Hemoglobin-Modified Core–Shell Fe3O4@Au Nanostructures for the Electrochemical Detection of Acrylamide

In this study, electrochemical detection of acrylamide using hemoglobin (Hb)-modified core–shell Fe3O4@Au nanostructures was conducted. Fe3O4 nanoparticles (~4.9 nm) and core–shell Fe3O4@Au (5.0–6.4 nm) nanostructures were successfully synthesized by the thermal decomposition method. Electrochemical investigation revealed that the optimum amount of Hb of 2 mg/mL could be immobilized in 0.1 M acetate buffer solution (pH = 6). Moreover, the detection of acrylamide using Fe3O4@Au/Hb was evaluated by the cyclic voltammetry technique. A linear calibration curve (R = 0.98) in the concentration range of 0.1 to 1.0 μM could be achieved with an estimated limit of detection, limit of quantification, and sensitivity of 0.136 μM, 0.453 μM, and 0.4411 μA/μM, respectively. Furthermore, the developed biosensor exhibited high selectivity in the presence of ascorbic acid, melamine, and caffeine. The developed biosensor was applied to the detection of acrylamide in coffee samples and validated using the standard high-performance liquid chromatography (HPLC) method. The concentration of acrylamide in coffee samples was determined to be 37.450 and 35.377 ppm using electrochemical measurement and HPLC, respectively.


Introduction
Acrylamide, a carcinogen and neurotoxin, is primarily present in various sugar-containing and carbohydratecontaining foods processed at high temperatures [1,2]. However, the current analytical tools used to detect acrylamide in food samples, such as liquid chromatography-tandem mass spectrometry [3], highperformance liquid chromatography (HPLC) [4], headspace solid-phase microextraction, and gas chromatography-flame ionization detection [5], have several drawbacks, including high cost and sample preparation complexity. Therefore, the development of methods for detecting acrylamide that are easy, inexpensive, and fast is important to monitor healthy foods in the community. One of the methods employed to overcome the previously mentioned problems is the development of a biosensor, which is often combined with electrochemical techniques because of its high selectivity and sensitivity [6]. Recently, hemoglobin (Hb)based biosensors have attracted considerable interest in the development of methods for detecting acrylamide [7][8][9].
Hb is a redox-active protein involving four polypeptide chains, and each polypeptide chain has one Fe 3+ electroactive heme group. Hb electroactivity is associated with the reversible conversion of Hb-Fe 3+ into Hb-Fe 2+ . The Hb functional groups are not easily oxidized or reduced in plain bare electrodes because of the slow transfer of electrons caused by the large threedimensional structure of Hb [8]. This large structure leads to the possibility of the Hb orientation being inappropriate for the surface of the electrode and increases the distance between the center of the heme and the surface of the electrode [10]. Moreover, the polarity difference between the nonpolar groups on the surface of the carbon electrode and polar groups in one of the Hb functional groups results in weak adsorption [11]. To produce biosensors with proteins (such as Hb), the electrochemical contact between active redox proteins (Hb) and transducers (electrodes) needs to be maintained. Biosensors can respond directly to oxidation in Hb; however, electron transfer occurs slowly, resulting in a weak signal response [12]. The problem of slow electron transfer caused by protein adsorption and electrode passivity is overcome using various electro-mediators, including Ag, Au, Pt, Pd, Ir, Rh, Ag/Au, Pt/Ag, and Pt/Ag [9].
In this study, electrochemical biosensors were developed by combining screen printing technology and nanotechnology. Nanoparticles can play an important role in improving sensor performance because of their large specific surface area, excellent conductivity, and compatibility. The use of nanoparticles for electrochemical sensors has been widely reported [13][14][15][16]. Among all types of nanoparticles, superparamagnetic iron oxide nanoparticles, particularly Fe3O4NP, have attracted considerable attention because of their unique magnetic properties and their capability to be easily chemically modified to increase biocompatibility and dispersibility [17]. Meanwhile, the synthesis of noble metal nanostructures is interesting because of their chemical, physical, and catalytic properties [18]. In this context, bimetallic nanostructures exhibit significantly improved optical, catalytic, and electrical properties of each monometallic constituent [19]. A previous study reported that Au-Fe3O4NP was more active than the single-component AuNP or Fe3O4NP [20]. The increased activity can be attributed to the partial charge transfer between Au and Fe3O4 at the nanoscale particle interface [20].
