Electrochemical Determination of Tyrosine by a Silver Nanoparticle – Hollow Carbon Sphere Modified Glassy Carbon Electrode (GCE)

Abstract The accurate and sensitive determination of tyrosine is of paramount value. In this work, we reported the synthesis and characterization of a new nanohybrid composed of silver nanoparticles and hollow carbon nanospheres (Ag/HMCS) for the amperometric sensing of tyrosine. The Ag/HMCS nanohybrid was used to modify electrodes and to fabricate an amperometric sensor for tyrosine at a working potential of 450 mV in aqueous 0.1 M phosphate buffer at pH 7.0. Under optimal conditions, the sensor determined tyrosine from 0.08 to 100 μM with a detection limit of 0.02 μM.

Foods rich in tyrosine including whole grains, pork, cheese, egg, fish, and beans are positive for human health (Jongkees et al. 2015;Vrshek-Schallhorn et al. 2013).Tyrosine is often added to food, pharmaceutical formulations, and health care products (Donaldson and Samuel 1946;Poustie and Rutherford 2000).Considering the significance of tyrosine in dietary and health, it is vital to establish a sensitive and accurate method in food.
These methods, which include high-performance liquid chromatography (HPLC), capillary electrophoresis, chromatography, and chemiluminescence, are sensitive and selective (Braal et al. 2019;Feng et al. 2015;Li et al. 2023;Wang et al. 1992).However, most have the disadvantages of expensive equipment, time-consuming protocols, and cumbersome pretreatments.Electrochemical methods, in contrast, are sensitive, portable, and low cost, and thus are widely used in biological analysis (Wu et al. 2020).Tyrosine is an electroactive compound suitable for electrochemical analysis (Negut and Staden 2021;Rahman et al. 2015;Varmira et al. 2018;Zribi et al. 2020).
Hollow carbon nanospheres (HCSs), a family of carbon nanomaterials, have drawn considerable attention in energy storage, absorption, catalysis, and sensing (Deshmukh et al. 2010;Tao et al. 2019).Similar to other carbon-based, HCSs offer low density, good conductivity, intrinsic hydrophobicity, and chemical stability (Ghimire et al. 2017).Significantly, HCSs integrate the advantages of porous carbon materials and spherical colloids with high surface area, good conductivity, accessible interior space and large pore volume (Ghimire et al. 2017;Zhou et al. 2018).These features allow HCSs to provide broad electrochemical applications (Chen et al. 2018;Li et al. 2013;Liu et al. 2018).3-Aminophenol-formaldehyde resin spheres were employed as the carbon precursor to prepare N-HMCS by a dissolution-capture procedure (Du et al. 2020).The obtained N-HMCS exhibit high surface area, a carbon shell with rich mesoporous, and high capacitance.
Additionally, the pore characteristic and specific surface area of N, S self-doped hollow-sphere porous carbon (NS-HPC) were optimized by activating puffball spores with KOH (Shang et al. 2021).The enhanced electrochemical performance and surface wettability are ascribed to the large specific surface area (1431 m 2 g À 1 ), well-developed porous structure, and N, S self-doped heteroatoms.
The introduction of metal nanoparticles into HCSs to design functional nanomaterials for electrochemical sensor has been recently reported (Tao et al. 2019).Ultrathin Ag/g-C 3 N 4 nanoparticle hybrids were prepared by a photo-assisted technique (Zou et al. 2019) for the determination of tyrosine.The Ag doping reduced the interfacial resistance, accelerated electron transfer, and enhanced the catalytic activity.The Ag/g-C 3 N 4 hybrids displayed higher response in comparison to bulk g-C 3 N 4 .
Ag nanoparticles were embedded into hollow porous carbon spheres (HPCS) by thermal carbonation for the preparation of a highly sensitive H 2 O 2 sensor (Ma et al. 2021).The Ag NPs provided numerous active sites with improved electrochemical response.Ag NPs offer low cost, high conductivity and good catalytic activity in fabricating electrochemical sensors (Guan et al. 2018;Qin et al. 2022;Saidu et al. 2020).Based upon the literature, the electrochemical determination of tyrosine using a Ag NP and HCS composite is anticipated to provide excellent performance.
Herein, a novel Ag/HMCS nanohybrid was prepared by a low-cost synthesis.The HMCS was synthesized using mesoporous silica as the template and resorcinol and formaldehyde polymer oligomers as the carbon source.The Ag NPs were produced by the reduction of silver nitrate with NaBH 4 to obtain the Ag/HMCS nanohybrid.The Ag/HMCS/GCE provides excellent electrochemical performance for the determination of tyrosine with high repeatability, stability, reproducibility, and selectivity.The device was optimized for the determination of tyrosine in milk and eggs with a suitable linear dynamic range and limit of detection (LOD), providing a new approach for carbon nanostructured materials in electrochemical analysis.A schematic for the preparation of Ag/HMCS nanocomposite and the modified electrode is shown in Scheme 1.

