An amperometric H2O2 biosensor based on hemoglobin nanoparticles immobilized on to a gold electrode

The nanoparticles (NPs) of hemoglobin (Hb) were prepared by desolvation method and characterized by transmission electron microscopy (TEM), UV spectroscopy and Fourier-transform IR (FTIR) spectroscopy. An amperometric H2O2 biosensor was constructed by immobilizing HbNPs covalently on to a polycrystalline Au electrode (AuE). HbNPs/AuE were characterized by scanning electron microscopy (SEM), cyclic voltammetry (CV) and electrochemical impedance spectra (EIS) before and after immobilization of HbNPs. The HbNPs/AuE showed optimum response within 2.5 s at pH 6.5 in 0.1 M sodium phosphate buffer (PB) containing 100 μM H2O2 at 30°C, when operated at –0.2 V against Ag/AgCl. The HbNPs/AuE exhibited Vmax of 5.161 ± 0.1 μA cm−2 with apparent Michaelis-Menten constant (Km) of 0.1 ± 0.01 mM. The biosensor showed lower detection limit (1.0 μM), high sensitivity (129 ± 0.25 μA cm−2 mM−1) and wider linear range (1.0–1200 μM) for H2O2 as compared with earlier biosensors. The analytical recoveries of added H2O2 in serum (0.5 and 1.0 μM) were 97.77 and 98.01% respectively, within and between batch coefficients of variation (CV) were 3.16 and 3.36% respectively. There was a good correlation between sera H2O2 values obtained by standard enzymic colorimetric method and the present biosensor (correlation coefficient, R2 =0.99). The biosensor measured H2O2 level in sera of apparently healthy subjects and persons suffering from diabetes type II. The HbNPs/AuE lost 10% of its initial activity after 90 days of regular use, when stored dry at 4°C.


