Portable-Equipment-Assisted Colorimetric Hydrogel Kits Based on Quercetin-Reducing Silver Nanoparticles for Hg²⁺ Tests in Aquatic Products

Abstract

As a highly hazardous heavy metal, mercury ion (Hg²⁺) poses a serious threat to human health through the intake of polluted aquatic products and water. Accordingly, the development of a simple, sensitive, and efficient method for Hg²⁺ detection is of significant importance. Herein, a novel silver nanoparticle (Qu-CD-AgNPs) was successfully synthesized using an inclusion complex of Qu and β-cyclodextrin, which forms an Ag–Hg alloy upon interaction with Hg²⁺, markedly enhancing its oxidase-like activity to oxidize 3,3′,5,5′-tetramethylbenzidine (TMB), generating a Hg²⁺ concentration-dependent color change. On this basis, a colorimetric detection platform for Hg²⁺ was fabricated by integrating the hydrogel-based Qu-CD-AgNPs + TMB system and a self-developed RGB-values-analysis app for portable equipment. Under the optimized conditions, the platform shows a linear range between 3 and 100 μM and a detection limit as low as 0.023 μM, as well as excellent selectivity and anti-interference performance. This work promises a broad application prospect in water-quality monitoring and the inspection of aquatic products.

1. Introduction

Mercury is a highly toxic environmental pollutant that is apt to persist in ecosystems and therewith to bioaccumulate and biomagnify up the food chain, posing severe risks to human health. Among chemical forms of mercury, Hg²⁺ draws growing awareness due to readily binding to small functional groups such as methyl, phenyl, and thiol moieties within organisms, causing cellular function disruptions, leading to neurological damage and other diseases. Therefore, strengthening Hg²⁺ monitoring to reduce exposure risk is critical to mitigating threats to human health. However, as far as we know, the current Hg²⁺ detection methods mainly include atomic absorption spectroscopy, inductively coupled plasma mass spectrometry, and Raman spectroscopy, which generally require expensive instrumentation and complex operations. , Although highly sensitive, the inherent property of conventional detection methods makes them really unsuitable for on-site determination of mercury in environmental and biological samples, thus influencing the rapid assessment of contamination levels, guidance for remediation, and compliance management.

Relatively speaking, taking advantage of simplicity, convenience, speed, and low-cost, the portable Hg²⁺ detection strategies based on nanomaterials have attracted extensive attention in recent years. − In particular, various silver particles (AgNPs) have been widely applied to colorimetric, Raman scattering or fluorescence assays for determining Hg²⁺ levels in water and aquatic products. − Current studies have shown that AgNPs prepared and modified via different routes have an intrinsic affinity with Hg²⁺ to form Ag–Hg alloys, enhancing their oxidase-mimicking activity, which, even in the absence of hydrogen peroxide, generates superoxide anion radicals to convert the achromatous 3,3′,5,5′-tetramethylbenzidine (TMB) into its blue oxidized product (oxTMB) with a maximum absorbance at 652 nm. Meanwhile, within a certain concentration range, this chromogenic reaction correlates with the Hg²⁺ concentration, enabling the construction of AgNPs-TMB-based sensors for quantitative colorimetric detection of Hg²⁺. Consequently, on the basis of novel AgNPs, developing new Hg²⁺ detection methods with rapid, sensitive, and user-friendly features is of pressing practical significance.

In recent years, with a prevailing trend in the environmental sustainability of nanomaterials, the green synthesis of AgNPs, especially by reduction of natural compounds, has gradually become an important strategy. Quercetin (Qu), a kind of plant-derived flavonoid compound with low water solubility, which, beyond its antibacterial and antioxidant applications, has also been used in the fabrication of various nanomaterials, including silver nanomaterials and nano-composites. − Additionally, considering that β-cyclodextrin (β-CD) possesses a hydrophilic outer surface and a hydrophobic inner cavity containing sufficient space for Qu, encapsulating Qu with β-CD may enhance its stability and aqueous solubility. More interestingly, the above inclusion complex can act simultaneously as the reducing agent, dispersant, and proppant and is responsible for the green synthesis of AgNPs, which presents a promising strategy. − Moreover, according to our knowledge, the approach to directly synthesizing AgNPs in aqueous media through the β-CD encapsulated Qu has not been reported.

