recorded on an UV-2450 UV-vis recording spectrophotometer (Shimadzu, Japan) and a Tensor 27 Bruker instrument (Bruker, Japan), respectively. The TEM images were recorded using a Philips CM30 transmission electron microscope with accelerating voltages of 80 and 150 kV. Samples were prepared by drop casting solution on carbon coated copper grids and dried at room temperature. All pH measurements were performed at 25.0 ± 0.1 °C by using a Metrohm 713 pH/ion-meter with a standard uncertainty of 0.1 mV (Metrohm, Switzerland).

Synthesis of hemoglobin-capped AuNCs

All glassware was thoroughly cleaned with aqua regia (HNO₃/HCl, 1 : 3) and rinsed extensively with ethanol and Milli-Q water (resistivity >18 MΩ cm) prior to use. The generation of blue emitting Hb-AuNCs was mainly based on previously established synthetic methods.^20,21 In brief, a 5 mL aqueous solution of HAuCl₄ (2.8 mM, 37 °C) was reacted with 5 mL of human adult hemoglobin (Hb) (7 mg mL⁻¹, 37 °C), under vigorous stirring. After 10 min, 1.0 mL of NaOH solution (1 M) was introduced and the reaction was allowed to proceed under vigorous stirring at 37 °C for 24 h. After centrifugation (12 000 rpm, 10 min, 4 °C) to remove any suspended particles and byproducts with larger particle sizes, the blackish green solution of the as-prepared Hb-AuNC was collected and kept at 4 °C before use. The concentration of Au nanoclusters was calculated by the concentration of the Hb in Hb-AuNCs solution.²⁴ The concentration 5.5 × 10⁻⁵ M of Hb-Au nanoclusters was measured spectrophotometrically using a molar absorptivity of 131 936 M⁻¹ cm⁻¹ at 280 nm. The particle size and shape of Hb-AuNPs were evaluated by transmission electron microscope (TEM). The TEM image of AuNCs@Hb is shown in Fig. 1. It can be seen that the AuNCs are well-dispersed with spherical shapes with an average size of 2.5 ± 0.6 nm.

Fig. 1 TEM images of Hb-AuNCs.

Preparation of biosensor

A GCE (d = 3 mm) was polished to a mirror-like surface with 0.3 and 0.05 μm aluminum slurry, respectively. Subsequently, the electrode was rinsed with 1 : 1 HNO₃–H₂O (v/v), ethanol and doubly distilled water in an ultrasonic bath for 2–3 min for each wash and dried under nitrogen at room temperature. Chitosan (Chit) was dissolved in 1.0% (v/v) acetic acid solution and stirred for 2 h to form 0.5% (w/v) solution at room temperature. After filtering out of the un-dissolved materials, the pH was adjusted using 1.0 M NaOH. Then the Hb-AuNPs/Chit composite biosensor was prepared with the following procedure: 8 μL of the colloidal Hb-AuNPs solution was cast at on the surface of GCE, and the electrode was dried in air. Finally, 12 μL of the as-prepared chitosan solution was cast on the surface of fabricated modified electrode and dried in air.

Results and discussion

FTIR study

Since proteins have conformation sensitive spectral signature in the infrared region of the electromagnetic radiation, the FTIR was employed as a suitable technique to identify the conformational sensitive binding sites with the nanoparticle. Fig. 2A shows a comparison between the FTIR spectra of Hb (a) and Hb-AuNCs (b). Compared with the pure IR spectra of Hb (a), the intensity of amide I (1650 cm⁻¹) of Hb immobilized on Hb-AuNCs (b) is decreased, and amide II (1533 cm⁻¹) is disappeared, in the expense of the appearance of a rather sharp band at 1436 cm⁻¹ (Fig. 2A). The results indicated that the gold nanoclusters present in the prepared Hb-GNCs induce a conformational change of the secondary structure of the protein.²⁰ Moreover, the 1436 cm⁻¹ band is due to the vibration of tryptophan (Trp) that becomes drastically larger when nanoclusters are prepared at high pH.²⁵

Fig. 2 (A) FT-IR spectra of Hb and AuNC@Hb films and (B) UV-vis absorption spectra of Hb and Hb@AuNCs in 0.05 M PBS buffer of pH, 7.4.

