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. 2026 Apr 6;35:103830. doi: 10.1016/j.fochx.2026.103830

Trypsin-functionalized Ag—Au nanoflowers for enhanced sensing of hydrogen sulfide in diverse foods: dual mechanisms of steric hindrance-mediated biothiols exclusion and silver-driven 30-fold cascade amplification

Hongyu Chen a,b,⁎, Yafang Wang a, Xinrui Sun a, Nannan Wang a, Wenping Zhu a, Manman Sun a, Qingfeng Li a, Yahong Chen a
PMCID: PMC13091321  PMID: 42006642

Abstract

Combining the excellent optical properties, spatial structure specificity and 30-fold cascade signal amplification strategy of the trypsin-stabilized silver‑gold nanoflowers (Try-AgAuNFs), a novel nanosensing system was constructed for improving the sensing of hydrogen sulfide (H2S). It is worth noting that the high loading capacity of Ag can be used both for the design of the cascade signal amplification strategy, and to effectively increase the active sites of the probe, making it suitable for real-time detection of H2S. Excitingly, the Try on the surface of Try-AgAuNFs increases the spatial steric hindrance of the probe, effectively preventing biothiols and other amino acids from interacting with Try-AgAuNFs, thereby enabling reliable H2S detection and food-freshness evaluation across diverse samples. This work not only presents a novel and highly stable method for synthesising Try-AgAuNFs, but also reveals that surface-bound protein ligands can enhance probe selectivity through steric hindrance control.

Keywords: Trypsin-stabilized silver‑gold nanoflowers, Cascade signal amplification strategy, Spatial structure specificity, Synergistic effect, Real-time H2S sensing

Highlights

  • •

    The novel trypsin-stabilized silver‑gold nanoflowers (Try-AgAuNFs) were prepared by ultrafast synthesis method.

  • •

    A novel hydrogen sulfide (H2S) nanosensing system was designed using Try-AgAuNFs as the signal transduction medium.

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    The system was based on spatial structure specificity and 30-fold cascade signal amplification strategy.

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    The 30-fold cascade signal amplification strategy makes the system superior to the routine method in sensitivity.

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    The proposed method has been applied to the analysis of H2S in multi-category food samples.

1. Introduction

Hydrogen sulfide (H2S) is commonly found in various foods, particularly those rich in proteins and amino acids, such as eggs, fish, and chicken. H2S undergoes enzymatic decomposition or thermal decomposition in foods to form cysteine or peptides containing cysteine. Elevated levels of H2S both damage food flavor and exert toxic effects on the human body, potentially leading to severe poisoning or even death. Meanwhile, H2S is also recognized as the third endogenous gaseous signaling molecule after nitric oxide (NO) and carbon monoxide (CO) and it is characterized by its distinctive rotten-egg odor (Zhang et al., 2025). It plays a crucial role in various physiological processes, including cell apoptosis, inflammation, vasodilation, ischemia-reperfusion injury, and neural regulation (Huang et al., 2022; Myszkowska et al., 2021; Sun et al., 2025; Suzuki et al., 2021). However, abnormal H2S levels are associated with a series of diseases, including diabetes, hypertension, liver cirrhosis, acute kidney injury, Alzheimer's disease, Down syndrome, and cancer (Brglez, 2021; Disbrow et al., 2021; Pei et al., 2025; Zhang et al., 2021). As such, developing rapid, highly sensitive, and selective methods for the quantitative detection of H2S is of great significance for ensuring food safety and protecting public health.