It is noteworthy that the deep integration of various sensors based on colorimetry and RGB data analysis software for portable equipment have been successfully applied for on-site rapid detection of possible analytes, such as biogenic amines, dexamethasone, and ascorbic acid. − Furthermore, in practical scenarios relevant to the rapid detection of water bodies and aquatic products, instruments such as UV–vis spectrophotometers are generally unavailable. Therefore, to further enhance the convenience of the Hg²⁺ detection system, a preferred route is to acquire images of the chromogenic reaction with the camera of portable equipment, and then quantify the Hg²⁺ concentration via the aid of dedicated image-analysis software based on RGB values, which will greatly improve the practicality and broaden the scope of application.

Herein, a new type of Qu-cyclodextrin silver nanoparticles with oxidase-like activity was successfully prepared using the inclusion complex of Qu and β-CD. A colorimetric detection platform for Hg²⁺ was fabricated by integrating the hydrogel-based Qu-CD-AgNPs + TMB system and a self-developed RGB-values-analysis app for portable equipment, providing a new approach to the direct determination of Hg²⁺ in environmental water samples and aquatic products. The portable hydrogel kit will offer the portability of rapid on-site analysis, favorable cost-effectiveness, and broad adaptability, exhibiting strong prospects for practical application in agricultural fields such as water environmental monitoring and aquatic product testing.

2. Materials and Methods

2.1. Chemicals

Quercetin (Qu), β-CD, AgNO₃, Hg (NO₃)₂·H₂O, citric acid (C₆H₈O₇·H₂O), sodium citrate (C₆H₅Na₃O₇·2H₂O), ethanol (C₂H₆O), CuSO₄, Zn­(NO₃)₂·6H₂O, MgSO₄, KCl, FeCl₃, MnSO₄·4H₂O, BaCl₂, CaCl₂, and lead­(II)­acetate ((CH₃OO)₂Pb) were all of analytical grade and purchased from Shanghai Chemical Reagent Co., Ltd., (Sinopharm Group). Agarose and 3,3′,5,5′-tetramethylbenzidine (TMB) were purchased from Aladdin (Shanghai).

2.2. Characterizations

Microstructural morphology was characterized using a transmission electron microscope (TEM) (Thermo Fisher Scientific, Talos F200X G2, America), for which the samples were prepared by dropping the sample suspension onto a carbon-coated copper grid and drying at room temperature, operating at an accelerating voltage of 200 kV. Zeta potential was measured with a laser particle size analyzer (Brookhaven, 90PlusPALS, America) at room temperature. Each sample was measured in triplicate to ensure accuracy. The crystal structure information on the samples was obtained by collecting X-ray diffraction (XRD) patterns using an XRD instrument (Bruker D8 ADVANCE, Germany), which was equipped with Cu Kα radiation (λ = 1.5418 Å), setting the tube voltage and current to 40 kV and 40 mA, respectively. The patterns were recorded in the range of 10°–90° with a scanning rate of 5°/min. Optical property tests were performed using a UV–visible spectrophotometer (Shimadzu, UV-2600i, Japan) in the wavelength range of 200–800 nm using deionized water as the baseline reference, and a multi-mode microplate reader (Infinite E plex, Swiss) measuring at an absorbance wavelength of 652 nm. Under room temperature, the electron spin resonance (ESR) spectra were recorded using an ESR spectrometer (Bruker, EMXplus-6/1, Germany) in which the typical spectrometer parameters were set as follows: X-band microwave frequency of 9.8 GHz, microwave power of 6.3 mW, modulation frequency of 100 kHz, and modulation amplitude of 1.0 G. X-ray photoelectron spectroscopy (XPS) analysis was performed using an X-ray photoelectron spectrometer (AXIS ULTRA DLD, SHIMADZU, Japan) equipped with a monochromatic Al K α X-ray source (pass energy of 50 eV, energy step size of 0.050 eV and spot size of 400 μm).