UV-vis spectroscopy

It was important to know whether heme-protein maintained its natural state in composite film. It is well known that position of the Soret absorption band of prosthetic heme group provided information about possible denaturation of heme-proteins.²⁵ The UV-vis absorption spectroscopy was then employed for the conformation study of Hb in composite film (Fig. 2B). It is quite obvious from Fig. 2B that the Soret band of Hb alone (curve a) is located at about 412 nm, but in the presence of AuNCs (curve b), it is shifted to 403 nm while its intensity is significantly decreased. These results indicate that the formation AuNCs in the presence of Hb leads to the unfolding of the protein skeleton.²⁰ In addition, the increase in intensity of a band around 215 nm (related to the α-helix structure of the protein) and the disappearance of bands located at 280 nm (due to the phenyl group of Trp and tyrosine residues), and 540 and 575 nm (related to oxy-band or Q-band) confirm that the microenvironment surrounding heme in Hb-AuNCs is different from that of native Hb.^20,26

EIS study

EIS is a commonly used method to probe the interface information of the impedance changes during the stepwise-assemble process of the surface modified electrode.^27–29 In EIS the semicircular part at higher frequencies corresponds to the electron transfer limited process and the linear part at lower frequencies corresponds to the diffusion process. The value of charge transfer resistance (R_ct) can be estimated according to the diameter of the semicircle of the Nyquist plots at the high frequency region, which controls the electron transfer kinetics of redox probe at the electrode surface and reflects the interfacial electron transfer ability.

Fig. 3A shows the impedance spectra represented as Nyquist plots (Z′′ vs. Z′) for bare GCE (a) and Hb-AuNCs/Chit/GCE (b) in 5 mM Fe(CN)₆^3−/4− containing 0.1 M KCl solution with the frequency sweep from 105 to 0.1 Hz. Here Z′ and Z′′ are the real and the imaginary variables of impedance. The representative Randles circuit for the Nyquist plots is shown in inset of Fig. 3A, where R_s, CPE, R_ct and W represent solution resistance, a constant phase element corresponding to the double layer capacitance at the electrode surface, the charge transfer resistance and the Warburg impedance, respectively. The diameter of the semi-circle is proportional to the charge transfer resistance; hence, the electron transfer properties can be determined accordingly. The straight line portion represents the Warburg impedance which takes into account the frequency dependence on diffusion transportation to the electrode surface. As is shown, there is a very low charge transfer resistance for Fe(CN)₆^3−/4− at bare GCE (curve a). However, after modifying the GCE with Hb-AuNCs/Chit film, the R_ct increases dramatically to about 2000 Ω (curve b), as obtained from the corresponding Randles circuit, indicating that the biocomposite film hinders the charge transfer process.

Fig. 3 (A) Nyquist plots for GCE (a) and Hb-AuNCs/Chit/GCE (b). (B) Plot of impedance vs. ω^−1/2, in the presence of 5 mM [Fe(CN)₆]^3−/4− and 0.1 M KCl at a frequency range of 10⁵ to 0.1 Hz.

It should be noted that the plot of Z′ at Warburg portion vs. ω^−1/2 (Fig. 3B) can also be applied to determine R_ct. The obtained value of R_ct from the intercept of the linear Z′ vs. ω^−1/2 plot²⁷ was found to be 1998 Ω, which nicely confirms the obtained value of 2000 Ω from the corresponding Nyquist plot.