A variety of methods have been developed for H2S detection, including gas chromatography (Yang et al., 2026), high-performance liquid chromatography (Koike et al., 2017; Shi et al., 2021), surface-enhanced Raman scattering (Chen et al., 2024; Nie et al., 2025), electrochemical analysis (Chen et al., 2025; Kim et al., 2025; Ranjana et al., 2025; Shang et al., 2025; Zhu et al., 2025), and optical methods (Chen & Krishna, 2025; Jia et al., 2025; Li et al., 2025; Shen et al., 2025; Tu et al., 2025; Xie et al., 2025). Among these, the optical methods, especially the colorimetric methods, have attracted great attention from researchers as a result of their simplicity, ease of operation, and convenience. Colorimetric detection of H2S typically relies on its strong reducing ability and its high affinity toward metal-based nanomaterials. For example, Wang and co-workers developed a novel colorimetric nanosensor utilizing the unique optical properties of gold nanorods, where H2S triggers the production of I2 from IO3−, leading to etching of the nanorods (Su et al., 2025). While this sensor exhibits a rapid response time (15 min), its specificity toward H2S is limited, as other strong reducing agents (e.g., ascorbic acid (AA), glutathione (GSH)) in complex samples can also react with IO3− to generate I2, resulting in signal interference. Huang's research group designed a new colorimetric nanosensing platform based on the cerium-diacetyl ferrocene dual-metal framework (FcCe-MOF-NH2) and the strong redox reaction between H2O2 and H2S (Liu et al., 2024). Despite exhibiting a wide linear response range, this approach still lacks specificity for the presence of strong reducing substances (such as AA, GSH, etc.). Yin's research group constructed a new H2S colorimetric nanosensor based on the excellent optical properties of Au/AgI dimeric nanoparticles (Au/AgI NPs) and the strong interaction between H2S and AgI (Zeng et al., 2018). Similarly, Huang's research group developed a new H2S colorimetric nanosensing method based on the excellent peroxidase-like activity of platinum nanoparticles (Pt NPs) and the strong interaction between H2S and Pt NPs (Liang et al., 2024). Shang's team designed a colorimetric sensing platform based on copper-doped indium-based metal-organic frameworks (Cu/In-MOFs), leveraging both their peroxidase-like activity and high reactivity toward H2S (Shang et al., 2024). Although these systems demonstrate promising sensitivity and practicality, their detection mechanisms rely primarily on the strong interaction between S2− and metal-based materials, leading to cross-reactivity with biothiols such as cysteine (Cys), homocysteine (Hcy), and GSH (Halawa et al., 2025; Xiao et al., 2015). Moreover, most of these sensors detect only conventional colorimetric signal changes, limiting their overall sensitivity. As such, designing nanomaterials capable of eliminating interference from reducing agents and endogenous biological sulfides would substantially enhance the specificity of H2S detection. Furthermore, integrating signal amplification strategies and enabling real-time response capabilities could substantially improve both the sensitivity and timeliness of H2S sensing.

Gold nanoflowers possess high extinction coefficient, multiple reactive sites and strong surface plasmon resonance absorption properties in the long wavelength region, which make them suitable for the construction of colorimetric sensing nanoprobes. In general, two major strategies are employed for the synthesis of gold nanoflowers, namely the seed-mediated method (Li et al., 2022; Zhao et al., 2009) and the one-step synthesis method (Heli & Rahi, 2016). While the one-step synthesis method is simple and convenient, it often suffers from poor control over particle dispersion. In contrast, the seed-mediated approach produces gold nanoflowers (Se-AuNFs) by depositing small gold nanocrystals onto preformed gold seeds, allowing better control of morphology and uniformity. Despite better controlling the dispersion of Se-AuNFs, it requires the pre-synthesis of certain-sized gold nanomaterials as gold seeds, thereby increasing procedural complexity (Boca et al., 2010). Notably, gold nanoflowers obtained via both approaches typically possess exposed active sites that are readily affected by biothiols in complex matrices, which severely limits their practical applicability. As such, it is of great significance to prepare new metal-based nanoflowers with good dispersion, template protection, high stability, and suitable for applications in complex real samples.