Apparatus
Field-emission scanning electron microscopic images were obtained employing a model S-4800II instrument.A Tecnai 12 instrument was used to capture the transmission electron microscopic images of Ag/HMCS.The high-resolution transmission electron microscopy images were collected using a Tecnai G 2 F30 instrument.The crystal structures of Ag/HMCS were characterized by X-ray diffraction on a D8 advance (Bruker).All electrochemical measurements were performed in a three-electrode system controlled by a CHI 660E electrochemical workstation (Shanghai Chenhua Instruments).

Synthesis of the Ag/HMCS nanomaterials
The HMCS were prepared based upon a modified St€ ober method (Du et al. 2020).10 mg of HMCS were evenly dispersed in 20 mL of deionized water and 30 mL of 0.1 mM AgNO 3 were added with stirring for 30 min.6 mL of 0.1 M NaBH 4 were added dropwise with stirring for 30 min.The products were isolated by centrifugation, washed with deionized water and ethanol 3 times, and dried in a vacuum oven.Three Ag/HMCS nanomaterials with different volume ratios of AgNO 3 and NaBH 4 (denoted by Ag/HMCS-1, Ag/HMCS-2 and Ag/HMCS-3) were prepared as shown in Table S1.

Preparation of the tyrosine sensor
The GCEs were polished with Al 2 O 3 paste, rinsed with deionized water, sonicated successively in deionized water, nitric acid, and acetone, and dried under nitrogen.The Ag/HMCS nanomaterial (2 mg) was dissolved in 1 mL of deionized water and a uniform suspension was formed following 30 min of sonication that was deposited onto the polished GCE and dried at room temperature.The modified GCEs were stored at 4 � C when not in use.

Characterizations of the Ag/HMCS nanomaterials
The morphology of Ag/HMCS was investigated by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and scanning tunneling electron microscopy (STEM).Figure 1(a) shows the Ag/HMCS are uniform spheres with an average diameter of 200 nm.The SEM and STEM images demonstrate that the HMCS have hollow structures with the Ag NPs distributed upon the surface.The Ag/HMCS composition and elemental distribution were determined by EDX. Figure S1 shows the prepared nanomaterial was primarily composed of C, O, and Ag.In addition, C and O were uniformly distributed and the Ag NPs were on the HMCS surface (Figure 1).These results verify the fabrication of the Ag/HMCS.
Raman spectroscopy was employed used to characterize the order and graphitization of HMCS and Ag/HMCS (Figure 2(b)).The D bond at 1355 cm À 1 is associated with the vibrations of disordered carbon atoms and defects (sp 3 ), while the G bond at 1594 cm À 1 is due to the vibration of ordered carbon atoms (sp 2 ).The intensity ratio of D and G band (I D /I G ) is used to evaluate the structural disorder.In comparison with the value of 0.79 for HMCS, Ag/HMCS showed a relatively high value of 0.82, implying increased disorder, suggesting the introduction of Ag NPs reduces the crystallinity (Zou et al. 2019).