Introduction
Hydrogen peroxide (H 2 O 2 ) is an incompletely reduced metabolite of oxygen that has a diverse array of physiological and pathological effects within living cells, depending on the extent, timing and location of its production. Characterization of the cellular functions of H 2 O 2 requires measurement of its concentration selectively in the presence of other oxygen metabolites with spatial and temporal fidelity in live cells [1]. H 2 O 2 is one of the reactive oxygen species (ROS) amongst others like superoxide anion (O 2 •− ), hydroxyl radical (OH • ), hydroxyl ion (OH − ). The higher concentration of H 2 O 2 is associated with diabetes, atherosclerosis, and ageing, as it generates free hydroxyl radicals, which cause oxidative damage to the tissue components such as lipids and proteins beside DNA [2,3]. For the measurement of H 2 O 2 in biological fluids, several sensitive methods based on horseradish peroxidase (HRP) and artificial substrates (such as Amplex Red and 3,5,3 ,5 -tetramethylbenzidine) or on ferrous oxidation in the presence of xylenol orange (FOX) have been developed [4]. The rapid and accurate determination of H 2 O 2 is very important, as it is not only the product of the reactions catalyzed by many highly selective oxidases, but also employed in various fields such as food, pharmaceutical, biology, medicine, industry and environmental analysis [5]. Hyperglycemia is one of the major ways of inducing ROS and endothelial dysfunction in patients with diabetes [6]. The imbalance between the production of ROS and the endogenous antioxidant mechanism to counteract the effect of ROS or repair the resulting damages generate oxidative stress [7]. The determination of H 2 O 2 in diabetics helps in evaluating their oxidative stress status. By maintaining a good diabetic control and implementing strategies including supplementation of antioxidant micronutrients (vitamins E and C, β-carotene) will reduce the oxidative stress and thus help in reducing the risk of chronic complications [8]. Several methods, such as titrimetry [5], spectrometry [9], chemiluminescence [10], fluorimetry [11], chromatography [12], and electrochemical techniques [13,14] have been reported for this purpose. Amongst these techniques, the electrochemical techniques are preferable, because of their simplicity, low cost, high sensitivity, rapidity, and selectivity. The proteins containing heme groups, such as hemoglobin (Hb), myoglobin, and cytochrome c possess peroxidase-like for reduction of H 2 O 2 , due to the electroactive center of heme [15][16][17]. Amongst these, Hb is more suitable for H 2 O 2 biosensor, because of its known structure, commercial availability at low cost and is relatively more stable. Thus the biosensors using native Hb were more specific with reduced fabrication cost [18,19]. The native Hb has been immobilized on to various types of nanocomposites for construction of H 2 O 2 biosensor such as Hb/Pluronic P123-nanographene platelet (NGP) [20], Hb/collagen microbelt [21], Hb/Ag sol films [22], Hb/AuNPs/L-Cysteine (L-cys)/p-aminobenzene sulphonic acid (p-ABSA)/Pt disk [24], Hb/collagen-multiwall carbon nanotubes (c-MWCNT) [24], DNA-Hb/Au [25]. The various supports used for the H 2 O 2 biosensors are: platinum nanoparticles (NPs) (PtNPs)/reduced graphene oxide (RGO)/chitosan (CS)/ferrocene (Fc) [28], self-assembled dipeptide (DP)-gold NPs (AuNPs)/HRP/glassy carbon electrode (GCE) [29], graphene capsules (GRCAPS)/HRP/indium tin oxide (ITO) [30], HRP/poly(aniline-co-N-methyllanthionine) (PAN-PNMThH) [31], electrochemically reduced graphene oxide (ERGO)/GCE [32], 3,3 ,5,5 -teramethylbencidine (TMB)/HRP/polydimethylsiloxane (PDMS)/tetraethylorthosilicate (TEOS)/silicon oxide NPs (SiO 2 NPs) [33], turnip tissue paper (TTP)/SPCE [34], GPtNPs [35], PtRu/3DGF [36], cytochrome c (Cytc)/nickel oxide nanaoparticles (NiONPs)/c-MWCNT/polyaniline (PANI)/Au [37]. However, these methods involve the complex strategies for construction of working electrode and have poor detection limit and narrow linear range. Hence, there is a need for development of an efficient and reliable analytical device that can provide high sensitivity, facile operation, and quick response to the H 2 O 2 molecule. In recent years, protein NPs have been employed in the construction of improved biosensors due to their exceptional electronic, optical, mechanical, and thermal properties [26]. AuE offers many attractive features such as good conductor of electricity, low cost, low background current, and easy availability [27]. The present work describes the construction of an H 2 O 2 biosensor based on covalent immobilization of HbNPs on to AuE, its characterization, evaluation, and application for amperometric determination of H 2 O 2 in sera of diabetic patients.

Experimental procedures Materials
Hb, Tris buffer, NAD + , D-Glucose, uric acid, urea, glucose, ascorbic acid, potassium chloride, silica gel from SRL, Mumbai; sulphuric acid from Qualigens Fine Chemicals, Mumbai were used. Au wire (diameter: 2 mm) for the preparation of AuE was purchased from a local jewellery shop. Sera samples of apparently healthy subjects and persons suffering from diabetes type II were collected from local PGIMS hospital. All other chemicals were of analytical reagent (AR) grade. Double distilled water (DW) was used throughout the experimental studies.

