In this study, we developed a pioneering non-enzymatic electrochemical sensor utilizing a flexible porous graphene electrode modified with ZnO nanoparticles (ZnO/fPGE sensor) to assess hypoxanthine (HXA) content in pork at post-mortem time. The ZnO/fPGE sensor was synthesized via hydrothermal method and direct laser writing with a CO2 laser on a polyimide film at ambient conditions. Its characterization was analyzed by Raman, Fourier-transform infrared spectroscopy, field-emission scanning microscopy, energy-dispersive x-ray spectroscopy, and cyclic voltammetric techniques. Linear response, the limit of detection, and sensitivity to the HXA were enhanced with the values of the range from 1.5 to 150, 0.14 µM, and 6.6 µA μM−1 cm−2, respectively. Effective resistance to common physiological interferences (such as uric acid, ascorbic acid, dopamine, glucose, and xanthine) was indicated, and additionally, the determination of HXA concentration in real samples with good selectivity is attributed to the synergistic effects between ZnO nanoparticles and fPGE. Therefore, the ZnO/fPGE has provided a favorable electrical environment for developing high-performance electrochemical biosensors to determine hypoxanthine in pork meat.

In recent years, ensuring the quality and freshness of meat and aquatic products like fish has been increasingly receiving much attention worldwide. After being slaughtered, the metabolism of organic compounds confidently occurs during storage time, causing unexpected food spoilage. As reported in the previous studies, hypoxanthine (HXA), a product of the degradation pathway of adenosine triphosphate, is considered a useful indicator to determine food freshness,1,2 and accumulated in the tissues of dead animals. Hence, the determination of HXA has become an indispensable factor in the food industry.

In this regard, numerous well-known analytic methods have been applied such as high-performance liquid chromatography,3,4 capillary electrophoresis,5 and mass spectrometry.6,7 Unfortunately, the disadvantages of these methods as time-consuming, costly, complicated experimental systems with the support of experts, and low sensitivity, are a large challenge for the food industry. Consequently, there are increasingly stringent requirements for rapid, economical, portable, and accurate analytic techniques to determine the HXA concentration in place, meeting the current demands of the meat consumption industry. Studies have demonstrated that the enzymatic biosensors based on xanthine oxidase dramatically lean on pH, temperature, and humidity as well as low stability and weak immobilization. These factors are responsible for the decreased sensitivity of the device. Up until now, electrochemical bio-sensing techniques have been achieving positive breakthroughs with much special interest from researchers and enterprises owing to their high sensitivity, selectivity, and easier utilization.8–12 A great deal of effort has been realized to develop highly precise electrochemical biosensors for HXA, typically non-enzyme biosensors.12–15 In addition, the biosensors modified by nanomaterials, metal or metal oxide nanoparticles,12 metal-organic frameworks,13 carbon materials,14 and graphene15 have demonstrated their effectiveness in the improvement strategy.