2.3. Preparation of Qu-CD-AgNPs

2 mL of the quercetin-ethanol solution (10 mg·mL^–1) was added to 18 mL of the β-CD solution (40 mg·mL^–1), vigorously stirring for 2 h in a 55 °C water bath away from light. After filtering through a 0.22 μm membrane, 1.5 mL of the quercetin-β-CD solution was diluted with deionized water to 3 mL, and subsequently, 2 mL of the 5 mg·mL^–1 AgNO₃ solution was added and continued to be stirred in the dark for 2 h at 55 °C. Finally, the Qu-CD-AgNPs were obtained by washing and freeze-drying.

2.4. Colorimetric Detection of Hg²⁺ and Assessment of the Oxidase-like Activity

According to a slightly modified protocol as reported, the oxidase-like activity of Qu-CD-AgNPs was investigated. First, a 0.1 mol·L^–1 citrate-sodium citrate buffer solution of pH 4, 3 mM of Hg (NO₃)₂ solution, 10 mmol·L^–1 of TMB solution, and Qu-CD-AgNPs dispersions of 1 mg·mL^–1 were prepared. Next, the above solutions were mixed in different combinations and reacted for about 10 min, and then the absorbance values were measured using a multimode microplate reader to verify the oxidase-like activity of the Qu-CD-AgNPs + Hg²⁺ system. In detail, 20 μL of Qu-CD-AgNPs and 20 μL of Hg (NO₃)₂ were added to 140 μL citrate-sodium citrate buffer; subsequently, a specific concentration gradient of TMB was added to this mixture. Over 10 min, the change in optical density at a wavelength of 652 nm was measured, and the catalytic activity of Qu-CD-AgNPs was evaluated to determine the kinetic parameters based on the Michaelis–Menten eq . The kinetic parameters (kinetic constant K _m and maximum velocity V _max) were calculated as follows

$$\frac{1}{V}=\frac{K_{\mathrm{m}}}{V_{\max }}·\frac{1}{[\mathrm{S}]}+\frac{1}{V_{\max }}$$ 1

where V is the reaction rate, V _max is the maximum reaction rate, [S] is the substrate concentration, and K _m is the Michaelis constant. Calculation of substrate concentration using Lambert–Beer’s law eq .

$$A=\epsilon bc$$ 2

where A is the absorbance at 652 nm, ε is the molar absorption coefficient, b is the optical range, and c is the substrate concentration. The molar absorption coefficient of oxTMB at 652 nm is 39,000 M^–1·cm^–1.

Moreover, to assess the property of Hg²⁺ detection of the Qu-CD-AgNPs + Hg²⁺ system, we mixed the prepared 0.1 mg·mL^–1 Qu-CD-AgNPs dispersion, 0.1–1 mM TMB solution, and pH 4.0 citrate-sodium citrate buffer with various concentrations of Hg (NO₃)₂ solutions at 3, 5, 10, 20, 40, 60, 80, and 100 μM, respectively. The Qu-CD-AgNPs/Hg²⁺/pH 4.0 citric acid-sodium citrate buffer solution: TMB system uses a ratio of 1:1:7:1.

The limit of detection (LOD) was calculated according to the IUPAC-recommended equation

$$\mathrm{L}\mathrm{O}\mathrm{D}=3\sigma /S$$

where σ is the standard deviation of three blank measurements and S is the slope of the calibration curve.

2.5. Influence of Metal Ions

Common metal ions (Cu²⁺, Zn²⁺, Mg²⁺, Ba²⁺, Fe³⁺, Mn²⁺, Ca²⁺, K⁺, Pb²⁺) in the aquatic environment, with an 8 times the concentration of Hg²⁺ (concentration of Hg²⁺ termed 400 μM), were chosen to be added to the same detection system to observe the color changes and measure the absorbance value. Furthermore, a coexisting ion interference test was carried out in the above system, in the presence of Hg²⁺ combined with nine kinds of metal ions. Additionally, the detection of Hg²⁺ was performed in the presence of differing concentrations of three organic species, glutathione (GSH), l-cysteine (CYS), bovine serum albumin (BSA), humic acid (HA), and tissue protein extracts (TPE), demonstrating the system’s robustness.