Herein, leveraging a 30-fold cascaded signal amplification strategy, the unique structure of trypsin-stabilized silver‑gold nanoflowers (Try-AgAuNFs), and the synergistic formation of strong Au—S and Ag—S bonds, we developed a novel colorimetric nanosensing platform for enhanced H2S detection (Scheme 1). The prepared Try-AgAuNFs exhibit strong surface plasmon resonance properties, high Ag loading capacity, and high stability, making them suitable for the detection of H2S in complex samples. Compared with try-stabilized gold nanoflowers (Try-AuNFs), the Ag-enriched surface of Try-AgAuNFs provides enhanced sensitivity toward H2S. Upon exposure to H2S, the material undergoes sequential etching through strong Ag—S and Au—S interactions, leading to pronounced changes in its absorption signal, achieving a 30-fold cascaded signal amplification effect, which significantly improves the overall sensitivity of H2S detection. In addition, compared with Try-AuNFs, Try-AgAuNFs enable real-time detection of H2S through the strong synergistic interaction of Ag—S and Au—S bonds. In particular, it is worth noting that Try-AgAuNFs can effectively address the interference arising from biothiols (such as Cys, Hcy, GSH) as Try-AgAuNFs that are synthesized by using Try as a template have a large steric hindrance, which prevents biothiols and other natural amino acids from approaching the surface of Try-AgAuNFs and interacting with them, significantly enhancing the selectivity of the sensing system. Meanwhile, UV–Vis, TEM, and XPS were employed to elucidate the etching mechanism of Try-AgAuNFs for the first time, which is governed by their steric hindrance and the H2S-triggered cascade signal amplification strategy, thereby providing new insights into H2S-based sensing applications.

Scheme 1.

Scheme 1

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Schematic representation of the ultrafast synthesis of Try-AgAuNFs (A). The sensing mechanism of Try-AgAuNFs toward H2S based on 30-fold cascade signal amplification and Try-AgAuNFs-mediated structure-modulation strategy (B).

2. Experimental section

2.1. Reagents and instruments

Threonine, tyrosine, serine, arginine, glycine, histidine, Cys, Hcy, GSH were purchased from Sigma-Aldrich (Shanghai, China). Xanthine, HAuCl4·3H2O, tannic acid, hydroxylamine hydrochloride (NH2OH·HCl), NaBH4, AA, dopamine (DA), Na2S were obtained from Aladdin (Shanghai, China). Urea, Na2HPO4, NaH2PO4, NaCl, KCl, MgCl2, CaCl2, etc. were purchased from Sinopharm Chemical Reagent Co., Ltd. Try was purchased from Beijing Bailingwei Technology Co., Ltd. The commercial assay kit for H2S was bought from Suzhou Grace Biotechnology Co., Ltd. (Suzhou, China). Different pH phosphate buffer solutions (PBS) were prepared using 10 mM Na2HPO4 and NaH2PO4. All chemical reagents can be directly used without further purification. Ultra-pure water (18.2 MΩ cm) was purified using the Millipore-Q system.

Transmission electron microscopy (TEM) images and high-resolution TEM (HRTEM) images were measured using a Talos F200X system (FEI, U.S.A.). Energy-dispersive X-ray spectroscopy elemental mapping of monometallic nanohybrids was obtained by high-angle annular dark-field scanning TEM (HAADF-STEM). Fourier transform infrared (FT-IR) spectra were collected using a Nicolet Nexus 670 infrared spectrometer (Nicolet Instrument Co., U.S.A.). The X-ray photoelectron spectroscopy (XPS) was conducted on the Thermo Scientific K-Alpha XPS system (Thermo Scientific, USA). The ultraviolet–visible absorption spectroscopy (UV–Vis) of Try-AgAuNFs was measured using the Lamda 35 spectrophotometer (Perkin-Elmer, USA).

2.2. The synthesis of H-AuNFs

H-AuNFs were successfully synthesized by a simple, ultra-fast and template-free method. Briefly, 5 mL of HAuCl4 solution (0.35 mM) was mixed with an appropriate amount of NH2OH·HCl (40 mM) and allowed to react for 3 min. The pH of the resulting solution was then adjusted to 9–10 using 1 M NaOH. The color of the solution changed from colorless to greenish-brown within seconds and remained stable, indicating the formation of H-AuNFs.