Direct electrochemistry of Hb-AuNCs and electrocatalysis of H₂O₂

In order to compare the electrochemical properties of Hb and Hb-AuNCs film, the cyclic voltammograms of Hb/Chit/GCE and Hb-AuNCs/Chit/GCE in 0.1 M PBS of pH 7.4 at a scan rate of 50 mV s⁻¹ were recorded. As shown in Fig. 4 curve (a), when the electrode was modified with Hb/CHIT film, only a unsymmetrical quasi reversible weak peak for Fe(III)-porphyrin ↔ Fe(II)-porphyrin redox reaction with a ΔE_p of about 174 mV was observed, indicating that the direct electron transfer rate of Hb with GCE is very slow and only few Hb molecules closest to the electrode surface could exchange electrons. However, at the Hb-AuNCs/Chit/GCE an enhanced redox peak with much lower ΔE_p of 130 mV was observed (curve b). The formal potential (E°′), estimated as (E_pa + E_pc)/2, where E_pa and E_pc are the anodic and cathodic peak potentials, respectively, is −0.285 vs. Ag/AgCl. It is interesting to note that this value is more positive than the corresponding literature reported values for Hb/C60-NCNTs/CHIT/GCE (−0.335 V vs. SCE),³⁰ Hb/RTIL/PDDA-G (−0.326 V vs. SCE),³¹ Nafion/Hb/PAM-P123/GCE (−0.317 V vs. SCE),³² Hb/AgNPPdop@CNP/GCE (−0.398 V vs. SCE),³³ Hb/HNTs/ILs-modified electrodes (−0.313 V vs. Ag/AgCl);³⁴ while, it is more negative than that reported for GCE/CM-DDAM@Hb (−0.186 V vs. Ag/AgCl)³⁵ and Hb/Chit-[bmim]PF₆-Gr/GCE (−0.206 V vs. SCE).³⁶

Fig. 4 Cyclic voltammograms of GC/Hb/Chit and GC/AuNC@Hb/Chit in absence (a and b) and presence (a′ and b′) of a 12.0 μM H₂O₂ in 0.1 M PBS of pH 7.4 at a scan rate of 50 mV s⁻¹.

As is obvious from Fig. 4, here the charging current increased, due to the increased active surface and 3D structure of Hb-AuNCs which confers a much higher electrochemical activity to the modified electrode. As can be seen, at Hb-AuNCs/Chit film, the electrochemical response of Hb is greatly enhanced, indicating that the AuNCs play a key role as conductive holders and accumulators of the redox active centers (RACs) at the surface of glassy carbon electrode. Thus, as expected, the capability of AuNCs in improving the electron transfer rate of Fe(II)/Fe(III) redox reaction resulted in enhanced sensitivity of the biosensor due to increased density of the RACs, relative to the simple Hb/Chit modified electrode.

In order to compare the electrocatalytic activity of Hb/Chit/GCE and Hb-AuNCs/Chit/GCE, their responses to the reduction of H₂O₂ was explored. Typical cyclic voltammograms of Hb/Chit/GCE and Hb-AuNCs/Chit/GCE in 0.1 M PBS solution of pH 7.4 in the presence of 12.0 μM of hydrogen peroxide at a scan rate of 50 mV s⁻¹ are presented in Fig. 4, curves (a′) and (b′), respectively. As is obvious from curve (a′), the addition of H₂O₂ to the electrochemical cell resulted in an increase in the reduction peak at −0.35 V accompanied by a distinct decrease in the oxidation peak, emphasizing a typical electrocatalytic reduction process for H₂O₂. Moreover, as expected, the electrocatalytic current at Hb/Chit/GCE for H₂O₂ (curve b′) found to be much lower than that at Hb-AuNCs/Chit/GCE (curve a′), which is attributed to the small RACs density at Hb/Chit/GCE.

As is obvious from Fig. 4, in the presence of hydrogen peroxide, the Fe(III) ↔ Fe(II) onset potential was slightly delayed. This is most probably due to the higher electron transfer resistance of active centers which causes the absorption hydrogen peroxide on the Fe(III) active center to some extent. The results thus obtained demonstrated that the presence of AuNCs at the electrode surface results in the increased efficiency of immobilization. The high efficiency of AuNCs in charge transfer can uphold number of Hb molecules that can be reduced/oxidized, simultaneously. According to the literature,³⁷ the heme can react with H₂O₂ to first form an intermediate (compound 1 in Fig. 5), which in turn, possesses a high catalytic activity towards H₂O₂. Accordingly, a plausible catalytic pathway is explained in Fig. 5.