2.6. Preparation of a Portable Hydrogel-Based Sensor and Application in Aquatic Animal Samples for Hg²⁺ Detection

Based on the optimized reaction conditions, the specific preparation steps for the hydrogel sensor were as follows: An optimal 1% agarose gel was prepared using the aforementioned pH 4.0 citric acid-sodium citrate buffer solution. We melted a 2% (mass fraction) agarose aqueous solution, then cooled it to approximately 50 °C. Subsequently, the aforementioned pH 4.0 citric acid-sodium citrate buffer solution at a 1:1 (volume ratio), 1% (volume ratio) of a 1 mg·mL^–1 Qu-CD-AgNPs dispersion liquid, and 10% (volume ratio) of a 20 mM TMB solution were thoroughly mixed with the above agarose solution to prepare the 1% concentration of mixed agarose solution. The mixture was added to a 24-well plate and then solidified, and then stored at 4 °C. To establish a portable signal reading and analysis platform, 400 μL of Hg²⁺ solution at different concentrations was added to the agarose hydrogel, and after 15 min, the absorbance at 652 nm was read using a microplate reader. After capturing images of the color-changed agarose hydrogel using a portable device, we extracted the RGB values from the images using custom software, then fitted the standard curve based on the obtained RGB values. Oysters, clams, and shrimps sourced from the agricultural market on Wenhua Road in Zhifu District, Yantai City, Shandong Province were selected as the actual samples to demonstrate the feasibility of this system, and the sample preparation was performed according to a previously reported method with a minor modification. , In short, for every sample, 5 g of homogenized shell-removed tissue was filtered through a 100-mesh gauze and then subjected to centrifugation at 13,000 rpm for 10 min. Subsequently, a specific volume of Hg²⁺ solution (100 μM) was added to the 10 mL supernatant for the spiked recovery test. All samples were added to the hydrogel reagent kit. After a 15 min reaction, the absorbance at 652 nm was measured using a microplate reader, and then the RGB values of the captured images were analyzed via our custom software. According to the above-plot standard curve, the corresponding concentrations were calculated to determine the spiked recovery rate.

2.7. Statistical Analysis

The experiment was replicated thrice for all samples, and one-way analysis of variance was performed using IBM SPSS Statistics 24. A P-value less than 0.05 was considered statistically significant. All graphs were plotted using Origin 2018, and the results of the experiments were expressed as the mean standard deviation (mean ± SD).

3. Results and Discussion

3.1. Characterization of Qu-CD-AgNPs

The detailed preparation is shown in Figure A. As illustrated in Figure B, the characteristic absorption peak of Qu-CD-AgNPs was at approximately 298 nm, consistent with the usual absorption region of other kinds of nano-silver nanoparticles, compared to those of Qu (373 nm) and Qu-β-CD (370 nm). In addition, the surface zeta potential of Qu-CD-AgNPs was at – 28.9 mV, which is a striking difference with that of Qu-β-CD (+15.75 mV), implying the existence of the nano-silver (Figure C). Moreover, taking into account both the similar absorption peaks of Qu and Qu-β-CD and the positive surface potential of Qu-β-CD matching that of β-CD and the difference with Qu, the formation of the inclusion complex of Qu-β-CD was also confirmed. To further verify the morphologies of Qu-CD-AgNPs, the TEM images of them displayed plenty of irregularly ellipsoidal particles around 90 nm, with an uneven spread, in line with an average hydration size of about 155 nm as DLS analysis (Figure D,E). Furthermore, the HAADF imaging showed strong signals of Ag, besides weaker signals of C, O and N, which may originate from functional groups in Qu or β-CD, or inescapable O₂, or residual NO₃ ^– (Figure F). As depicted in Figure G, the high-resolution TEM images confirmed that the lattice spacing distance of Qu-CD-AgNPs was between 0.2544 and 0.2211 nm, corresponding to the [111] and [220] facets, respectively, which was also verified by XRD results (Figure H). , Furthermore, regarding the XRD spectrum, the refraction peaks were observed at 38.1°, 44.3°, 64.4°, and 77.5°, corresponding to the [111], [200], [220], and [311] planes, respectively, which confirms a face-centered cubic silver nanocrystal structure, in good agreement with the reported nano-silver nanoparticles (Figure H). ,, Collectively, the results provided solid evidence for the successful synthesis of Qu-CD-AgNPs.