In this study, Hb-modified core-shell Fe3O4@Au nanoparticles were prepared for the electrochemical detection of acrylamide. The observed decrease in the current responses due to the formation of Hb-acrylamide (Hb-AA) adduct was used as the signals. A linear calibration curve in various concentrations of acrylamide could be achieved, indicating that the developed method was promising for the detection of acrylamide. Wako Pure Chemical Inc., Osaka, Japan), melamine (C3H6N6, 99%; Wako Pure Chemical Inc., Osaka, Japan), and ascorbic acid (C6H8O6, 99%; Wako Pure Chemical Inc., Osaka, Japan) were purchased in reagent grade. Meanwhile, distilled water was obtained with a resistivity >18.2 MΩ·cm. Transmission electron microscopy (TEM) images were obtained using a Jeol JEM-2100F (University of Ulsan, Ulsan, South Korea). Inductively coupled plasma-optical emission spectro-metry (ICP-OES) measurement was conducted using a SPECTROBLUE ICP-OES (Ametek; University of Ulsan, Ulsan, South Korea). X-ray diffraction (XRD) measurement was conducted using a Rigaku D/Max 2500V/PC scanning in the 2θ range of 30° to 90° (University of Ulsan, Ulsan, South Korea). Scanning electron microscopy (SEM) images were obtained using a Zeiss EVO M10 (DropSens μStat 400 S/N: UST0157400; Universitas Indonesia, Depok, West Java, Indonesia).

Synthesis of Fe3O4@AuNP and Fe3O4@Au/Hb.
Fe3O4NP was synthesized by the thermal decomposition method according to the method proposed by Kim et al. with some modifications [21]. Fe3O4NP was prepared using the iron oleate complex as the precursor. This complex was synthesized by dissolving FeCl3·6H2O (5.4 g) and sodium oleate (18.25 g) in 26.67 mL ethanol, 30 mL distilled water, and 70 mL hexane. This mixture was heated at 70 ℃ for 4 h. After the reaction was completed, the reddish brown top layer containing the organic compound was separated and washed with distilled water several times to remove NaCl and unreacted monomers. The iron oleate complex (18 g) and oleic acid (3.56 mL) were dissolved in 1-octadecene (100 g) through vigorous stirring. Then, the reaction temperature was increased to 320 °C at a heating rate of 3.3 °C/min and maintained at this temperature for 30 min under atmospheric nitrogen. During the reaction, the brownish red color turned to dark brown. After cooling the mixture to room temperature, the products were washed with ethanol and hexane and dried.
Fe3O4@AuNP was prepared according to the procedure proposed by Sun et al. with some modifications [16] by dropwise mixing of 10 mL chloroform containing 40 mg Fe3O4NP and 2 mmol oleylamine with various concentrations of gold solutions. All variations were stirred for 20 h. Ethanol was added to the precipitation. Then, the precipitation was washed with hexane and ethanol several times and dried under vacuum. The product was dissolved in an aqueous solution containing 0.1 M cetyltrimethylammonium bromide and 0.1 mM sodium citrate. After 10 min sonication, Fe3O4@AuNP formed a pink solution. Characterization was performed using TEM, XRD, and inductively coupled plasma-mass spectrometry (ICP-MS). To prepare Fe3O4@Au/Hb, 1 mg Fe3O4@AuNP was incubated in Hb solutions at 37 °C for 4 h. Various buffers (i.e., acetate buffer solution and phosphate buffer solution (PBS)), pH values (i.e., 4, 5, 6, 7, and 8), and Hb concentrations (i.e., 2, 4, 6, 8, and 10 mg/mL) were investigated to achieve high electrochemical signal currents.
