ZIF-8/Graphene Oxide Nanocomposite Modified Electrode for Electrochemical Detection of Trimethylamine as a Freshness Indicator in Crayfish

1. Introduction

Aquatic products are rich in high-quality proteins, unsaturated fatty acids, and various micronutrients, constituting an important component of the human dietary structure. However, due to their high moisture content, fragile muscle tissue, and strong endogenous protease activity, aquatic products are highly susceptible to microbial contamination and enzymatic degradation during harvesting, storage, transportation, and processing [1]. Such spoilage not only causes substantial economic losses but may also produce harmful substances such as biogenic amines and volatile basic nitrogen, posing direct threats to consumer health. Therefore, establishing rapid and accurate methods for evaluating the freshness of aquatic products is of great significance for ensuring food safety and improving industrial efficiency.

At present, freshness evaluation methods for aquatic products are mainly divided into two categories: sensory evaluation and physicochemical detection. Sensory evaluation relies on human senses such as sight, smell, and touch to comprehensively assess sample color, odor, and elasticity. Although simple and intuitive, the results are highly dependent on the experience and subjective judgment of the evaluators, resulting in poor repeatability and comparability [2]. In contrast, physicochemical detection methods based on the concentration changes of specific freshness characteristic compounds can provide objective and quantitative data support, and have become the primary means for evaluating the freshness of aquatic products [3]. Common freshness indicators include total volatile basic nitrogen (TVB-N), trimethylamine (TMA), biogenic amines, and purine metabolites.

Among various physicochemical detection techniques, electrochemical sensing technology has shown broad application prospects due to its operational simplicity, rapid response, high sensitivity, low cost, and ease of miniaturization [4]. In recent years, electrochemical methods have been successfully applied to the detection of various spoilage markers in aquatic products, including biogenic amines (such as histamine and putrescine) [5,6], TVB-N [7], purine compounds [8], and trimethylamine [9].

Trimethylamine (TMA) is a volatile nitrogen-containing compound generated from the reduction of trimethylamine N-oxide (TMAO) by microbial trimethylamine reductase, possessing a strong fishy odor. As storage time extends, the TMA concentration increases significantly, and its content is closely related to the degree of spoilage. When the concentration exceeds 10 ppm, it is generally regarded as a decisive criterion for aquatic products entering the spoilage state [10,11].

Existing electrochemical detection technologies for TMA are mainly divided into gas-phase detection and liquid-phase detection. Gas-phase electrochemical sensors are usually based on metal oxide semiconductors (such as ZnO, In₂O₃, WO₃) or conductive polymers [12,13]. Although this approach enables non-contact, real-time monitoring, it generally suffers from high operating temperatures (typically 200–400 °C), high energy consumption, and susceptibility to cross-interference from environmental humidity and other volatile organic compounds [14]. In comparison, liquid-phase electrochemical detection directly analyzes sample extracts, generating current signals through the catalytic oxidation or reduction of TMA at the liquid–solid interface of modified electrodes. This method operates under ambient temperature and pressure, and the current response usually exhibits a good linear relationship with TMA concentration, ensuring high quantitative accuracy [15]. In recent years, liquid-phase electrochemical detection of TMA has achieved a series of advances. For example, Bourigua et al. [15] constructed a TMA biosensor based on a polypyrrole–FMO3 composite membrane; Chakravorty et al. [16] utilized molecularly imprinted polypyrrole–MoS₃ nanosheets for trimethylamine N-oxide detection; Gao et al. [17] proposed an electrochemiluminescence sensing strategy for TMAO; and Choi et al. [18] employed trimethylamine dehydrogenase immobilized on a gold nanoparticle electrode for TMA detection.