Preparation of HbNPs
The HbNPs were prepared by desolvation method. To the Hb solution (1 mg/ml), 5 ml of absolute ethanol was added dropwise at a rate of 0.1-0.2 ml/min under continuous stirring at a speed of 500 rpm. The desolvating agent encouraged the aggregation of protein molecules into small NPs by eliminating water molecules amongst them. In the same way, process was followed by addition of 1 ml of 1% glutaraldehyde solution into the reaction mixture. The mixture was kept under the similar stirring conditions at 4 • C for 24 h to ensure complete cross-linking of HbNPs. Such a high concentration of glutaraldehyde is likely to provide intermolecular cross-linking of HbNPs molecules through Schiff's base. Besides, above all the enzyme HbNPs thus formed were thiol functionalized by adding 0.02 g/ml cysteamine solution (0.02 g/ml) to HbNPs suspension under constant stirring for 5-6 h. Last, HbNPs were separated from the supernatant by centrifuging NPs suspension at 1200×g for 10 min at 4 • C, followed by redispersion of HbNPs in 0.1 M phosphate buffer (PB) (pH 7.3) and sonication for 5 min. Moreover, it is assumed that α-NH 2 group of cysteamine reacts with excess unreacted -CHO groups of glutaraldehyde cross-linked Hb molecules to form Schiff's base. Thus, the glutaraldehyde cross-linked HbNPs get functionalized with -SH groups. These -SH functionalized HbNPs were stored at 4 • C until use [38].

Characterization of HbNPs
HbNPs were characterized by recording their TEM images, UV and visible spectra and FTIR spectra. The size and shape of HbNPs were measured by a high-resolution transmission electron microscope, Zeiss EM 912, at an accelerating voltage of 120 kV. TEM samples of the HbNPs were prepared by placing the product solution on to the carbon-coated copper grids, allowing the solvent to evaporate in air. UV absorbance of HbNPs dispersed in 0.1 M sodium PB (pH:7.0) was recorded in the range from 200 to 600 nm at an interval of 50 nm, using UV spectrophotometer (make: Shimadzu, Japan, model 1700). FTIR spectra of the HbNPs were measured by FTIR spectrometer (Thermo Scientific, U.S.A.) using the standard KBr method over a range of 500-4000 cm −1 .

Preparation of HbNPs modified Au electrode
Thiol groups on the surface of HbNPs provide a facile way for attachment of HbNPs on the surface of Au electrode (AuE). Before immobilization, the cleaned bare AuE was scanned over the 0-0.5 V range in freshly prepared 0.2 M H 2 SO 4 until the voltammogram characteristic of the clean polycrystalline Au was established. The polycrystalline AuE was placed in the HbNPs suspension under mild stirring at 4 • C for 12 h to get covalent coupling between thiol-functionalized cross-linked HbNPs and polycrystalline AuE via Au-thiolated bond through dehydration process ( Figure 1). The HbNPs/AuE were rinsed with 0.1 M of PB (pH 7.0) carefully and stored in PB buffer at 4 • C, when not in use. The characterization of HbNPs/Au electrode was done by SEM and electrochemical impedance spectra (EIS). Below is a representation of the chemical reaction that describes the attachment of the HbNPs to the AuE.

Optimization of H 2 O 2 biosensor
To optimize the working conditions of the biosensor, effects of pH, incubation temperature, time of incubation, and substrate (H 2 O 2 ) concentration on biosensor response were studied. To determine the optimum pH, it was varied

Applications of H 2 O 2 biosensor
Blood samples (1 ml each) were drawn from apparently healthy males and females (20 each), and persons with diagnosis of diabetes type II at Pt B.D.S. PGIMS, Rohtak Hospital and centrifuged at 2000 rpm for 10-15 min and their supernatant (serum) was collected. H 2 O 2 content in sera was determined by the present biosensor in the similar manner as described above for its response measurement, under its optimal working conditions except that H 2 O 2 was replaced by serum sample. The H 2 O 2 content in serum was interpolated from standard curve between H 2 O 2 concentrations compared with current (in mA) prepared under optimal assay conditions of biosensor ( Figure 2).

Characterization of HbNPs
The average size of HbNPs as measured by TEM was between 5 and 100 nm (Figure 3a (Figure 3b). In comparison with the FTIR spectrum of the Hb and HbNPs, the major shift was observed in the carbonyl, hydroxyl, and amino groups of protein, which is due to ethanol, glutaraldehyde, and cysteamine treatment of HbNPs during their preparation.