Electrochemical detection of acrylamide. Various concentrations of standard acrylamide solutions (0.01-0.1 µM) were added to 1 mL Fe3O4@AuNP/Hb. After sonication, the precipitate (Fe3O4@AuNP/Hb-AA) was separated using external magnets and redispersed in 10 mL of 0.1 M PBS pH 6. A 40 µL volume of these solutions was measured using the cyclic voltammetry (CV) technique on a screen-printed carbon electrode (SPCE) in the potential range −0.8 V to +0.8 V at a scan rate of 50 mV/s. These steps were repeated three times. The selectivity of the developed biosensor was evaluated against other compounds that are commonly found in coffee samples, including caffeine, melamine, and ascorbic acid, at the concentration of 0.1 µM in 0.1 µM acrylamide solutions.
Coffee sample measurements. The Lampung coffee powder sample was treated according to the technique proposed in Ref. [22] with some modifications. First, 1.1 g coffee powder and 10 mL n-hexane were added to the sample and mixed in a vortex for 5 min. The residue was separated and dried in an oven at 60 °C for 1 h. Then, the residue was sonicated in a mixture of 10 mL acetone and 50 µL distilled water for 20 min. The acetone layer was filtered using filter paper and evaporated in a water bath. Next, the residue was dissolved in 10 mL of a mixture solution of acetonitrile and distilled water (5:95, v/v), shaken, and filtered with a 0.45 µm filter disk. The acrylamide content in coffee samples was measured using the CV technique under the optimum conditions. Validation of the method was performed using the HPLC technique [23]. A 20 µL volume of various concentrations of acrylamide standard solutions (10-250 ppm) and coffee samples were measured using the HPLC system. The mobile phase used was a mixture solution of acetonitrile and distilled water (5:95, v/v). The flow rate was 1 mL/min at the wavelength of 284 nm.

Results and Discussion
Synthesis of Fe3O4@AuNP. The XRD pattern of the as-synthesized Fe3O4 NP ( Figure 1) shows peak broadening, which is generally observed when the particle size is small. The diffractogram shows poor crystallinity, and peaks are hardly visible above the noise because of the nanoparticles of various oxygen contents (multiple phases) [24]. However, the signals can be assigned to a spectrum of spinel. The TEM image (inset of Figure 1) shows that particles of ~5 nm could be formed.
Then, the prepared Fe3O4 nanoparticles were used as the precursors to synthesize Fe3O4@AuNP by mixing with HAuCl4·3H2O solution. Fe3O4 could act as the core, whereas the gold solutions could act as the shell precursors. The same concentrations of the core were used with various concentrations of the shell, as shown in Table 1. The color changes the mixture from brownish black to pink indicated the formation of a gold shell. The TEM images (Figure 2a-2d) indicate that all variations of gold (shell) and Fe3O4 (core) ratios formed monodisperse particles with narrow particle size distributions (standard deviation of less than 10%). Further characterization using XRD spectrum ( Figure  2e) indicated that the centered cubic phase of Fe3O4NP was coated with a layer of crystalline gold.
To prepare the water-soluble nanoparticles, the dried Fe3O4@AuNP was dissolved in an aqueous solution containing 0.1 M cationic surfactant combination (CTAB) and 0.1 mM sodium citrate. Sodium citrate can attach to Au and stabilize nanoparticles by giving a negative charge to the surface of Fe3O4@AuNP. Sun et al. reported that sodium citrate alone cannot stabilize Fe3O4@AuNP in water, whereas the CTAB with citrate can stabilize Fe3O4@AuNP by forming a double layer on the surface of nanoparticles [25]. This CTAB/citrate method can dissolve AuNP encapsulated by oleylamine, but cannot be used for Fe3O4NP. The dissolution of the nanoparticles indicated that the nanoparticles have been encapsulated by Au or Fe3O4@AuNP is successfully formed. Furthermore, confirmation using the ICP-MS data ( Table 1) showed that the use of various concentrations of gold solutions as the precursors produced different mole ratios of Fe3O4 as the core and gold as the shell.