Fig. 5 Possible mechanism of electrocatalytic reduction of hydrogen peroxide.

Effect of scan rate

Fig. 6A shows the cyclic voltammograms of Hb-AuNCs/Chit/GCE modified electrode in 0.05 M PBS of pH 7.4 at different scan rates from 20 to 500 mV s⁻¹. It can be seen that the redox peak currents increase linearly with scan rates over the range studied (Fig. 6B), indicating that the pair of redox waves originates from the surface confined molecules. The respective linear regression equations between the oxidation and reduction peak currents and scan rate were calculated as: I_pa (μA) = 3.739ν (V s⁻¹) + 0.188 (R² = 0.993) and I_pc (μA) = −4.697ν (V s⁻¹) − 1.002 (R² = 0.994), respectively. This observation suggested that the redox process of Hb over the prepared sensor was surface-controlled. Meanwhile, the redox peak potential was shifted slightly with increasing scan rate so that the peak-to-peak separation was increased gradually, clearly indicating a quasi-reversible electrochemical process.

Fig. 6 (A) Cyclic voltammograms of the modified electrode in a 0.05 M PBS of pH 7.4 at various scan rates of 20, 50, 100, 150, 220, 300, 400 and 500 mV s⁻¹. (B) Variation of peak currents vs. potential scan rate.

The redox peak potentials exhibited linear relationship with natural logarithm of scan rate (ln ν) in the range from 20 to 500 mV s⁻¹ with the regression equations E_pa (V) = 0.068 ln ν − 0.083 and E_pc (V) = −0.049 ln ν − 0.511, respectively. By using the above regression equations for E_pa and E_pc, the electrochemical parameters of Hb in the hybrid film were evaluated according to the following Laviron's equations:^38,39

where, α is the electron transfer coefficient, n is the number of electron transferred, ν is the scan rate, and E°′ is the formal potential, k_s is the electron transfer rate constant and ΔE_p is the peak-to-peak potential separation and R, T and F have their conventional meanings. The values of α and k_s were then calculated as 0.52 and 0.85 s⁻¹, indicating a fast electron transfer rate with high efficiency.

By further use of the effect of the scan rate on the electrochemical response of Hb-AuNCs/Chit/GCE (Fig. 6B) and use of the following standard equation:³⁸

where Q is the charge involved in the reaction which is equal to nFAΓ, n is the number of electron transferred (n = 4 for Hb), F is the Faraday constant, and A is the electrode area (cm²), the surface coverage (Γ) of the electroactive Hb in the modified electrode was estimated for the GCEs modified with Hb-AuNCs/Chit and of Hb/Chit as 3.8 × 10⁻⁹ and 3.2 × 10⁻¹⁰ mol cm⁻², respectively. These values were found to be larger than that of the theoretical monolayer coverage of 1.89 × 10⁻¹¹ mol cm⁻²,⁴⁰ suggesting that more than one layer of Hb is participating in the electron transfer process and also show that the RACs of Hb-AuNCs nanomaterial immobilized electrode is more than that of simple Hb one, under the same dispersed concentration of Hb.¹⁵

Effect of pH

The solution pH is essential to the electrochemical behaviors of proteins. Because its proton participated in the redox process, the immobilized Hb shows a strong dependence on solution pH (Fig. 7). As is obvious from Fig. 7, in the pH range of 3.5–10.0, the peak potentials shifted negatively and the maximum peak currents obtained at pH 7.4. Therefore, a PBS solution of pH 7.4 was use in further studies. The electrochemical reaction can be expressed as: HbFe(III) + H⁺ + e⁻ ⇌ HbHFe(II). Thus, the reason for the above mentioned pH effect might be the influence of the protonation states of trans ligands to the heme iron and amino acids around the heme, or the protonation of the water molecule coordinated to the central iron.⁴¹

Fig. 7 CVs of proposed biosensor in 0.05 M PBS of different pHs at a scan rate 50 mV s⁻¹.