2.3. The preparation of try-AgAuNFs

Try-AgAuNFs were synthesized through a two-step procedure. In the first step, Try-AuNFs were prepared using a method previously developed by our group (Chen, Li, & Wu, 2025). Briefly, 5 mL of HAuCl4 solution (0.35 mM) was mixed with an appropriate volume of Try solution (2 mg/mL) at room temperature until homogeneous. After 5 min, an appropriate amount of NH2OH·HCl (40 mM) was added to the above solution. The pH value of the solution was adjusted to 9–10 using NaOH (1 M). Finally, the color of the solution changed from colorless to blue and remains stable, indicating the formation of Try-AuNFs. The concentration of Try-AuNFs was calculated based on the initial HAuCl4 content used in synthesis. In the second step, 2 mL of the as-prepared Try-AuNFs were mixed with an appropriate amount of AgNO3 solution (20 mM) at room temperature (25 ± 1 °C) until uniformly dispersed. After 10 min, 10 μL of NH2OH·HCl (25 mM) was added to the above solution. The solution turned deep blue within 30 s and remains stable. Finally, the obtained Try-AgAuNFs solution was stored in the refrigerator (4 °C) for further use.

2.4. Construction of the H2S sensing platform based on the cascaded signal amplification strategy

The high-specificity H2S sensing platform was constructed based on Try-AgAuNFs as follows. A series of H2S solutions with varying concentrations (20 μL each) were mixed with PBS buffer (20 mM, pH 9.0) containing Try-AgAuNFs (0.35 mM) to a final volume of 400 μL. The resulting mixtures were analyzed by UV–Vis spectroscopy, and each measurement was performed in triplicate to ensure reproducibility. The selectivity of the sensing system toward H2S was evaluated by replacing H2S with other ions or biological molecules (Mg2+, Na+, Zn2+, K+, Ca2+, glucose, urea, xanthine, AA, histidine, arginine, glycine, threonine, Cys, Hcy, GSH).

2.5. Preparation and analysis of multi-category food samples

Fresh lean pork, prawn, eggs, tofu, apples, dragon fruit, onions, and Tsingtao beer were purchased from Wanguoyuan Supermarket (Zhoukou, China). The pork samples were processed as follows. Fresh pork was divided into 50 g portions, sealed individually, and stored at 4 °C for 0, 1, 3, 6, or 8 d. At each designated time point, 20 mL of ultrapure water was added directly to the sample, and the mixture was rapidly homogenized in an ice-water bath to fully release H2S from the tissue. The homogenate was then centrifuged at 10,000 rpm for 15 min at 4 °C, and the supernatant was filtered through a 0.22 μm aqueous filter membrane. The filtrate was immediately used for H2S detection. The prawn samples were processed in a similar manner. Briefly, 50 g of the edible portion of the prawn was accurately weighed, mixed with 20 mL of ultrapure water, and rapidly homogenized in an ice-water bath. The homogenate was centrifuged at 10,000 rpm for 15 min at 4 °C, and the supernatant was filtered through a 0.22 μm aqueous filter membrane. The resulting filtrate was immediately used for H2S measurement. The egg samples were processed as follows. Fresh eggs were boiled in water for 10 min until fully cooked, allowed to cool, and then shelled. The yolks were removed, and only the egg whites were retained. A 50 g portion of egg white was accurately weighed, mixed with 20 mL of ultrapure water, and rapidly homogenized in an ice-water bath. The homogenate was centrifuged at 10,000 rpm for 15 min at 4 °C, and the supernatant was filtered through a 0.22 μm aqueous membrane filter. The filtrate was immediately used for H2S measurement. For tofu samples, 50 g of tofu was accurately weighed, mixed with 20 mL of ultrapure water, and rapidly homogenized in an ice-water bath. After centrifugation at 10,000 rpm for 15 min at 4 °C, the supernatant was collected and filtered through a 0.22 μm aqueous membrane filter. The filtrate was immediately used for H2S detection. For apple samples, 50 g of fresh pulp was accurately weighed, mixed with 20 mL of ultrapure water, and rapidly homogenized in an ice-water bath. The homogenate was centrifuged at 10,000 rpm for 15 min at 4 °C, and the supernatant was filtered through a 0.22 μm aqueous membrane filter. The filtrate was immediately used for H2S determination. For dragon fruit samples, 50 g of fresh pulp was accurately weighed, mixed with 20 mL of ultrapure water, and rapidly homogenized in an ice-water bath. The homogenate was centrifuged at 10,000 rpm for 15 min at 4 °C, and the supernatant was filtered through a 0.22 μm aqueous membrane filter. The filtrate was immediately used for H₂S determination. For onion samples, 50 g of the fresh edible portion was accurately weighed, mixed with 20 mL of ultrapure water, and rapidly homogenized in an ice-water bath. The homogenate was centrifuged at 10,000 rpm for 15 min at 4 °C, and the supernatant was filtered through a 0.22 μm aqueous membrane filter. The filtrate was immediately used for H2S determination. For Tsingtao beer, an appropriate volume of the sample was directly filtered through a 0.22 μm aqueous membrane filter, and the filtrate was immediately used for H2S determination.