1.

Characterization of Qu-CD-AgNPs. (A) Detailed synthesis process of the Qu-CD-AgNPs; (B) UV spectra of Qu, Qu-β-CD, and Qu-CD-AgNPs; (C) potential diagram of Qu-β-CD and Qu-CD-AgNPs; (D) TEM image and particle size distribution diagram of Qu-CD-AgNPs. Data are represented as mean ± SD; (E) TEM image of Qu-CD-AgNPs; (F) HAADF image of Qu-CD-AgNPs and elemental distribution energy spectrum (EDS) diagram of Qu-CD-AgNPs; (G) high-resolution HRTEM image of Qu-CD-AgNPs; (H) XRD pattern of Qu-CD-AgNPs.

3.2. Steady-State Kinetic and Catalytic Mechanism of Hg²⁺-Enhanced Oxidase-Mimicking Activity of Qu-CD-AgNPs

In the same citrate-sodium citrate buffer solution (pH 4.0), our comparison analysis of three systems (Qu-CD-AgNPs + TMB, TMB + Hg²⁺, and Qu-CD-AgNPs + TMB + Hg²⁺) found that neither blue coloration nor obvious absorption at 652 nm was observed in both the Hg²⁺ + TMB system and the Qu-CD-AgNPs + TMB system; in contrast, a pronounced blue color appeared in Qu-CD-AgNPs + TMB + Hg²⁺ system, accompanied by a strong absorption at 652 nm (characteristic peak of oxTMB) (Figure A). It suggested that, even if H₂O₂ was not added, Hg²⁺ could enhance the intrinsic oxidase-like activity of Qu-CD-AgNPs, and then TMB was oxidized to blue oxTMB, presumably resulting from the reactive oxygen species caused by the formation of an Ag–Hg alloy. For more precise identification of the main reactive oxygen species, the ESR spectra were employed, presenting the distinct signals of superoxide anions (O₂ ^·–), which were generated from the dissolved oxygen in solution, also similarly to the previous findings (Figure B). In order to further evaluate the mechanism of Hg²⁺-enhanced oxidase-like catalysis of Qu-CD-AgNPs, we analyzed the XPS spectra and the corresponding Gaussian curve fitting spectra of the high-resolution spectrum of Hg 4f and Ag 3d. Figure C shows the presence of both Hg (Hg 4f) and Ag (Ag 3d) in the Qu-CD-AgNPs + Hg²⁺ system, demonstrating a strong interaction between Hg²⁺ and Qu-CD-AgNPs. Furthermore, two dominant peaks of Hg 4f_7/2 and Hg 4f_5/2 are evident in the high-resolution spectrum of Hg 4f (Figure D), which could be obviously deconvoluted into four components: two peaks at 101.0 and 105.1 eV corresponding to Hg⁰ and the other two characteristic peaks at 101.7 and 105.7 eV corresponding to Hg²⁺, in accordance with the previous research. Moreover, compared to two high-resolution spectra of Ag 3d (Figure E), the spectral lines of the Qu-CD-AgNPs + Hg²⁺ system could also be deconvoluted into four components, in which two peaks at 368.0 and 373.9 eV were assigned to Ag⁺; meanwhile, the other two fitting peaks (368.8 and 374.7 eV) corresponded to Ag⁰. Together with the results of XPS spectra, we confirmed the formation of an Ag–Hg alloy, on account of the high affinity of Hg²⁺ and nano-silver. Furthermore, owing to the formation of Ag–Hg amalgam, the transfer of electrons from Ag–Hg amalgam will be much easier than that from nano-silver particles, which originate from the fact that Hg possesses the smaller absolute electronegativity than Ag, leading to a decrease of electronegativity on surface of Qu-CD-AgNPs. , Herein, we suggested that the Hg²⁺-enhanced oxidase-mimicking activity of Qu-CD-AgNPs is mainly caused by the evident promoting effect on electron transfer due to the emergence of the Ag–Hg amalgam; apart from that, it has been partly attributed to the fact that Qu-β-CD largely increased the dispersibility of Qu-CD-AgNPs.