It cannot be denied that graphene and its derivatives have been an ideal material for the evolution of biosensors thanks to its outstanding properties.16 Many publications demonstrated superior support of this material in the detection of xanthine and HXA, especially reduced graphene oxide (rGO), which contains functional oxygen groups and hydrophilic.17–20 By exploiting the exceptional electrical conductivity of rGO, Wang et al., created an enzyme-free biosensor based on rGO and polyglycine modified glassy carbon electrode that effectively and simultaneously detects xanthine and HXA with a linear range of 1–340 µM and a detection limit of 0.2 µM for HXA. Another non-enzymatic biosensor using a reduced graphene oxide/chitosan/chromium oxide nanocomposite-modified glassy carbon electrode was developed by Ghanbari and, which simultaneously detects dopamine, uric acid, xanthine, and hypoxanthine.20 Nevertheless, the majority of graphene and rGO are utilized as materials to modify the conventional electrodes, thus deeper and wider research is still needed to build them as a primary functioning electrode. Notably, the technology of photothermal conversion of polyimide film into a continuous 3D porous graphene structure by pulsed laser irradiation in air is known as laser-induced graphene (LIG). The LIG has high electrical conductivity and large specific surface area, flexibility in patterning, non-chemicals, fast fabrication, and low cost,21 leading to fully potential graphene production at a large scale.22,23 Compared with graphene fabricated by other methods, the LIG improves charge transfer efficiency, better conductivity, and contains a lot of active sites for functionalization, which are considered requisites for biosensors.22,24 Furthermore, several works have shown an outstanding physicochemically synergistic effect between the LIG and metal oxide nanoparticles, which plays an interesting role in positively pushing up electrochemical reactions. Nayak et al. reported a non-enzyme CuO/LIG electrochemical sensor to detect glucose in whole blood, serum, sweat, and urine with a detection limit of 0.1 µM, linear range of 1 μM–5 mM, and high selectivity.25 Santos et al.26 demonstrated that ZnO rods synthesized by electro-plating on the LIG are capable of fast electrochemical electron transfer with a rate constant of 1.24 × 10−2 cm/s for the system [Ru(NH3)6]2+/3+, and a suitable LIG/ZnO internal electrical contact is thus achieved. Herein, LIG offers more active sites for facile functionalization with metal oxides to form heterogeneous electrochemical sensing materials possessing efficient electrocatalytic activity.27 On the other hand, metal oxide nanomaterials (MONs) have also shown great promise in the biosensor domain due to possessing prominent physicochemical and catalytic properties, especially better biocompatibility, and easily tunable size and shape.28–30 Paid much more attention compared to other MONs, ZnO semiconductor with a direct bandgap and a high isoelectric point, high chemical stability, and good biocompatibility, resulting in strong adsorption and fast electron-transfer kinetics, has been investigated with various morphologies.31 The non-enzymatic H2O2 sensor based on a graphene oxide/ZnO modified electrode had a linear range from 0.02 to 22.48 µM, with a correlation coefficient R2 of 0.995.32 Another non-enzyme sensor for cortisol detection in saliva using ZnO-graphene was developed, which had a very low detection limit of 0.15 nM.33 Shaikshavali et al. reported an electrochemical sensor based on a carbon glass electrode and ZnO nanoparticles integrated on multiwall carbon nanotubes (ZnO/MWCNT) for the detection of epinephrine in human serum and pharmaceutical formulations. The linear range and detection limit of that sensor were 0.4–2.4 and 0.016 µM, respectively.34 A non-enzyme xanthine bio-sensor using an RGO/ZnO nanocomposite on a zinc foil fabricated by Zhang et al. revealed a sensitivity of 2.10 µA μM−1 cm−2 and a detection limit of 1.67 µM.35 To our best knowledge, a ZnO nanoparticles (NPs)/graphene composite serving as a catalyst in the enzyme-free biosensor has still been an unexplored topic for the aim to determine the freshness of meat.

Inspired by the above-mentioned reasons, we fabricated a biosensor for the detection of hypoxanthine using laser-induced graphene, which was synthesized directly on a polyimide film to form flexible porous graphene and ZnO. The as-prepared ZnO/fPGE exhibited high electrocatalysis to HXA due to the large surface area of the electrode as well as the synergistic effect between ZnO and porous graphene. It is worth highlighting that our study can be the first one that utilizes a ZnO nanoparticle-modified laser-induced Graphene (ZnO/fPGE) electrode for analysis of the HXA. The results show that the ZnO/fPGE sensor operated effectively as demonstrated by high sensitivity, wide linear range along with good reproducibility and selectivity. Moreover, the sensor has been validated and successfully applied for determining the HXA content in real samples.

Hypoxanthine, xanthine, uric acid, phosphate buffered saline (PBS), potassium ferricyanide K3[Fe(CN)6], potassium ferrocyanide K4[Fe(CN)6], KCl, NaOH, methanol, and Zn(CH3CO2)2 · 2H2O were purchased from Sigma-Aldrich. All chemicals were of analytical grade and used without further purification. Deionized water was used for all our experiments.

The surface morphology and structural characterization of the samples were determined by a field emission scanning electron microscopy (FESEM, Hitachi S4800) and Bruker D8 advanced x-ray powder diffractometer with Cu-Ka radiation (k = 1.5406 Å); Raman scattering data were collected using a LabRAM HR evolution confocal Raman microscope (Horiba Instruments Inc). Fourier-transform infrared spectroscopy (FT-IR) spectra were specified by Nicolet iS10 spectrometer (Thermo Scientific, Waltham, MA, USA).

An Autolab electrochemical workstation (Metrohm PGSTAT30 Potentiostat Galvanostat Electrochemical System) was conducted to execute electrochemical measurements on a three-electrode cell system. ZnO/porous graphene, graphene, and Ag/AgCl operate as a working electrode, a counterelectrode, and a reference electrode, respectively.

Preparation of the polyimide (PI) substrate: A 125 µm-thick polyimide substrate with a dimension of 210 × 297 mm2 was cut into 30 smaller PI sheets. The size of each sheet is defined as 42 × 49 mm2. The parameters of power, processing speed, and pulse frequency of the CO2 laser beam were set in turn at 5.6 W, 13%, and 500 PPI. The PI sheets were cleansed in alcohol solution three times by ultrasonic vibration. Ultimately, the sheets were transferred to a vacuum oven to fully dry.

Fabrication of graphene: Graphene film was fabricated on the polyimide substrate by placing the PI sheets in a laser machine and then setting optimal fabricating conditions of the CO2 laser beam, typically, the power of 3.12 W, processing speed of 6.6%, and pulse frequency of 1000 PPI. The diameter of the working electrode obtained is 1.8 mm.