Electrochemical properties of the Ag/HMCS/GCE
The electrochemical response of tyrosine upon the modified GCEs was investigated by cyclic voltammetry (CV) using 200 lM Tyr in 0.1 M phosphate buffer solution (PBS) at pH 7.0.Figure 3 shows the bare GCE and HMCS/GCE provide no distinct redox response.However, the currents on the Ag/HMCS-1/GCE, Ag/HMCS-2/GCE, and Ag/HMCS-3/GCE were approximately 1.6, 2.8 and 2.0 times higher than upon the HMCS/GCE due to the Ag NPs which increase the number of catalytic sites and  enhance electron transfer.In addition, the combination of HMCS with a large specific surface with the Ag nanoparticles enhances the sensitivity for tyrosine.
The Ag doping ratio has a large influence upon the electrochemical properties.The largest oxidation peak occurs on the Ag/HMCS-2/GCE at a peak potential of 0.45 V, showing that the Ag/HMCS-2 provides the best performance (Figure 3).The peak current of Ag/HMCS-3/GCE decreased, perhaps due to excess Ag hindering the HMCS catalytic sites.Thus, subsequent work focused upon Ag/HMCS-2; and hence Ag/HMCS refers to Ag/HMCS-2 in the following discussion.
The influence of the Ag/HMCS concentration up on the response to tyrosine was investigated in 0.1 M PBS. Figure S2 shows the optimum response was obtained using 2.0 mg mL À 1 .This concentration of Ag/HMCS suspension was used in the subsequent experiments.

Influence of scan rate on the electrochemical properties of tyrosine
The influence of scan rate between 50 to 350 mV s À 1 upon the peak current of the analytes (200 lM tyrosine in pH 7.0 PBS) on the Ag/HMCS/GCE was characterized by cyclic voltammetry (CV) (Figure S3(a)).As the scan rate increased, the oxidation peak current was higher with irreversible oxidation peaks.Figure S3(b) shows the linear relationship between scan rate and peak current described by I p (lA) ¼ 4.246 V 1/2 -29.955 (R 2 ¼0.9906).The results indicate that the oxidation of the target on Ag/HMCS/GCE was an irreversible diffusion-controlled processes.

Optimization of experimental conditions
The influence of pH upon the electrochemical performance of tyrosine on the Ag/HMCS/GCE was investigated using cyclic voltammetry (CV) (Figure 4).The current and peak potentials varied with the pH with a maximum at pH 7.0 (Figure 4(b)) which was employed in subsequent experiments.
The peak potential (E pa ) shifted negatively with pH, suggesting that protons directly participated in the oxidation of tyrosine.A linear relationship was obtained between peak potential and pH described by E pa (V) =-0.0306 pH þ 0.709 (R 2 ¼ 0.9925).The Nernst equation (Liu et al. 2020) was used to show that the ratio of electrons and protons transferred during the oxidation of tyrosine on Ag/HMCS/GCE is 1:1, which is consistent with the literature.
Accumulation is an effective approach for improving the sensitivity.In order to evaluate the influence of accumulation potential upon the determinations of tyrosine, the peak current was measured in pH 7.0 0.1 M PBS at applied potentials between 0.35 V and 0.55 V.The maximum peak current was obtained from 0.35 V to 0.45 V. Hence, 0.45 V was deemed to be the optimum value.

Analytical performance of Ag/HMCS/GCE
Chronoamperometry, a more sensitive method than cyclic voltammetry, was employed for the determination of tyrosine on the Ag/HMCS/GCE.Figure 5(a) shows the current-time response for the successive addition of tyrosine to continuously stirred pH 7.0 0.1 M PBS using a potential of 0.45 V.A sharp and clear response was observed for each tyrosine injection with the steady-state current response attained within 3 s, demonstrating rapid oxidation.Furthermore, the peak current increased linearly with the tyrosine concentration from 0.08 to 100 lM (Figure 5(b)) described by I (lA) =-0.0544 c (lM) þ 1.1222 (R 2 ¼0.9908).The detection limit was determined to be 20 nM based upon a signal-to-noise ratio of three.
The performance of this sensor was compared with the literature as shown in Table 1.The Ag/HMCS/GCE provides a wide linear range and low detection limit for tyrosine due to the introduction of Ag NPs into the HCS which provides improved performance due to synergistic properties of the nanomaterials.