2.

(A) UV–vis absorption spectra of different systems; (B) ESR spectra; (C) XPS survey spectrum; (D) high-resolution XPS spectra region of Hg 4f; (E) high-resolution XPS spectra of Ag 3d with or without Hg²⁺; (F) steady-state kinetic analysis of the Qu-CD-AgNPs/Hg²⁺ system: Michaelis–Menten curve; (G) Lineweaver–Burk linear diagram.

To further determine the catalytic features of the Qu-CD-AgNPs + TMB + Hg²⁺ system, we performed the steady-state kinetic analysis subsequently. As depicted in Figure F, the reaction rate improved with increasing TMB concentration from 200 to 800 μM, and then tended toward stability, indicating an excellent fitting to the typical Michaelis–Menten model (R ² = 0.99). Subsequently, the Lineweaver–Burk analysis revealed a good linear relationship between the reciprocal of the initial rate and the TMB concentration, with a fitting equation of y = 1879.91905x + 1.89966 (R ² = 0.99) (Figure G). Moreover, for the Qu-CD-AgNPs + Hg²⁺ system, the Michaelis constant (K _m) and maximum reaction velocity (V _max) are 0.99 mM and 8.77 × 10^–9 M·s^–1. According to previous literature reports, the K _m values for Ag₂S@GO and FA@Ag NPs were 0.1 mM and 0.12 mM, respectively, and thus the Qu-CD-AgNPs exhibit a lower K _m value, which represents a higher affinity with the substrate, compared to other silver-based nanoenzymes. , From these results, the prepared Qu-CD-AgNPs demonstrate an excellent oxidase-mimicking activity and high affinity toward the substrate TMB, enabling rapid and highly sensitive detection of Hg²⁺. In addition, the experiment parameters of this Hg²⁺ detection were further optimized for the Qu-CD-AgNPs + TMB + Hg²⁺ platform (See Supporting Information and Figure S1); then the applied conditions (pH 4.0, 1.0 mM TMB and 10 min) were setup for the subsequent detection of Hg²⁺.

3.3. Colorimetric Sensing Performance of Hg²⁺

Aiming to evaluate the reliability and application feasibility, we analysis the performance of the Qu-CD-AgNPs + TMB + Hg²⁺ platform for detecting Hg²⁺. As demonstrated in Figure A, when the Hg²⁺ concentration increases gradually from 3 to 100 μM, the color of the Qu-CD-AgNPs + TMB + Hg²⁺ system constantly deepens, from light blue to deep blue, also with a concomitant increase in absorbance at 652 nm. Furthermore, the fitting of results revealed an excellent linear correlation between Hg²⁺ concentration and the absorbance at 652 nm within a valid range of 3–100 μM due to the signal saturation above 100 μM, described by a linear equation of y = 0.01327x + 0.00414 with R ² = 0.996 (Figure B). Additionally, the LOD of Hg²⁺ reached a low of 0.023 μM (detailed calculation process in Supporting Information S2), which is prominently lower than the WHO limit in drinking water or food (6 μg·L^–1, approximately 0.03 μM), and the European Union standard in fishery products and fish muscle (0.5 mg·kg^–1 and 1 mg·kg^–1, respectively), indicating the robust colorimetric performance for Hg²⁺ determination and an adequate capability of practical applications within certain limits. ,, For the freeze-dried Qu-CD-AgNPs powder storage at 4 °C, we found a gradually decrease in colorimetric performance over time, which eventually stabilized at around 3.7% within 15 days (Figure S2). In spite of this modestly weakened performance, the prepared nano-silver indicated a fair degree of stability of Hg²⁺ colorimetric detection. Furthermore, the preparation repeatability among three independently prepared batches was also inspected by comparing their critical parameters, including K _m, V _max, and the linear range; and the similar performance demonstrated the consistency of the Qu-CD-AgNPs + TMB + Hg²⁺ platform (see Supporting Information and Figure S3).