Cleaning: After completing synthesizing the graphene on the PI substrate to create a flexible porous graphene electrode, we used a squeeze to remove any remaining impurities on the fPGE electrode.

A simple approach was applied to form a ZnO-modified flexible porous graphene electrode. ZnO nanoparticles (NPs) were synthesized by the hydrothermal method with reasonable conditions. In detail, Zn(CH3CO2)2 · 2H2O, NaOH, and methanol precursors with the mole ratio of 1:1:25 were separately dissolved in the distilled water (with the mole ratio methanol and distilled water is 1:1) and then mixed and stirred for 1 h at room temperature and pressure. The milky white solution was poured into a white Teflon. The fPGE electrode was placed in the reaction Teflon. A steel autoclave covered such a Teflon before transferring it to a hydrothermal oven. The hydrothermal process was implemented at 200 °C for 4 h. The electrode was taken out and washed several times with distilled water. Finally, the electrode was incubated at 100 °C for 5 h to reinforce adhesion between ZnO nanoparticles and fPGE.

The electrochemical behavior of the fPGE and ZnO/fPGE fabricated electrodes was studied using cyclic voltammetry (CV) in 5.0 mM [Fe(CN)6]3−/4− a solution containing 0.1 M KCl and in HXA 50 µM (PBS 1×, pH 7.0). Differential Pulse Voltammetry (DPV) experiments were carried out in a solution of PBS (1×, pH 7.0) containing hypoxanthine, xanthine, and uric acid. DPV was recorded from −0.4 to 0.6 V with a pulse amplitude of 50 mV and pulse with 50 ms.

Pork tenderloins were purchased from a local supermarket and certified as fresh. The meat was packaged, vacuum-sealed, and stored at a temperature of 20 °C before being transported and tested in the laboratory. It was then thawed at room temperature. After 5 h, 5 g of meat was finely chopped and homogenized in deionized water (15 ml) using a homogenizer (IKA Ultra Turrax workstation). Subsequently, the homogenate was centrifuged at 5000 rpm for 15 minutes and the supernatant was collected and filtered using a polytetrafluoroethylene chromatography syringe filter (0.45 µm Pore). The HXA content in the real sample was measured by the ZnO/fPGE sensor using the chronoamperometric technique at room temperature, applying a constant potential (1.0 V). The practical application of the ZnO/fPGE electrode was studied by the analysis of HXA content in pork samples using the standard addition method. 0.15 ml of solution extracted from real meat was added to each portion of 20 ml PBS (1×, pH 7.0), and then was stirred well (200 rpm). Three samples of HXA solution with known concentrations of 40, 80, 120, and 190 µM were added to three different beakers containing the meat-extracted solution mentioned earlier. HXA aliquots were added at a time interval of 50 seconds to achieve a steady current.

Morphological characterization of the as-prepared ZnO, fPGE, and ZnO/fPGE samples observed by FESEM is presented in Fig. 1. The ZnO NPs, in general, are in spherical shape with ∼20 nm in size [Fig. 1(a)]. Figure 1(b) shows the porous structure of fPGE, in which the diameter and thickness of pores are up to micrometers and 100 nm. The high conductivity of the film is enhanced originating from forming an interconnected network between pore walls. Figure 1(c) indicates the good dispersion of ZnO NPs on the walls of the pores of the graphene in the ZnO/fPGE structure as expected.

FIG. 1.

Morphology of (a) ZnO NPs, (b) fPGE, and (c) ZnO/fPGE.

FIG. 1.

Morphology of (a) ZnO NPs, (b) fPGE, and (c) ZnO/fPGE.

Close modal

The composition and percentage of elements in the ZnO/fPGE, which was chosen as a representative, were determined using SEM-EDS (Fig. S1). The ZnO/fPEG only contains C, O, Zn elements, and the atomic percentages of C, O, and Zn are 75.68, 20.1, and 4.22 atm %, respectively. Apparently, the atomic percentage of O is ∼4.7 times greater than that of Zn, it can be confirmed the adsorption of oxygen atoms onto the surface of graphene.