All the sample filtrates were measured under the same detection conditions, and the detection steps were consistent with the standard H2S detection steps in Section 2.4. Finally, 5 μM and 15 μM H2S standard solutions were added to each of the sample filtrates to check the recovery rate of the method. Each experiment was repeated three times.

3. Results and discussion

3.1. The preparation and characterization of try-AgAuNFs

In this work, Try-AuNFs were synthesized using Try as both a stabilizing template and a morphological inducer, with NH2OH·HCl serving as the reducing agent. Subsequently, NH2OH·HCl was used to further reduce AgNO3, allowing Ag to be loaded on the surface of Try-AuNFs. Through this two-step process, a novel protein-protected, acid-base-resistant (pH 5–14) nanocomposite, Try-AgAuNFs, was successfully prepared for the selective detection of H2S in complex real samples. To prove the successful synthesis of Try-AgAuNF under the optimal experimental conditions (Figs. S1-S2), the Try-AgAuNFs were characterized by using TEM, HRTEM, HAADF-STEM, XPS, and FT-IR. TEM images revealed that the Try-AgAuNFs exhibited a well-defined flower-like morphology (Fig. 1A) with good dispersion and uniform particle size (average diameter: 68.2 ± 4.6 nm) (Fig. S3). The HRTEM image further showed clearly resolved lattice fringes with an interplanar spacing of 0.234 nm, corresponding to the (111) plane of face-centered cubic gold (Fig. 1B) (Priecel et al., 2016). This further indicates the successful synthesis of Try-AgAuNFs. Additionally, the HAADF-STEM results clearly revealed the presence of Au, Ag, and S elements (Figs. 1D-F), indicating that Try (source of sulfur) was successfully incorporated as a stabilizing template during synthesis. XPS analysis was subsequently performed to further verify the elemental composition and oxidation states of Try-AgAuNFs. The XPS spectra of Try-AgAuNFs revealed distinct Au 4f peaks at 87.6 eV (Au 4f5/2) and 84.1 eV (Au 4f7/2), confirming that metallic Au0 is the dominant species (Fig. 1G). Similarly, the Ag 3d peaks at 374.3 eV (Ag 3d3/2) and 368.3 eV (Ag 3d5/2) indicate the predominance of metallic Ag0 (Fig. 1H). Additional peaks corresponding to C 1 s (285.0 eV), N 1 s (399.9 eV), O 1 s (532.2 eV), and S 2p (285.0 eV) (Fig. S4) further verify the successful incorporation of Try in the synthesis of Try-AgAuNFs. The FT-IR spectrum provided additional evidence of the interaction between Try and the nanostructure. As shown in Fig. 1I, the amide I band of Try shifted from 1647 cm−1 to 1655 cm−1 (Akavaram et al., 2021), indicating a conformational change in the protein backbone resulting from coordination between Try and Au. Collectively, these results confirm that Try-AgAuNFs were successfully synthesized, with Try effectively coating and stabilizing the nanoparticle surface.

Fig. 1.

Fig. 1

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TEM (A), HRTEM (B), HAADF-STEM images (C), and the corresponding elemental mappings (D, E, F) of Try-AgAuNFs. XPS high-resolution spectra of Au 4f (G), Ag 3d (H) of Try-AgAuNFs. (I) FT-IR spectrum of Try-AgAuNFs (black line) and Try (red line). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

The stability of Try-AgAuNFs under various temperature and pH conditions was systematically evaluated. As shown in Fig. S5A, the maximum absorption wavelength of Try-AgAuNFs remained nearly unchanged across temperatures ranging from 5 °C to 55 °C and pH values between 5.0 and 14.0, indicating excellent thermal and pH stability. Finally, the storage stability was further assessed at 4 °C. Over a 50-d period (Fig. S6), the maximum absorption wavelength of Try-AgAuNFs exhibited negligible variation, confirming their excellent long-term stability, which is essential for reliable biosensing applications.