3.

(A) UV–visible absorption spectra of the colorimetric assay for different concentrations of Hg²⁺ ions; (B) curve of the Qu-CD-AgNPs + TMB system for different concentrations of Hg²⁺; (C) selective detection of Hg²⁺ against common metal ions; (D) coexisting ion assay.

As far as we know, diverse metal ions, for instance, Zn²⁺, Mn²⁺, Cu²⁺, Mg²⁺, Fe³⁺, Ba²⁺, K⁺, Ca²⁺, and Pb²⁺, are common in the aquatic environment and aquatic animals, as potential interferents for detecting Hg²⁺, thus recognizing the specificity of the Qu-CD-AgNPs + TMB + Hg²⁺ platform. In spite of eightfold concentrations of Hg²⁺, no interfering ion can enhance the peroxidase-like activity of Qu-CD-AgNPs to produce blue oxTMB (Figure C). Ulteriorly, the coexisting ion assay revealed that all samples appeared nearly uniform blue and almost the same absorbance value at 652 nm, with no significant difference (P > 0.05), exhibiting the negligible effect of interfering ions (Figure D).

To further assess the anti-interference capability of the sensor, the effects of GSH, CYS, BSA, HA, and TPE were investigated. High concentrations (20–200 μM) of these organic species significantly suppressed the Hg²⁺ response; however, their interference was negligible at low concentrations (2 μM). This inhibition is likely due to the strong coordination between sulfhydryl groups and Hg²⁺, protein-based surface adsorption, or the reducing nature of these compounds (Figure S4).

Together with the aforementioned results, it is proven that our constructed Qu-CD-AgNPs + TMB + Hg²⁺ platform exhibits excellent specificity and selectivity for Hg²⁺ detection. Compared with existing literature on silver-based Hg²⁺ detection sensor which revealed a range of LODs from 0.001 to 0.070 μM, our proposed platform has similar performance to them. Although what is noteworthy is that other reported nanozyme-based methods, such as platinum nanoparticles with peroxidase-like activity and gold nanoparticle with peroxidase-like activity, showed lower LOD, reaching to 5.1 nM and 5.4 nM, respectively, they are relatively expensive and need to add H₂O₂ to the reaction system, causing inconvenience for developing a test kit. , By embracing the non-H₂O₂ reaction system and an innovative self-developed RGB-values-analysis app, the Qu-CD-AgNPs + TMB + Hg²⁺ platform is more capable of analyzing practical samples.

3.4. Application of the Portable Hydrogel-Based Sensor in Actual Samples

Similar to the findings in the solvent system of Qu-CD-AgNPs + TMB + Hg²⁺, under the optimized experimental conditions, the fabricated hydrogel-based sensor also exhibited progressively deeper blue coloration with increasing Hg²⁺ concentration and fitted a linear equation of y = 0.02454x + 0.05461 with R ² = 0.982, reasonably showing different slopes (Figure A). Using portable equipment imaging to extract RGB values (Figure B), a linear relationship was established between Lg­[(G + B)/R] and Hg²⁺ concentration, which was fitted to y = 0.02001x + 0.021234 with R ² = 0.993 (Figure C).

4.

(A) Fitting curve of the absorption peak at 652 nm for the Qu-CD-AgNPs hydrogel; (B) correlation between RGB value from 3 to 100 μM; (C) linear correlation between Hg²⁺ concentration and logarithmic RGB values.