In this study, a laser source with the wavelength of λ = 532 nm (2.33 eV) and laser power under 1 mW was prioritized to apply. Raman spectroscopy is a well-established tool that enables the determination of crystal structure characteristics in a graphene film. The graphene Raman spectrum has three important feature peaks at 1335, 1566, and 2678 cm−1 for D, G, and 2D, respectively (Fig. 2), whereas the emergence of the D peak is conjectured to be due to the presence of defects and/or impurities in hexagonal sp2 structure. The G peak is related to the in-plane vibration mode of sp2 hybridized carbon atoms, while the 2D peak is the result of a two-phonon lattice vibration process in graphene structure.36,37 According to some studies, the 2D and G peak intensity (I2D/IG) ratio depends on the number of graphene layers, in particular, the ratio >1 for single-layer graphene and the ratio <1 for multi-layer graphene.38 In addition to the I2D/IG ratio, the number of graphene layers is also deduced from the position and shape of the 2D peak and its FWHM (full width half maximum). The FWHM can be obtained from the Lorentz fitting of the 2D peak. From Fig. 2, calculation values of the I2D/IG, ID/IG (the D and G peak intensity), and FWHM for the fPGE and ZnO/fPGE are 0.67 (<1), 0.77, ∼89, and 0.7, 0.92, 86 cm−1, respectively. These results imply that as-synthesized fPGE is a multi-layer graphene film. The quality of graphene in fPGE can be predicted based on the ID/IG ratio. The higher this ratio, the better the quality of porous graphene and vice versa. Obviously, the ID/IG ratio of ZnO/fPGF is higher than that of fPGE, confirming that ZnO NPs were deposited into fPGE, which enhances the number of defects on the graphene surface. As a result, the ZnO was successfully attached to the fPGE film.

FIG. 2.

Raman spectra of fPGE and ZnO/fPGE.

FIG. 2.

Raman spectra of fPGE and ZnO/fPGE.

Close modal

Moreover, a peak at 428 cm−1 observed in the Raman spectrum of the ZnO/fPGE corresponds to the Raman E2 (high) active mode of hexagonal wurtzite structure ZnO,39 affirming the existence of the ZnO NPs in the ZnO/fPGE.

Figure 3 presents the FTIR spectra of fPGE and ZnO/fPGE. The peaks of fPGE at 1721, 1498, and 1371 cm−1 in turn characterize the stretching vibration of C=O, the aromatic ring stretching, and the vibration of the methyl group.40 The peaks between 1250 and 950 cm−1 are due to the vibrations of aromatic C–H in-plane bend and the peaks in the region from 900 to 675 cm−1 are caused by the vibrations of aromatic C–H out-plane bend.41,42 After the ZnO NPs were introduced on the fPGE, the peaks at 3365, 1705, 1479, and 1393 cm−1 characterize the stretching vibration of OH, C=O, aromatic ring, and the methyl group. It means that the ZnO/fPGE electrode possesses many negatively charged functional groups (–OH) and electron-enriched oxygen atoms. Similar to the FT-IR spectrum of fPGE, the peaks in the range of 1250–950 cm−1, and 900–675 cm−1 of the ZnO/fPGE correspond to the vibrations in the aromatic C–H in-plane, and the out-plane bend, respectively. Compared to the FT-IR spectrum of fPEG, an additional peak at 555 cm−1 in the ZnO/fPEG spectrum is possibly due to the metal–oxygen stretching vibration, relating to the formation of ZnO wurtzite structure.42 Besides, one can observe the shifts and enhancement of the vibration peaks in the ZnO/fPGE spectrum toward lower wavenumbers compared to the C=O vibration peaks in fPGE, namely, from 1721 to 1705 cm−1 and from 1498 to 1479 cm−1. This shift may stem from the interaction between the carboxyl group at the boundary of the graphene oxide surface and the ZnO NPs. The results obtained from the FT-IR spectra are completely consistent with those in the Raman spectra, demonstrating the successful deposition of ZnO NPs on the fPGE surface.

FIG. 3.

FT-IR spectra of the fPGE and ZnO/fPGE.

FIG. 3.

FT-IR spectra of the fPGE and ZnO/fPGE.

Close modal

Electrochemical behaviors of the fPGE and ZnO/fPGE electrodes were monitored by cyclic voltammetry (CV) technique with a scan rate of 60 mV/s in 5 mM [(Fe(CN)6]3−/4−/0.1 M KCl solution. In Fig. 4(a), it can be observed that the peak potential separation of ZnO/fPGE (ΔEp = 183 mV) is smaller than that of fPGE (ΔEp = 189 mV), while the intensity of the anodic peak current (Ipa) increases by about 1.17 times. As a consequence, the electron transfer kinetics and electrocatalytic activity of the ZnO/fPGE electrode are faster and better.43,44

FIG. 4.

CV of (a) fPGE, ZnO/fPGE in 5 mM [(Fe(CN)6]3−/4−/0.1 M KCl solution with the scan rate of 60 mV/s; [(b) and (c)] CV of fPGE and ZnO/fPGE at the different scan rates (20–100 mV/s), and (d) the plot of Ipa and Ipc vs ν1/2.

FIG. 4.

CV of (a) fPGE, ZnO/fPGE in 5 mM [(Fe(CN)6]3−/4−/0.1 M KCl solution with the scan rate of 60 mV/s; [(b) and (c)] CV of fPGE and ZnO/fPGE at the different scan rates (20–100 mV/s), and (d) the plot of Ipa and Ipc vs ν1/2.