Interferences
The influence of substances that may interfere with the determination of tyrosine were investigated, including Na þ , K þ , Ca 2þ , glycine (Gly), uric acid (UA), glucose (Glu), and oxalic acid (OA).Figure 6 shows no significant current changes were observed when the potential interferences were added to 100 lM tyrosine.Hence, the Ag/HMCS/GCE showed good selectivity for the analyte.

Repeatability, reproducibility, and stability
The repeatability, reproducibility, and stability of the electrode were studied under the optimal conditions.The repeatability was characterized by consecutive measurements of 20 lM,100 lM, and 180 lM tyrosine with the same electrode in pH 7.0 0.1 M PBS.The corresponding relative standard deviations (RSD) were 2.26%, 1.76% and 0.85% (n ¼ 10), indicating good repeatability (Figure S4(a)).
The reproducibility was examined by recording the currents of thirty sensors immersed into the above three solutions.The relative standard deviations were less than 3%, demonstrating excellent reproducibility (Figure S4(b)).
The stability of Ag/HMCS/GCE was investigated by leaving a modified electrode in the air and determine tyrosine weekly in pH 7.0 PBS. Figure S4(c) shows that after six weeks the current was 93.4% of the initial response.This experiment demonstrates suitable long-term stability.

Determination of tyrosine in food samples
The determination of Tyr milk and eggs was performed using the Ag/HMCS/GCE by the method of standard addition (Table 2).The samples were prepared by simple dilution with pH 7 0.1 M PBS.The recoveries were between 97.9% and 103.5% under the optimum conditions, illustrating favorable analytical performance.

Conclusion
Ag/HMCS was synthesized by chemical reduction to prepare an electrochemical sensor for tyrosine.The morphology, crystal structures and elemental composition were characterized and demonstrate that the Ag/HMCS composites have abundant active sites, large surface areas, and enhanced conductivity.The developed Ag/HMCS sensor provided a wide linear range from 0.08 to 100 lM, a low detection limit of 0.02 mM, high selectivity, good stability, and suitable repeatability.Due to its favorable analytical figures of merit, this sensor is promising for the determination of tyrosine.

Figure 1 .
Figure 1.(a) Scanning electron microscopic and (b) transmission electron microscopic images of the Ag/HMCS.Scanning tunneling electron microscopic images of the (c) Ag/HMCS and element mapping of (d) Ag, (e) C, and (f) O.

Figure 2 .
Figure 2. (a) X-ray diffraction patterns of the HMCS and Ag/HMCS.(b) Raman Spectra of the HMCS and Ag/HMCS.

Figure 4 .
Figure 4. (a) Cyclic voltammograms on the Ag/HMCS/GCE in the presence of 200 lM tyrosine at various pH values in 0.1 M PBS.(b) Relationship between the oxidation peak current and pH.(c) Influence of pH in 0.1 M PBS upon the peak oxidation potential of tyrosine on the Ag/HMCS/GCE.(d) Influence of applied potential upon the response toward 200 lM tyrosine in 0.1 M PBS.

Figure 5 .
Figure 5. (a) Amperometric response of the Ag/HMCS/GCE to tyrosine in pH 7.0 0.1 M PBS at 0.45 V. (b) Calibration relationship of current as a function of tyrosine concentration at the Ag/HMCS/GCE.

Figure 6 .
Figure 6.Response of the Ag/HMCS/GCE to tyrosine in the presence of potential interferences.The concentrations of tyrosine (Tyr) and interferents are 100 lM.Definitions: Gly: glycine, UA: uric acid, Glu: glucose, and OA: oxalic acid.

Table 1 .
Comparison of the developed Ag/HMCS/GCE to literature modified electrodes for the determination of tyrosine.: multiwalled carbon nanotubes, WGE: paraffin-impregnated graphite electrode, and PANI: polyaniline. MWCNT

Table 2 .
Determination of tyrosine in food using the developed Ag/HMCS/GCE.