According to the above equation based on RGB values, the feasibility of the hydrogel test kit was evaluated using oyster, clam, and shrimp by determining Hg²⁺ recoveries via a spike and recovery protocol. For oyster samples (Figure A,B), the linear regression was y = 0.0215x + 0.25484 over 3–100 μM with R ² = 0.990; for shrimp (Figure C,D), y = 0.02006x + 0.26463 over 3–100 μM with R ² = 0.998; and for clam samples (Figure E,F), y = 0.02239x + 0.20186 over 3–100 μM with R ² = 0.992. Moreover, the results show spiked recoveries of 95.5–105.2% with relative standard deviations of 0.26–1.75% (Table ). Additionally, storage stability testing of the hydrogel-based sensor was conducted, which revealed that the Hg²⁺-sensing response retained around 97.8% of its initial absorbance after 15 days of storage at 4 °C, indicating satisfactory storage stability and repeatability (Table S2).

5.

RGB analysis of aquatic products; (A) correlation between RGB values for the oyster sample; (B) linear correlation between Hg²⁺ concentration and logarithmic RGB values for the oyster sample; (C) correlation between RGB values for the shrimp sample; (D) linear correlation between Hg²⁺ concentration and logarithmic RGB values for the shrimp sample; (E) correlation between RGB values for the clam sample; (F) linear correlation between Hg²⁺ concentration and logarithmic RGB values for the clam sample.

1. Detection of Hg²⁺ in Aquatic Products by the Spiked Recovery Method.

samples	spiked concentration (μM)	found (μM)	recovery (%)	relative standard deviation, RSD (%, n = 3)
Oyster	20	20.07	100.30	1.75
	40	39.33	98.33	0.53
	60	59.50	99.17	1.27
Shrimp	20	19.10	95.5	1.05
	40	40.03	100.08	0.52
	60	60.43	100.72	0.34
Clam	20	21.03	105.2	0.73
	40	40.57	101.42	0.62
	60	58.34	97.28	0.26

In this case, these findings verify that the hydrogel kit reliably quantifies Hg²⁺ in real samples, and the colorimetric method relying on the Hg²⁺-enhanced oxidase-like activity of Qu-AgNPs is feasible and robust, with satisfactory accuracy and precision, holding broad application prospects.

4. Conclusion

In this study, a neotype oxidase-like Qu-CD-AgNP was successfully prepared, and a Qu-CD-AgNP + TMB + Hg²⁺ colorimetric detection system was established to identify its discoloration mechanism. This platform exhibits high sensitivity, high selectivity, and excellent anti-interference capability for the determination of Hg²⁺. On this basis, a convenient hydrogel-based sensor coupled with portable equipment-enabled quantitative analysis provides a simple and efficient solution for the rapid detection of Hg²⁺ in aquatic products. Overall, we believe that our work plays a valuable role in convenient visual detection of Hg²⁺ and will promise broad application prospects in water-quality monitoring and the inspection of aquatic products.

Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.6c05867.

• Qu-CD-AgNPs optimization of sensing conditions; stability of Hg²⁺ colorimetric detection of the freeze-dried Qu-CD-AgNPs powder over time; preparation repeatability of the nanozyme; interference from organic matter; comparison of features between reported nanozyme-based Hg²⁺ sensors; stability of the hydrogel kit; calculation of Michaelis–Menten kinetic parameters; and calculation of the LOD (PDF)

• Operation of the self-developed RGB analysis software for quantitative determination of Hg²⁺ concentration (MP4)

Qinghao Ge: data curation, methodology and writing original draft. Changzhuo Zheng: editing, data curation and formal analysis. Chaoqun Hou: validation, editing and data curation. Xinyan Zhao: editing and data curation. Jun Chen: editing. Xiaolong Han: editing and funding acquisition. Juan Ding: writing review, editing and supervision. Yang Pu: editing, review and funding acquisition. Qinghao Ge and Changzhuo Zheng are co-first authors.

This work was supported by The Shandong Provincial Natural Science Foundation (grant number ZR2022MC116), Shandong provincial education system government-sponsored study abroad program (2023) and Ludong University research initiation funds (ly2014041).

The authors declare no competing financial interest.