Close modal
The CV curves at different scan rates are represented in Figs. 4(b) and 4(c). A [(Fe(CN)6]3−/4− redox couple is considered as a redox probe to investigate the electrochemical properties.43 The ratio between anodic and cathodic peak response currents of fPGE and ZnO/fPGE was determined to be 1.04 and 0.99, respectively, the redox reaction on both electrodes is thus reversible.39,40 The electroactive surface area is calculated according to the Randled–Sevcik equation,44,45 as follows:
Ip=2.69×105n3/2AD1/2ϑ1/2C,
(1)
where Ip and n, in turn, stand for the peak response current and the value of electron transfer (n = 1) in the [(Fe(CN)6]3−/4−redox system), A is defined for the electroactive surface area, D is the diffusion coefficient of the [(Fe(CN)6]3−/4− (6.8 × 10−6 cm2 s−1), and ν and C render scan rate (V s−1) and the concentration of reaction, respectively. Figure 4(d) shows that the anodic and cathodic peak currents (Ipa, Ipc) of fPEG and ZnO/fPGE are linear with the square root of the scan rate. This result indicates that the oxidation of [(Fe(CN)6]3−/4−is a diffusion-controlled process.43–45 From the slope of Ipa − ν1/2, the electroactive surface area of fPGE and ZnO/fPGE was determined to be 0.099 and 1.2 cm2, respectively, which is in turn 3.96 times and 4.8 times higher than the actual electrode surface area (0.0254 cm2). Indeed, the above results reveal that the fPGE and ZnO/fPGE electrodes have excellent porosity and unique mechanical flexibility. Due to the higher electroactive surface area of ZnO/fPGE compared with the fPGE, its catalytic activity for hypoxanthine oxidation is expected to significantly improve.

The HXA sensing performance of the as-prepared electrode was tested by the CV method. Figure 5 displays the electrochemical responses of 20 µM HXA on the fPGE and ZnO/fPGE electrodes at a scan rate of 50 mV s−1 in PBS (1×, pH 7.0). Compared with the fPGE electrode, a significant increase in HXA oxidation capacity can be seen with a current peak at 1.02 V, indicating an enhanced electron transfer rate on the ZnO/fPGE electrode. More noticeably, the ZnO sites deposited on the fPGE surface act as catalytically active sites to improve the electrochemical activity, attracting hypoxanthine molecules to the ZnO/fPGE electrode surface.33 The electrocatalytic efficiency for the HXA increases with the increase of the electroactive surface area, as mentioned in Sec. III B, through ZnO deposition. This can be explained through the presence of the hydroxyl group formed by the reduction of the carbonyl group (C=O), which was observed through the FT-IR spectrum in Fig. 3.33,46 Electrochemical properties and structural and morphological features investigated show that the ZnO/fPGE surface attracts the HXA, leading to enhancing the current response.

FIG. 5.

CV of HXA detected at fPGE and ZnO/fPGE electrodes in PBS (1×, pH 7.0).

FIG. 5.

CV of HXA detected at fPGE and ZnO/fPGE electrodes in PBS (1×, pH 7.0).

Close modal
Crucial information about the electrochemical mechanism between HXA and ZnO/fPGE electrode can be taken out from the relationship between the electrochemical signal and scan rate. Therefore, the influence of scan rate on Ep and Ip is investigated based on the CV curve [Fig. 6(a)]. It can be seen that this is an irreversible oxidation reaction, and the peak potential increases proportionally to the scan rate.43 A linear correlation for HXA, rendered through the anode peak current and the square root of the scan rate, was performed to determine whether the oxidation reaction was controlled by diffusion or adsorption [Fig. 6(b)]. If the graph is linear, the process can be controlled by the diffusion. Additionally, the slope of the linear curve ln Ip vs ln ν can also indicate whether the process is controlled by diffusion or adsorption.43–45 The graph representing the linear relationship between the natural logarithm of the anode peak current and the natural logarithm of the scan rate (R2 = 0.999) is shown in Fig. S2(a) with a slope of 0.497, close to 0.5, deducing that this process is driven by the diffusion.17,43 The number of transferred electrons (n) and the electron transfer coefficient (α) for the irreversible voltammogram system are determined by the Laviron equation through the relationship between the peak potential Ep and the natural logarithm of the scan rate,20,
EP=E0+RTαnFlnαnFkRT+RT1αnFlnν,
(2)
where T, F, R, and Ep stand for thermodynamic temperature (T = 298 K), the constant of Faraday (96.485 C/mol), gas constant (R = 8.3145 J/mol/K), and the potential of the oxidative peak, respectively, ln ν is the natural logarithm of scan rate, and E0 represents the standard potential. The Ep plot of HXA with respect to ln ν is illustrated in Fig. S2(b). The linear regression equation is expressed as follows:43 
EpHXA=0.03lnν+1.16;R2=0.991.
(3)
FIG. 6.