Amperometric study

To evaluate the practical applicability of the designed Hb-AuNCs/Chit/GCE system, H₂O₂ was selected as an important reagent to examine the biocatalytic ability of the Hb-AuNCs. Fig. 8A shows the steady-state current response of H₂O₂, at a constant electrode potential of −0.3 V. As seen, during the successive addition of hydrogen peroxide, a well-defined response is observed. The plot of response current vs. H₂O₂ concentration (Fig. 8B) was linear over the concentration range 55 to 700 nM, with a linear regression equation of I (μA) = −0.351 [H₂O₂] (nM) − 20.064 (R² = 0.9961) and a limit of detection (LOD) of 16.5 nM (at S/N = 3). As is obvious, the developed method shows high sensitivity and wide linear range, and possessing the potential to be one of the most commonly used analytical methods for detection of very low concentration of H₂O₂.

Fig. 8 (A) Amperometric response of biosensor at a rotating modified electrode in the presence of different H₂O₂ concentrations. (B) Plot of amperometric currents vs. H₂O₂ concentrations.

The apparent Michaelis–Menten constant (K^app_M), which provided to be an indication of the enzyme substrate kinetics, was calculated from the electrochemical version of the Lineweaver–Burk equation:⁴²

where I_ss is the steady current after the addition of substrate, C is the bulk concentration of the substrate, and I_max is the maximum current measured under the saturated substrate condition. K^app_M, obtained by the analysis of the slope and the intercept of the plot of the reciprocals of the steady-state current vs. H₂O₂ concentration, was found to be 124 μM, which is much smaller than those of 369 μM for the Hb/HNTs/ILs/GCE,⁴³ 896 μM for Hb/Chit film-modified carbon paste electrode⁴⁴ and 490 μM for the Hb/cationic clay-modified GCE electrode.⁴⁵ The small value of K^app_M indicates that the immobilized Hb entrapped on the proposed biosensor exhibits higher enzymatic activity and a high affinity for H₂O₂.

Stability, repeatability and effect of potential cycling

The modified electrode showed an acceptable stability and repeatability. When not in use, the modified electrode was stored in pH 7.4 PBS at 4 °C for about 3 weeks, and found to retain about 94% of its initial current response. The relative standard deviation (RSD) was 6.4% for nine successive determinations of a 500 nM H₂O₂ with the modified electrode. The fabrication of seven modified electrodes, made independently, showed a good reproducibility with RSD of 7.2% for the current determined in the presence of 50 nM H₂O₂. Continuous potential cycling between −0.8 and 0.1 V in 0.05 M PBS of pH 6.8 was used for the investigation on the stability of modified electrode. As it is seen from Fig. 9, after 80 continuous cycles at a scan rate of 50 mV s⁻¹, the peak heights of the cyclic voltammograms did not show any considerable change.

Fig. 9 Potential cycling of biosensor between −0.8 and 0.1 V in 0.05 M PBS of pH 6.8 at a scan rate of 50 mV s⁻¹: (a) first cycle, (b) after 80 continuous cycles.

Comparison of linear range and limit of detection of different H₂O₂ sensors

Table 1 compares the linear range and LOD of the proposed Hb-AuNCs/Chit/GCE with those of some of the best enzymatic^46–48 and nonenzymatic^49–60 electrochemical hydrogen peroxide sensors, very recently reported in the literature. As is obvious from the summarized data, the proposed amperometric biosensor can be ranked among a few of the best reported H₂O₂ sensors, from the viewpoints of linear range and LOD.^46,47,54,61 Moreover, the fabricated biosensor showed very fast response, acceptable stability, excellent sensitivity, and high electrocatalytic activity toward the reduction of hydrogen peroxide. To the best of our knowledge, this is the first time that the direct electrochemistry of Hb-AuNCs and its excellent electrocatalytic property towards reduction of H₂O₂ has been investigated in a sensing system. Meanwhile the results obtained from this work emphasized that the Hb-AuNPs/Chit film possesses a promising potential in fabricating the third generation of nonenzymatic electrochemical biosensors, bioelectronics, biocatalysis and biomedical devices in the future.