Although the above methods each have distinctive features, some systems still suffer from insufficient conductivity, limited active sites, or complex preparation procedures [16,17]. Therefore, the development of novel electrode modification materials remains a research priority. Metal–organic frameworks (MOFs) are porous crystalline materials featuring ultrahigh specific surface areas and abundant active sites [19,20,21]. In 2017, our group reported the application of MOF materials in the electrochemical detection of heavy metal ions [19]. In the following years, our group have constructed a curcumin-enhanced MOF electrochemical sensor for methyl parathion detection with satisfied results[20]; polyethyleneimine–MOF-235 composite was also utilized for silver nanoparticle detection [21]. Zeolitic imidazolate framework-8 (ZIF-8) is considered an ideal material due to its mild synthesis conditions and specific adsorption affinity for nitrogen-containing compounds [22]. However, the inherently poor electrical conductivity of conventional MOFs limits their electrochemical applications [22].

Graphene oxide (GO) is a two-dimensional carbon nanomaterial rich in oxygen-containing functional groups, exhibiting excellent hydrophilicity and favorable electron transfer capability [23,24]. Our group utilized a reduced graphene oxide hydrogel to suppress the phase transformation of iron oxides [23]. We also combined HKUST-1-loaded Ag/TiO₂ nanoparticles with GO for nicotine sensing [24]. Therefore, the combination of GO and ZIF-8 is expected to overcome the defects of single materials. Based on the above considerations, this study prepared ZIF-8@GO composite materials and constructed a novel electrochemical sensor for the rapid and sensitive detection of TMA in crayfish.

2. Experimental Section

Instruments and Reagents

Trimethylamine hydrochloride (TMA, ≥98%), graphene oxide (GO), 2-methylimidazole (Hmim), zinc nitrate hexahydrate (Zn(NO₃)₂·6H₂O), Nafion solution (5%, w/w), N,N-dimethylformamide (DMF), methanol, sodium hydroxide, potassium ferricyanide (K₃Fe(CN)₆), potassium chloride, and potassium phosphate salts were of analytical grade and used without further purification. Ultrapure water (resistivity ≥ 18.2 MΩ·cm) was used throughout the experiments. Fresh crayfish were purchased from a local aquatic market.

Electrochemical measurements were performed on a CHI760E electrochemical workstation (Chenhua, Shanghai) with a conventional three-electrode system: a glassy carbon electrode (GCE, 3 mm in diameter) as the working electrode, a platinum wire as the counter electrode, and an Ag/AgCl (saturated KCl) electrode as the reference electrode. The morphologies and structures of the materials were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), Raman spectroscopy, Fourier transform infrared (FTIR) spectroscopy, and thermogravimetric analysis (TGA).

Material Synthesis and Electrode Fabrication

The steps for material synthesis and electrode fabrication were shown in Figure 1.

Synthesis of ZIF-8 nanoparticles: ZIF-8 was synthesized according to a previously reported method with slight modifications [22]. Briefly, 0.811 g of 2-methylimidazole and 0.366 g of Zn(NO₃)₂·6H₂O were dissolved in 25 mL of methanol, respectively. After magnetic stirring to form homogeneous solutions, the two solutions were mixed and stirred at room temperature for 3 h. The resulting precipitate was collected by centrifugation (10,000 rpm, 10 min), washed three times with methanol, and dried under vacuum.

Synthesis of ZIF-8@GO composite: A certain amount of 2-methylimidazole (0.811 g) was first ultrasonically dispersed in the methanol (25 mL) to form solution. Meanwhile the Zn(NO₃)₂ solid powder (0.366 g) was dispersed in methanol (25 mL) to form solution. Subsequently, the 2-methylimidazole solution and Zn(NO₃)₂ solution was added into a glass beaker (100 mL volume). The mixed solution was stirred at room temperature for 3 h. The product was washed three times with DMF and dried under vacuum for 24 h to obtain the ZIF-8@GO composite.

The GCE was carefully polished with 0.3 μm and 0.05 μm alumina slurry on a polishing cloth to a mirror-like surface, followed by successive sonication in ultrapure water, 1:1 (v/v) nitric acid, acetone, absolute ethanol, and ultrapure water for 2 min each. The electrode was then electrochemically activated in 0.5 M H₂SO₄ by cyclic voltammetry until a stable voltammogram was obtained. To prepare the ZIF-8@GO/GCE, 1 mg of ZIF-8@GO was dispersed in 1 mL of isopropanol containing 10 μL of 5% Nafion solution under ultrasonication for 10 min to form a homogeneous suspension. Then, 8 μL of the suspension (2 μL per drop, four drops in total) was drop-cast onto the cleaned GCE surface and dried under an infrared lamp. Similarly, ZIF-8/GCE and GO/GCE were fabricated for comparison.