CV curves of the ZnO/fPGE sensor toward 150 µM HXA in PBS (1×, pH 7.0) at different scan rates (a), and the linear line of the Ip vs the ν1/2 (b).

FIG. 6.

CV curves of the ZnO/fPGE sensor toward 150 µM HXA in PBS (1×, pH 7.0) at different scan rates (a), and the linear line of the Ip vs the ν1/2 (b).

Close modal

From Eqs. (2) and (3), for the HXA system on the ZnO/fPGE electrode surface, the value of α is 0.5 for the irreversible system. This results in the calculated value of n being ∼1.66. Thus, it can be assumed that n = 2 for HXA and the mechanism of HXA oxidation at the electrode may be related to the exchange of two electrons and two protons. This result is good consistent with previous publications.13,17 As mentioned in the FT-IR and Raman spectrum analyses, the ZnO/fPGE electrode contains many negatively charged functional groups and has active centers on the surface that can potentially strongly enhance the interaction with the HXA, leading to enhancing electron transfer rate and electrocatalytic activity of HXA.

We used 1× buffer solution (PBS) with a pH in the range of 6.0 and 7.5 to study the simultaneous determination of UA, XA, and HXA in a neutral environment. In addition, estimating the effect of pH on electrochemical signals can find out the appropriate pH value and the ratio of electrons and protons related to the oxidation of HXA. DPV technique is extensively conducted in this concern because of its better sensitivity compared to the linear sweep methods and there is a minimization of the capacitive current in the DPV.13,46 The DPV curves of 50 µM HXA at pH = 6.0–7.5 are presented in Fig. 7. The peak current intensity gradually increases as increasing pH from 6 to 7 and gains the highest value at pH 7 [Fig. 7(a)]. The pH drastically decreases from pH 7 to pH 7.5 since the HXA is deprotonated to form an anionic molecule at high pH. Hoan et al. proposed that at high pH, HXA becomes negatively charged. Simultaneously, the electrode surface is negatively charged, attributed to the presence of negative functional groups as revealed in IR analysis. This combination leads to a reduction in the adsorption of HXA on the electrode and a subsequent decrease in the response current (Ip).17 Therefore, pH 7.0 was chosen for the next HXA analysis experiments. The oxidation peak potential was found to depend on pH [Fig. 7(b)] indicating the shifts to a less positive potential as pH increases, corresponding to the involvement of protons in the oxidation reaction. With a pH in the range of 6–7.5, the oxidation potential is a linear function of pH. The slope value of 0.051 V/pH is close to the Nerst theoretical value (0.059 V/pH), showing that the HXA oxidation involves the balance of numbers of electrons and protons.13 

FIG. 7.

DPV curves of HXA at ZnO/fPGE sensor in PBS (1×) with different pH values (6.0, 6.5, 7.0, 7.5) (a), and the linear line of buffer pH vs IP and EP (b).

FIG. 7.

DPV curves of HXA at ZnO/fPGE sensor in PBS (1×) with different pH values (6.0, 6.5, 7.0, 7.5) (a), and the linear line of buffer pH vs IP and EP (b).

Close modal

As reported, the adenosine triphosphate involved in meat disappears within 2–24 h after the animal death meanwhile, the concentration of substances produced by the meat decomposition process, such as hypoxanthine, xanthine, and uric acid, gradually accumulates according to storage time.47 To test the HXA selectivity of the ZnO/fPGE electrode, it needs to study the ability to selectively determine HXA without any influence of UA and XA. Indeed, a mixture involving three substances at the same concentration of 50 µM was prepared together in PBS (1×, pH 7.0), then measured by the DPV technique at the potential in a range of 0–1.1 V [Fig. 8(a)]. Three distinguishable anodic peaks corresponding to the oxidation process UA (0.302 V), XA (0.661 V), and HXA (0.971 V) are observed on the DPV measurement curves of the ZnO/fPEG electrode. The peak potential separation is 150 mV (UA-XA), and 200 mV (XA-HXA), which is large enough to detect these three analytes at the same time.13,17 It is worth mentioning that the HXA is an important ingredient to determine the freshness and flavor of meat; indeed, when xanthine is produced that meat is severely spoiled and cannot be used as human food.48 Therefore, we focus only on the hypoxanthine content in meat.

FIG. 8.

DPV curves of ZnO/fPGE sensor in 50 µM (UA, XA, and HXA) solution in PBS (1×, pH 7.0) (a), and chronoamperogram of the ZnO/fPGE sensor on sequential addition of 50 µM XA, 50 µM glucose (Glu), 50 µM ascorbic acid (AA), 50 µM dopamine (DA), and 50 µM uric acid in PBS (1×, pH 7.0) at 1.0 V (b).

FIG. 8.