3.2. Investigation into the response mechanism for H2S

As a result of the strong Au—S and Ag—S interactions and the protective Try layer on the Try-AgAuNF surface, the developed probe is well-suited for highly sensitive and selective real-time detection of H2S in complex samples. Given that biothiols such as Cys and Hcy are commonly present in food and may potentially interfere with H2S detection, a series of selectivity experiments were conducted to evaluate the probe's specific response. As shown in Fig. 2A, the UV–Vis spectra of Try-AgAuNFs in the presence of Cys or Hcy (curves b and c) were nearly identical to that of the original Try-AgAuNFs (curve a), indicating negligible interaction. However, when H2S was added to the Try-AgAuNFs solution, the UV absorption signal of Try-AgAuNFs changed significantly (curve d), confirming that H2S can be specifically recognized and detected by monitoring UV–Vis variations of Try-AgAuNFs. It is worth noting that unprotected H-AuNFs failed to achieve such specificity (Fig. 2B). As such, both Cys and Hcy interacted with the exposed gold surface, producing observable UV–Vis shifts. These findings suggest that the Try-protected Try-AgAuNFs exhibit superior selectivity toward H2S, and the enhanced steric hindrance is a critical strategy for achieving specific target recognition in complex environments.

Fig. 2.

Fig. 2

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(A) The UV–Vis absorption spectra of Try-AgAuNFs (a) treated with 30 μM Cys (b), 30 μM Hcy (c), 5 μM H2S (d) and 30 μM H2S (e). (B) The UV–Vis absorption spectra of H-AuNFs (a) treated with 30 μM GSH (b), 30 μM Cys (c) and 30 μM Hcy (d). (C) The UV–Vis absorption spectra of Try-AgAuNFs (a) treated with 30 μM penicillamine (b), 30 μM mercaptopropionic acid (c), 30 μM mercaptoethylamine (d). (D) The UV–Vis of Try-AgAuNFs (a) treated with 400 μM penicillamine (b), 400 μM mercaptopropionic acid (c), 400 μM mercaptoethylamine (d).

To elucidate the mechanism underlying the high selectivity of Try-AgAuNFs toward H2S, the effects of thiol-containing molecules with varying steric hindrance were investigated. Specifically, molecules with smaller steric hindrance than Hcy (such as mercaptopropionic acid) and molecules with larger steric hindrance than Hcy (such as penicillamine) were chosen to investigate their influence on the UV–Vis absorption of Try-AgAuNFs. As shown in Fig. 2C, neither penicillamine nor mercaptopropionic acid induced significant spectral changes, which might be attributable to their larger steric hindrance. Based on this, mercaptoethylamine with a smaller steric hindrance than mercaptopropionic acid was selected to react with Try-AgAuNFs. As shown in Fig. 2D, mercaptoethylamine could effectively interact with the Try-AgAuNFs probe, resulting in a noticeable UV–Vis response. These findings suggest that the steric protection provided by the surface-bound Try layer restricts access to the nanomaterial surface, allowing only small molecules such as H2S to approach and react via Au—S and Ag—S bond formation. All the above results indicate that the steric hindrance effect of the Try protective layer on the surface of Try-AgAuNFs enables the nanoprobes to be suitable for specific recognition of H2S in complex samples.