Sample Pretreatment

All the experiments in this work comply with the biological ethics standards (No. WHPU 20230048, Supplementary Material S1). The Fresh crayfish purchased were all stored under the laboratory self-made breeding conditions before the experiments (Supplementary Material S2). Before the experiments, all Fresh crayfish were euthanized by ultra-low temperature freezing, and then decapitated, shelled, and deveined. Approximately 100 g of muscle tissue was collected and homogenized at 4 °C. The homogenate was sealed in polyethylene bags and stored at −20 °C. Before analysis, the sample was thawed at room temperature.

For HPLC validation, the sample pretreatment was performed according to the national standard method (GB 5009.179-2016) with minor modifications. In brief, 10.0 g of homogenized sample was placed in a 50 mL centrifuge tube, and 20 mL of 5% trichloroacetic acid solution was added. After thorough mixing, the mixture was centrifuged at 4,000 r/min for 5 minutes. The supernatant was collected, and the residue was extracted twice with 15 mL and 10 mL of 5% trichloroacetic acid, respectively. All extracts were combined and diluted to 50 mL with 5% trichloroacetic acid, then filtered through a 0.22 μm membrane for HPLC analysis.

Electrochemical Measurements

All the electrochemical measurements were carried by CHI760E under room temperature. Electrochemical characterization of the modified electrodes was carried out in 5.0 mM K₃Fe(CN)₆ containing 0.1 M KCl by CV (potential window: −0.2 to 0.6 V, scan rate: 100 mV/s) and EIS (frequency range: 0.1 Hz to 100 kHz). Chronoamperometric measurements were also conducted to evaluate the effective surface area.

For TMA detection, DPV was performed in 0.1 M NaOH (pH 14.0) solution. The parameters were as follows: potential range 0 to 1.2 V, pulse amplitude 50 mV, pulse width 0.05 s, sampling width 0.0167 s, and pulse period 0.5 s. All experiments were conducted at room temperature.

3. Results and Discussion

3.1. Characterization of Electrode Materials

The morphologies of GO, ZIF-8, and ZIF-8@GO were investigated by SEM. As shown in Figure 2A, GO exhibited a typical layered structure with clear edges, but obvious interlayer stacking was observed. This phenomenon was consistent with previous research reports [23,24,25]. ZIF-8 displayed a rhombic dodecahedral morphology consistent with literature reports (Figure 2B), yet particle aggregation was evident. In the ZIF-8@GO composite (Figure 2C), ZIF-8 nanoparticles were uniformly distributed on the GO surface without phase separation, and the particle size was significantly reduced compared to pristine ZIF-8, indicating that GO effectively inhibited the agglomeration of ZIF-8. At the scale of 2 μm for the three materials in Fig 2C-E, the SEM images also exhibited a similar structure.

The crystal structures were analyzed by XRD (Figure 3A). GO showed a characteristic diffraction peak at 2θ = 26.58° corresponding to the (011) plane. ZIF-8 exhibited sharp peaks at 7.3°, 10.3°, 12.7°, 14.7°, 16.4°, 18.0°, 22.2°, 24.5°, and 26.7°, confirming its high crystallinity. The ZIF-8@GO composite retained all characteristic peaks of both components, demonstrating the successful synthesis of the composite without altering the crystal structure of ZIF-8. Raman spectroscopy (Figure 3B) revealed that the D and G bands of GO appeared at 1330 cm⁻¹ and 1579 cm⁻¹, respectively. All of the above characteristics have demonstrated the presence of graphene oxide [23,24,25]. In the composite (GO-ZIF-8), both bands shifted, and the I_D/I_G ratio increased, indicating enhanced structural defects and successful coupling between ZIF-8 and GO. FTIR spectra (Figure 3C) showed that ZIF-8@GO retained the characteristic absorption peaks of both GO and ZIF-8. The peaks at 2970 cm⁻¹ and 3137 cm⁻¹ corresponded to C–H stretching vibrations, while peaks at 1680 cm⁻¹, 1380 cm⁻¹, and 954 cm⁻¹ were attributed to C=O, C=N, and Zn–N bonds, respectively, further confirming the successful synthesis of the composite. TGA curves (Figure 3D) indicated that GO@ ZIF-8 exhibited better thermal stability than pure GO. The mass loss of the composite at 400 °C was 50%, lower than that of pure GO (63.9%), demonstrating that the introduction of ZIF-8 effectively inhibited the high-temperature decomposition of GO.