DPV curves of ZnO/fPGE sensor in 50 µM (UA, XA, and HXA) solution in PBS (1×, pH 7.0) (a), and chronoamperogram of the ZnO/fPGE sensor on sequential addition of 50 µM XA, 50 µM glucose (Glu), 50 µM ascorbic acid (AA), 50 µM dopamine (DA), and 50 µM uric acid in PBS (1×, pH 7.0) at 1.0 V (b).

Close modal

A large challenge of non-enzymatic HXA sensors is their resistance to interference, which affects biosensor performance in real sample monitoring. To evaluate the selectivity of the ZnO/fPGE sensors, some potential noises were added when performing amperometric detection at a potential of +1.0 V. The amperometric response was recorded when adding 30 µM hypoxanthine and interferences including 50 µM xanthine, 50 µM uric acid, 50 µM ascorbic acid, 50 µM glucose, and 50 µM dopamine, as shown in Fig. 8(b). The current response to the interferences is negligible compared to that of hypoxanthine. These results confirm the high selectivity of the ZnO/fPGE sensors for the detection of hypoxanthine in real samples.

As mentioned, the DPV method has been favorable for appraising the linear concentration range, sensitivity, and detection limit of the sensor. Figure 9(a) introduces the DPV curves on the ZnO/fPGE electrode with different HXA concentrations. The peak current increases linearly upon increasing HXA concentration in the range of 1.5–150 µM conforming a linear regression equation: Ip (μA) = 6.6 CHXA + 67.69 (R2 = 0.993). The limit of detection (LOD) value is 0.14 µM, which is calculated from the formula of 3.3Sd/S, where Sd is the standard deviation of blank signals [n = 4, RSD ∼0.49% (Fig. S3)]. The sensitivity equals 6.6 µA μM−1 cm−2, which is calculated by the formula of S/A, where S is the linear curve calibration slope in Fig. 9(b) and A is the surface area.

FIG. 9.

DPV curves of ZnO/fPGE sensor determine HXA with continuous addition of HXA with various concentrations (1.5–150 µM), in PBS (1×, pH 7.0) (a), and the linearity of the Ip vs the HXA concentration (b).

FIG. 9.

DPV curves of ZnO/fPGE sensor determine HXA with continuous addition of HXA with various concentrations (1.5–150 µM), in PBS (1×, pH 7.0) (a), and the linearity of the Ip vs the HXA concentration (b).

Close modal

In comparison with the published reports using non-enzymatic HXA sensors, from Table I it can be seen that the ZnO/fPGE sensor has a wider linear range and much lower detection limit than those of CoFe2O4/rGO,17 rGO-GCE,18 Ag/AgCl (4B-PGE)49 sensors. Although ZnIn2S4/UiO-66-NH2/GCE,50 poly-(l-arginine)/graphene composite19 sensors have a slightly smaller detection limit, the active range is too small compared to the sensor introduced in this work. The sensor with Ru (DMSO)4Cl2 nanoaggregated Nafion electrode47 has a larger active range, but the detection limit is nearly 20 times higher than the ZnO/fPGE sensor. From the above analysis, the ZnO/fPGE sensor offers an efficient biosensor design with great application potential in hypoxanthine detection.

TABLE I.

The sensing parameters of the ZnO/fPGE sensor compared with some reported non-enzymatic sensors for the detection of HXA. CS: Chitosan’ PGE: Pencil graphite electrode.a,b

Electrode materialsLinear range (μM)LOD (μM)References
CoFe2O4/rGO 2–10 0.506 17  
ZnIn2S4/UiO-66-NH2/GCE 0.3–40 0.1 50  
rGO-GCEa 0.43–7.39 0.141 18  
GCE/rGO/CS/Cr2O3b 2–300 0.85 20  
Ag/AgCl (4B-PGE) 6–30 1.09 49  
Ru (DMSO)4Cl2 nanoaggregated Nafion 50−300 2.37 47  
Poly-(l-arginine)/graphene composite 0.2–20 0.1 19  
ZnO/fPGE 1.5–150 0.14 This work 
Electrode materialsLinear range (μM)LOD (μM)References
CoFe2O4/rGO 2–10 0.506 17  
ZnIn2S4/UiO-66-NH2/GCE 0.3–40 0.1 50  
rGO-GCEa 0.43–7.39 0.141 18  
GCE/rGO/CS/Cr2O3b 2–300 0.85 20  
Ag/AgCl (4B-PGE) 6–30 1.09 49  
Ru (DMSO)4Cl2 nanoaggregated Nafion 50−300 2.37 47  
Poly-(l-arginine)/graphene composite 0.2–20 0.1 19  
ZnO/fPGE 1.5–150 0.14 This work 
a

GCE: Glassy carbon electrode; rGO: reduced graphene oxide.

b

CS: Chitosan; PGE: Pencil graphite electrode.