As Ag is loaded on the surface of Try-AgAuNFs, Try-AgAuNFs are an ideal candidate for constructing a cascaded signal amplification strategy, significantly enhancing both the sensitivity and efficiency of H2S detection. As shown in Fig. S7, compared with Try-AuNFs, Try-AgAuNFs can be used for real-time detection of H2S, indicating strong potential for practical applications. In addition, Try-AgAuNFs can achieve a 30-fold cascade signal amplification to detect H2S (Fig. S8), effectively improving the detection sensitivity. This is attributable to the high content of Ag loading on the surface of Try-AgAuNFs. Mechanistically, upon exposure to H2S, Try-AgAuNFs are initially etched via the formation of Ag—S bonds, resulting in a measurable change in their optical absorption. Subsequently, further etching occurs through Au—S bond formation, leading to an additional absorption shift. This sequential process constitutes an H2S-triggered cascade signal amplification mechanism, enabling highly sensitive and quantitative detection. To prove that the cascade signal amplification strategy can be achieved, the morphology of Try-AgAuNFs before and after the reaction was examined using TEM. When Try-AgAuNFs were analyzed in the absence of H2S, they maintained a good dispersion state and had a distinct flower-like structure (Fig. 1A). By contrast, upon exposure to H2S, both the flower-like structure and the size of Try-AgAuNFs significantly decreased (Figs. 3A-D), indicating that H2S effectively etched Try-AgAuNFs and achieved the cascade signal amplification strategy for detecting H2S. To further confirm that the cascade signal amplification strategy can be achieved, XPS analysis was conducted to examine the changes in the elemental composition of Ag and Au after the reaction between H2S and Try-AgAuNFs. As shown in Figs. 3E-F, the relative contents of Au and Ag decreased from 15.46% and 12.01% to 11.55% and 6.97%, respectively, following H2S exposure. This confirmed that H2S can trigger the cascade signal amplification strategy for etching Try-AgAuNFs through the Ag—S bond and Au—S bond interaction. Collectively, these findings demonstrate that the integration of the Try-derived steric hindrance effect and the cascade amplification mechanism enables highly selective, ultrasensitive, and real-time detection of H2S.

Fig. 3.

Fig. 3

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HAADF-STEM images (A) and the corresponding elemental mappings (B, C, D) of Try-AgAuNFs/H2S. XPS high-resolution spectra of Au 4f (E), Ag 3d (F) of Try-AgAuNFs/H2S.

3.3. Measurement of H2S

As shown in Fig. 4, under the optimal experimental conditions (Fig. S9), the absorbance of Try-AgAuNFs at 606 nm gradually decreased upon the increase in the concentration of H2S (0.25–30 μM), indicating that the decrease in absorbance was linearly related to the H2S concentration. The linear regression equation was A606 = 0.4153–0.0044 [H2S], with an excellent correlation coefficient (R2 = 0.9959) (Fig. 4B). In addition, the sensing system exhibited a distinct color change from dark blue to light blue (inset of Fig. 4B), enabling rapid visual identification of H2S concentrations. The detection limit of this method was determined to be 0.0789, which is lower than the detection limits of most reported methods (Table S1). Such a low detection limit can be attributed to the 30-fold cascade signal amplification strategy, the strong synergy of Au—S bonds and Ag—S bonds, the high stability of the material, and the high loading of Ag.

Fig. 4.

Fig. 4

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The UV–Vis absorption spectra (A) of the sensing system with different concentrations of H2S (from 0 to 30: 0, 0.25, 2.5, 5, 10, 20, 30 μM). (B) The corresponding calibration curve for probing H2S. [Try-AgAuNFs]: 0.35 mM.