Figure 2. TEM Images of (a-d) Fe3O4@AuNP with Various Ratios of Fe3O4/Au and (e) its Representative XRD Patterns
Modification of Fe3O4@AuNP with Hb. The SEM images of Fe3O4@AuNP before and after modification with Hb (Figure 3a and Figure 3b, respectively) show that the particle size significantly increases after modification. Notably, Hb has self-assembled on the surface of Fe3O4@AuNP to form the globular morphology of Fe3O4@AuNP. Confirmation using Fourier transform infrared spectroscopy after modification (Figure 3c) showed a vibration peak of amide I (1,600-1,700 cm −1 ) attributed to C=O vibration of the peptide bond and a vibration peak of amide II (1,500-1,620 cm −1 ) attributed to the combination of NH stretching vibrations and C-N group vibration peptides [26]. Slight shifts observed at the wavelength of both vibration peaks in amide I and amide II were attributed to the bond between Au and -NH2 groups. Electrochemical studies of Fe3O4@AuNP/Hb. The CV technique on SPCE was applied to analyze the electrochemical behavior of Fe3O4@AuNP/Hb. The voltammograms shown in Figure 4a indicate that an oxidation peak at ~0.1 V related to the oxidation of Fe 2+ to Fe 3+ was observed in all types of Fe3O4@AuNP/Hb (Table 1), except for nanoparticles with a Fe3O4/Au mole ratio of 1:3. When gold fully covered the Fe3O4NP surface, this peak disappeared. Optimization of the amount of Hb immobilized in Fe3O4@AuNP was performed with a Fe3O4/Au mole ratio of 1:3. Various concentrations of Hb were added to the nanoparticles. The cyclic voltammograms shown in Figure 4b indicate that the highest current peak at approximately 0.1 V was observed at the Hb concentration of 2 mg/mL. Therefore, the Fe3O4/Au mole ratio of 1:3 and Hb concentration of 2 mg/mL were used for the subsequent experiments.  Solutions of commercial coffee powder samples were prepared and analyzed using the developed method. A typical voltammogram of sample measurement is shown in Figure 6a. The average current of three measurements of 0.802 µA was obtained from the voltammograms. The conversion of 50 dilutions resulted in the acrylamide content in coffee samples of 37.450 ppm. According to the Food and Drug Administration, the tolerable amount of acrylamide intake is 2.6 µg/kg BW/day to avoid carcinogenic risk. If the average body weight of adult women and men is assumed to be approximately 40-80 kg, then the tolerable amount of acrylamide intake is 104-208 µg/day.
HPLC was utilized to validate the method. The chromatograms of various concentrations of acrylamide in the concentration range 10-2,500 ppm shown in Figure 7a indicate that the standard acrylamide has a retention time of 3.1 min. The intensity was linear to the concentrations (R 2 = 0.998) with the equation of y = 46.587x + 1,609.6. Meanwhile, the chromatograms of the coffee samples showed several peaks at different retention times, indicating that some other compounds were present in the coffee samples. The chromatograms showed that acrylamide is not the main constituent of coffee as the peak at the retention time of 3.1 min has a significantly lower visible intensity than the other peaks ( Figure 7b, red line). The comparison of this chromatogram with that of 100 ppm acrylamide standard solution (black line) and that with the addition of 100 ppm standard acrylamide in the coffee samples (blue line) confirmed that acrylamide was present in the coffee samples. Based on the calculated results, the sample concentration was determined to be 35.377 ppm.

Conclusion
Fe3O4 nanoparticles were successfully synthesized by the thermal decomposition method using the iron oleate complex as the precursor with a particle size of ~4.9 nm. Modification with CTAB and citrate increased the hydrophilicity of Fe3O4NP. Accordingly, Fe3O4NP has been successfully coated with various concentrations of gold solutions. Increasing the concentration of the gold solution, which is used as the precursor, increased the thickness of the gold shell. The optimum Fe3O4/Au ratio of 1:3 with 2 mg/mL Hb in 0.1 M PBS pH 6 yielded acrylamide responses with good linearity at a concentration range of 0 to 1.0 µM. The LOD, LOQ, and sensitivity of 0.136 µM, 0.453 µM, and 0.4411 µA/µM, respectively, could be achieved. Finally, the detection of acrylamide in coffee samples exhibited high sensitivity and applicability, indicating that the developed sensor is promising.