Electrochemical Investigation and Characterization

To systematically evaluate the electrochemical behavior of the obtained nanohybrid, electrochemical investigation and characterization were carried in [Fe(CN)₆]³⁻/⁴⁻ redox system. The electrochemical behaviors of different electrodes were investigated by CV and EIS in 5.0 mM K₃Fe(CN)₆/0.1 M KCl solution. As shown in Figure 4A, all modified electrodes displayed reversible redox peaks. The peak currents increased in the order of bare GCE < ZIF-8/GCE < GO/GCE < ZIF-8@GO/GCE, with ZIF-8@GO/GCE exhibiting the highest peak current, indicating significantly enhanced electrochemical performance.

EIS Nyquist plots (Figure 4B) showed that the semicircle diameter of ZIF-8@GO/GCE was the smallest among all electrodes, indicating the lowest charge transfer resistance (R_ct) and the fastest electron transfer rate. This is attributed to the synergistic effect between GO and ZIF-8: GO provides excellent conductivity, while ZIF-8 offers abundant active sites.

Chronoamperometric measurements (Figure 4C and Figure 4D) were performed to estimate the effective surface areas of the electrodes. According to the Anson equation, the slopes of the Q–t^1/2 curves for bare GCE, ZIF-8/GCE, GO/GCE, and ZIF-8@GO/GCE were 2.352 × 10⁻⁴, 4.851 × 10⁻⁴, 5.835 × 10⁻⁴, and 6.237 × 10⁻⁴ C/s^1/2, respectively. The highest slope for ZIF-8@GO/GCE confirmed its largest electrochemically active surface area and superior charge transfer efficiency.

Electrochemical Detection of TMA

In order to study the electrochemical detection capability of the as-prepared nanocomposite electrode toward TMA, the electrochemical responses of TMA on different electrodes were investigated by CV and DPV in 0.1 M NaOH solution containing 0.1 μmol/L TMA. As shown in Figure 5A, an obvious oxidation peak appeared at approximately 0.8 V on all electrodes. Notably, ZIF-8@GO/GCE exhibited the highest peak current, indicating significantly enhanced electrocatalytic activity toward TMA oxidation. The DPV results (Figure 5B) further confirmed that ZIF-8@GO/GCE could effectively amplify the TMA signal and accelerate electron transfer.

The effect of scan rate on the electrochemical behavior of TMA was studied. As shown in Figure 5C, the oxidation peak current increased linearly with scan rate in the range of 10–300 mV/s. The linear regression equation was I_p (μA) = 0.5635 v (mV/s) + 50.8077, with a correlation coefficient R² = 0.9936 (Figure 5D). The direct proportionality between peak current and scan rate suggests that the electrochemical oxidation of TMA on ZIF-8@GO/GCE is an adsorption-controlled process.

The above results demonstrate that the GO-ZIF-8/GCE exhibits significant superiority for the electrochemical detection of trimethylamine. This enhanced performance can be attributed to the favorable adsorption capacity of the ZIF-8 metal-organic framework toward organic molecules such as trimethylamine, coupled with the large specific surface area and excellent electrical conductivity of graphene oxide. The as-fabricated nanocomposite electrode synergistically integrates the complementary advantages of GO and MOF materials, thereby substantially improving the electrochemical sensing capabilities. This observation is also in good agreement with previously reported literature findings.