Table 1 Comparison of linear range and limit of detection of different electrochemical sensors reported for hydrogen peroxide determination^a

Electrode	Method	Linear range	LOD	Ref.
a GCE, glassy carbon electrode; MWCNTs, multiwall carbon nanotubes; Cat, catalase; NPs, nanoparticles; Gr, graphene; Mes, mesoporous; OA, oleylamine; HRP, horseradish peroxidase; GPE, graphite pencil electrode; CQDs, carbon quantum dots; NC, nanocomposite; GrP, graphene paper; NCs, nanoclusters; Chit, chitosan.
GCE/MWCNTs/[bmim][PF6]/CAT	Impedimetry	5.0 nM–1.7 μM	0.25 nM	46
GCE/MWCNTs/NiO/CAT	Impedimetry	19–170 nM	2.4 nM	47
GC/MWCNT-NiO/CAT	Amperometry	0.20–2.53 mM	19 mM	47
Ag–MnO2-MWCNTs	Voltammetry	5.0 μM–10.4 mM	1.7 μM	49
NanoAg@TiO2/GCE	Voltammetry	0.83–43.3 μM	0.83 μM	50
AuNPs-MesSi/GrO	Voltammetry	0.5 μM–50 mM	60 nM	51
GNPs/CNTs/self-doped PA hollow spheres	Chronoamperometry	5 μM–0.225 mM	0.4 μM	52
CdNPs/MWCNTs/GCE	Voltammetry	0.5–200 μM	0.1 μM	53
PtNPs/CNTs	Voltammetry	5.8 nM–1.1 mM	1.9 nM	54
OA-AuNWs/HRP	Voltammetry	20–500 μM	5 μM	48
OA-AuNPs/HRP	Voltammetry	20–500 μM	8 μM	48
AgNPs/CNTs/GrO	Voltammetry	0.1–100 mM	0.9 μM	55
PtNPs/GPE	Amperometry	5 μM–5.3 mM	2.8 μM	56
CQDs-Cu2O NC	Voltammetry	0.03–15.0 mM	22.8 μM	57
Ag–Si–Mo NC	Amperometry	50 μM–24 mM	0.1 mM	58
MnO2 NWs-GrP	Amperometry	0.1–45.4 mM	10 μM	59
HRP/CDs/LDHs/GCE	Voltammetry	0.1–23.1 μM	0.04 μM	60
Pt-AuNCs/Gr	Voltammetry	15 nM–8.73 μM	8.0 nM	61
Hb-AuNCs/Chit/GCE	Amperometry	55–700 nM	16.5 nM	This work

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Conclusions

In this work, direct electrochemistry of Hb-AuNCs was investigated when the protein capped gold nanoclusters were immobilized by Chit on a GCE. The AuNCs play a role as conductive holder and accumulator of redox active centers (RACs) at the surface of glassy carbon electrode. The high RACs density of the prepared Hb-AuNCs/Chit/GCE biosensor increased its sensitivity considerably as it was compared with a simple Hb/Chit/GCE. Excellent electrocatalytic properties of the modified electrode toward hydrogen peroxide indicated that the films possess a promising potential in fabricating the third generation nonenzymatic electrochemical biosensors, biocatalysis, bioelectronics and biomedical devices in the future.

Acknowledgements

The authors thank Iran National Elite Foundation (INEF), Research Council of Razi University and Center of Excellence in Sensors and Biosensors at Razi University for the support of this work.

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