To evaluate the reproducibility of the sensor, five ZnO/fPGE electrodes were prepared with the same conditions and tested using the DPV method for 150 µM HXA sample in PBS (1×, pH 7.0).

The peak current obtained in five measurements shows that a relative standard deviation (RSD) is about 3.1%, indicating good reproducibility of the ZnO/fPGE-modified electrode. The stability of the sensor was assessed over 20 days (Fig. S4). The modified electrode was preserved at ambient conditions and its response after 3, 10 and 20 days is 98.2%, 93.5%, and 85.2% of the initial response, respectively, giving relatively good stability 10 days of this electrode.

The response was recorded using the chronoamperometry method at a voltage of +1.0 V. Finally, the HXA concentration was calculated using the standard addition method (Fig. S5). The obtained results are quite consistent with the data from biochemical analysis with a deviation of less than 0.5%. Furthermore, the recovery test results show adequate performance of the fabricated electrode. The recovery rate is determined by comparing the concentration obtained from the samples with the spiked concentration and is used to demonstrate the accuracy of the biosensor. The results show that the recovery is 98.2% and 98.6% in Table II, which shows that the sensor performs well in terms of accuracy for determining HXA in real samples.

TABLE II.

Determination of hypoxanthine in pork meat sample.

SampleDetected (μM)Spiked (μM)Found (μM)Recovery (%)
Pork meat samples 26.64 ⋯ ⋯ ⋯ 
  20 45.98 98.6 
  30 55.62 98.2 
SampleDetected (μM)Spiked (μM)Found (μM)Recovery (%)
Pork meat samples 26.64 ⋯ ⋯ ⋯ 
  20 45.98 98.6 
  30 55.62 98.2 

In conclusion, we fabricated successfully a high-performance electrochemical biosensor based on ZnO nanoparticles modified porous graphene fPGE, using a “green,” simple and inexpensive direct laser writing method with a CO2 laser on polyimide film in an ambient atmosphere. The ZnO/fPGE electrode provided a favorable environment for direct non-enzymatic electrochemical reactions, resulting in forming an efficient biosensor. In addition, the exploitation of the synergistic effects between ZnO nanoparticles and fPGE rendered better electroactivity to HXA sensing with a linear range of 1.5–150 µM, LOD of 0.14 µM, and sensitivity of 6.6 µA μM−1 cm−2. Remarkably, this biosensor was efficiently applied for the detection of HXA in pork with good selectivity. This result paves the way for great practical application possibilities of the ZnO/graphene electrode system in food quality analysis.

See the supplementary material for the cleaning procedure of the sensor electrodes; Fig. S1: SEM-EDS elemental mapping (a); and EDS of ZnO/fPGE. The inset is the weight and atomic percentage of elements (b); Fig. S2: The linearity of the Ep vs the ln ν (a) and the linear line of the lnEp vs the ln ν (b); Fig. S3: CV curves of the ZnO/fPGE sensor in PBS (1×, pH 7.0); Fig. S4: Percentage of oxidation peak current vs the number of cycles. Inset is the percentage of oxidation peak current vs different ZnO/fPGE; Fig. S5: Chronometry responses to known HXA concentrations (added) of the commercially available meat (a), and the linear plot of current vs concentration of HXA (b).

This work was financially supported by the Ministry of Science and Technology, Vietnam, with the Grant Code NDT. 70e-ASIA/19, and nanowires manipulation was done under the support of RSF (Grant No. 22-19-00783).

The authors have no conflicts to disclose.

N. T. H. Le: Conceptualization (equal); Data curation (equal); Formal analysis (equal); Funding acquisition (equal); Investigation (equal); Methodology (equal); Project administration (equal); Resources (equal); Software (equal); Supervision (equal); Validation (equal); Visualization (equal); Writing – original draft (equal); Writing – review & editing (equal). N. X. Viet: Conceptualization (equal); Methodology (equal); Resources (equal); Supervision (equal); Validation (equal); Visualization (equal); Writing – review & editing (equal). N. V. Anh: Data curation (equal); Formal analysis (equal); Methodology (equal). T. N. Bach: Data curation (equal); Formal analysis (equal); Methodology (equal). P. T. Thu: Formal analysis (equal); Writing – original draft (equal). N. T. Ngoc: Data curation (equal). D. H. Manh: Writing – review & editing (equal). V. H. Ky: Data curation (equal). V. D. Lam: Project administration (equal); Writing – review & editing (equal). V. Kodelov: Formal analysis (equal); Methodology (equal); Software (equal). S. Von Gratowski: Formal analysis (equal). N. H. Binh: Formal analysis (equal); Methodology (equal). T. X. Anh: Formal analysis (equal); Methodology (equal).

The data that support the findings of this study are available within the article and its supplementary material.

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Supplementary Material