In order to achieve better analytical detection performance, we optimized the conditions for electrochemical detection, including the pH of the electrolyte (Figure 6A-B), the adsorption time (Figure 6C), and the loading amount of the GO-ZIF-8 electrode material (Figure 6D). The influence of solution pH on the TMA response was investigated in the range of 7.0–14.0. As shown in Figure 6A and Figure 6B, the oxidation peak current increased significantly with increasing pH, reaching a maximum at pH 14.0. Meanwhile, the peak potential shifted negatively with increasing pH, indicating that protons participate in the electron transfer process. Therefore, 0.1 M NaOH (pH 14.0) was selected as the optimal supporting electrolyte. Effect of accumulation time (Adsorption time): As shown in Figure 6C, the oxidation peak current of TMA increased with accumulation time and reached a plateau at 180 s. Prolonged accumulation did not further enhance the signal, likely due to saturation of active sites. Thus, 180 s was chosen as the optimal accumulation time. Effect of composite loading: The effect of ZIF-8@GO loading on the electrode surface was investigated. As shown in Figure 6D, the peak current increased with loading amount from 0.04 to 0.10 mg/cm², and then decreased at higher loadings. The decrease at excessive loading is attributed to the formation of a dense layer that hinders electron transfer. Therefore, 0.10 mg/cm² was selected as the optimal loading.

Under the optimized conditions, the quantitative detection performance of the ZIF-8@GO/GCE toward TMA was evaluated by DPV. As shown in Figure 7A, the oxidation peak current increased linearly with TMA concentration in the range of 5–105 μM. The linear regression equation was I_p (μA) = 3.7876 C (μM) + 8.8846, with a correlation coefficient R² = 0.9997 (Figure 7B). The limit of detection (LOD) was calculated to be 1.79 μM (S/N = 3), and the limit of quantification (LOQ) was 5.97 μM (S/N = 10). A comparison with other reported electrochemical methods for TMA detection is presented in Table 1, demonstrating that the proposed sensor exhibits competitive analytical performance. Moreover, compared with the methods reported in other literature (such as enzyme-assisted electrochemical methods or electrochemiluminescence methods), the work presented in this paper adopts direct electrochemical oxidation, which has significant advantages.

The stability of the sensor was evaluated by measuring the response to TMA over 10 consecutive days. As shown in Figure 8A, the average peak current remained stable at 6.65 ± 0.16 μA, with an RSD of 1.03%, indicating excellent long-term stability. The reproducibility was investigated using five independently prepared electrodes. As shown in Figure 8B, the RSD of the peak currents was 0.89%, demonstrating good fabrication reproducibility. The selectivity of the sensor was evaluated by introducing potential interfering substances, including Mg²⁺, Ca²⁺, K⁺, phosphate, chloride, glucose, amino acids, and fatty acids. As shown in Figure 8C, these interfering species showed negligible effects on the TMA response, confirming the good selectivity and anti-interference ability of the proposed sensor in complex matrices.

To validate the practical applicability of the sensor, it was applied to the determination of TMA in real crayfish samples. The samples were pretreated as described in Section 2.4, and the TMA content was determined by the standard addition method. As shown in Figure S3 and Table 2, the recoveries ranged from 96.56% to 101.32%, with RSDs below 3%. Furthermore, the results were consistent with those obtained by HPLC, confirming the accuracy and reliability of the proposed method for real sample analysis.

4. Conclusions

In this work, a novel electrochemical sensor based on ZIF-8@GO nanocomposite modified GCE was successfully constructed for the rapid and sensitive detection of trimethylamine in crayfish. The composite combined the high specific surface area and specific adsorption capability of ZIF-8 with the excellent conductivity of GO, exhibiting synergistic enhancement effects. The sensor displayed a wide linear range (5–105 μM), low LOD (1.79 μM), good reproducibility (RSD = 0.89%), excellent long-term stability (RSD = 1.03% over 10 days), and superior anti-interference ability. The successful application to real crayfish samples with satisfactory recoveries (96.56%–101.32%) and good consistency with HPLC results demonstrated the practical potential of the proposed sensor. This work provides a reliable analytical tool for the rapid evaluation of aquatic product freshness and holds promise for on-site food safety monitoring.