By SEM
The SEM images of the surface of bare Au and HbNPs/Au are shown in Figure 3c. The stepwise modification of electrode could be seen clearly from these SEM images. The SEM image of the bare Au showed a smooth and feature-less morphology. On immobilization of HbNPs, a globular structural morphology appeared due to the interaction between HbNPs and Au, which revealed larger and effective surface area.
By EIS EIS provides useful information on impedance changes on the electrode surface during the fabrication process. It was carried out to investigate immobilization of HbNPs on to Au electrode. The diameter of the semicircle at higher frequencies corresponds to the electron transfer resistance (R CT ) which controls the electron transfer kinetics of the redox probe at the electrode interface, while the linear part at lower frequencies corresponds to Warburg diffusion process. The Nyquist plot (Figure 3d) displays EIS of HbNPs/Au in 5 mM K 3 Fe(CN) 6 /K 4 Fe(CN) 6. The R CT value for native Hb was 200 which is lower than HbNPs/Au, i.e. 300 . These results showed that increased R CT value of HbNPs/Au electrode was due to the immobilization of HbNPs on to Au surface. This increase in R CT is attributed to the fact that most of the biological molecules, including HbNPs, are poor electrical conductors at low frequencies (at least <10 kHz, applied voltage: 0.2 V) and cause hindrance to electron transfer.

Evaluation of H 2 O 2 biosensor
There was a linear relationship between the biosensor response, i.e. current (in mA) and the H 2 O 2 concentration in the range 1.0-1200 μM, which is better than earlier biosensors, 10-150 μM [20], 10-120 μM [25],  Table S2). These high precisions reveal the good reproducibility and consistency of the present method. The H 2 O 2 level in sera of 20 apparently healthy subjects and persons suffering from diabetes type II, as measured by present biosensor were compared with those obtained by the standard Enzo kit method. There was a good correlation (R 2 =0.99) between these two methods (Figure 6a) showing the high accuracy of biosensor.

Interference study
The interference study was performed to assess the selectivity of the present biosensor. The HbNPs biosensor exhibited excellent anti-interference abilities with various major blood components such as citric acid, glutamic acid, uric acid, urea, and ascorbic acid at their physiological concentrations. The change in relative response in presence of citric acid, glutamic acid, uric acid, urea, and ascorbic acid was 4.20, 2.70, 1.50, 3.80, and 2.80%, respectively under the standard assay conditions. These changes (1.50-4.20%) had practically no effect on biosensor response. This practically no interference could be attributed to functioning of biosensor at lower applied potential (-0.2 V). Hence, the present biosensor could be highly selective/specific for H 2 O 2 .

Applications of H 2 O 2 biosensor
The H 2 O 2 content as measured by the present biosensor was in the range from 23.9 + − 0.04 to 45.6 + − 0.05 μM (n=20) for sera of apparently healthy persons, which is in normal established range and from 68.2 + − 0.05 to 143 + − 0.05 μM (n=20) for persons suffering from diabetes type II (Supplementary Table S3).

Storage stability of HbNPs/Au electrode
The HbNPs/Au electrode were tested every fifth day over a period of 90 days, while being stored dry at 4 • C. Figure  6b shows the % of initial response of HbNPs/AuE compared with storage time. The electrode lost 10% of its initial activity after 90 days of its regular use when stored dry at 4 • C. The storage stability of the present biosensor is better than earlier reported biosensors as 25 days [34], 30 days [37] which could be credited to covalent immobilization of HbNPs on to AuE. The present biosensor could be advanced with respect to use in biomedicine or clinical settings by miniaturizing it into commercial/portable model, which could be used at the bedside of the patient. The miniaturization means designing of an electronic chip for current measuring device (potentiostat) and transformation of HbNPs/AuE into a microelectrode (like screen-printed electrode), which could be dipped into one drop of biological sample and an adopter to connect the miniaturized current device with the microelectrode. The current device needs to be battery operated and finally calibrated according to the concentration of H 2 O 2 . However, biosensor has some drawbacks such as lack of substrate specificity, requirement of expensive Au wire for construction of working electrode and cold temperature for its storage.