3.4. Selective investigation

To evaluate the selectivity of the proposed method for H2S detection, its response toward various potentially interfering species commonly found in complex food samples was investigated under identical conditions. As shown in Fig. 5A, the change in absorbance caused by H2S was significantly higher than that of other ions and biological molecules, indicating that this method has high selectivity for H2S detection. It is worth noting that Cys and Hcy did not produce significant variations in the UV–Vis absorption of Try-AgAuNFs, which can be attributed to the strong steric hindrance conferred by the Try protective layer. Such steric hindrance prevents molecules with larger steric hindrance than H2S (such as Cys, Hcy) from approaching the surface of Try-AgAuNFs, thereby failing to result in changes in the ultraviolet absorption signal of Try-AgAuNFs. To validate the role of steric hindrance, we provided two lines of evidence showing that steric hindrance is responsible for the specific recognition of H2S by the Try-AgAuNFs probe. Firstly, from the perspective of material comparison, the protein-coated Try-AgAuNFs exhibited good selectivity toward H2S, with no obvious response to Cys or Hcy. In contrast, in the absence of protein, H-AuNFs showed clear interactions with both Cys and Hcy. This control experiment (Fig. 2A and Fig. 2B) indicates that try significantly increased the steric hindrance of the Try-AgAuNFs probe, thereby effectively improving its specific recognition of H2S. Secondly, from the molecular structure perspective, we further investigated the interactions between Try-AgAuNFs and a series of thiol-containing small molecules with different degrees of steric hindrance, including mercaptoethylamine, mercaptopropionic acid and penicillamine. The results shown in Fig. 2D revealed that only mercaptoethylamine, a relatively small molecular size, was able to interact with Try-AgAuNFs, whereas the bulkier thiol compounds, such as mercaptopropionic acid and penicillamine, did not show observable interactions with the probe. Collectively, these results indicate that the steric hindrance effect is the key factor enabling the high selectivity of Try-AgAuNFs toward H2S.

Fig. 5.

Fig. 5

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(A) The selectivity of the nanosensing system for detection of H2S (r: 30 μM) over relevant substances: blank (a), 300 μM Mg2+ (b), 300 μM Na+ (c), 300 μM Zn2+ (d), 300 μM K+ (e), 300 μM Ca2+ (f), 300 μM glucose (g), 300 μM urea (h), 300 μM xanthine (i), 300 μM AA (j), 300 μM histidine (k), 300 μM arginine (l), 300 μM glycine (m), 300 μM threonine (n), 300 μM Cys (o), 300 μM Hcy (p), 300 μM GSH (q). (B) Determination of H2S in raw pork samples over time with 8-fold dilution.

3.5. Analysis of multi-category food samples

During storage, raw pork undergoes gradual deterioration, and sulfur-containing proteins in the spoiled meat decompose to produce H2S. To evaluate the practical applicability of the developed sensing platform, Try-AgAuNFs were employed to monitor H2S generation in raw pork over different storage periods (1, 3, 6, and 8 d), aiming to achieve rapid evaluation of meat freshness. As shown in Fig. 5B, the absorbance of the pork samples stored for 1 d decreased slightly, indicating that only a small amount of H2S had formed at this stage. As the storage time increased from 1 to 6 d, the absorbance showed a substantial decline, suggesting continuous accumulation of H2S during pork spoilage and indicating that the samples had undergone obvious deterioration. After 8 d of storage, however, the absorbance increased relative to that at 6 d, indicating a decrease in H2S content, which may be attributable to changes in the microbial community during advanced spoilage, leading to further metabolic transformation or consumption of H2S. These results indicate that the Try-AgAuNFs nanoprobe developed in this study has good practical potential and can be effectively applied to the evaluation of fresh pork quality. To further evaluate the practicality of this method for H2S detection, the standard addition method was applied to determine H2S in pork (stored for 8 d), prawns, eggs, tofu, apples, dragon fruit, onions, and Tsingtao beer (Table 1). The recovery rate of H2S was 92.47–109.20%, and the RSD was 0.05–1.33%. To confirm the accuracy of this method, the concentrations of H2S in complex food matrices were measured using a commercial H2S detection kit as a reference. The results obtained with the developed nano-sensing system were consistent with those from the commercial kit (Table 1), indicating its effectiveness for H2S detection in complex food samples.

Table 1.

Determination of H2S in multiple food types.

Samples Added
(μM)		 Found
(μM)		 Recovery
(%)		 RSD
(n = 3, %)		 Found by H2S
assay kit (μM)
Pork – 4.32 – 0.55 4.51
5.00 9.32 100.00 0.23 9.38
15.00 19.31 99.95 1.27 19.21
Prawn – 3.18 – 1.28 3.02
5.00 8.18 100.00 0.49 7.81
15.00 19.09 105.01 0.84 18.23
Egg – – – 0.05 –
5.00 4.68 93.60 0.11 4.91
15.00 14.62 97.47 0.37 15.12
Tofu – – – 1.07 –
5.00 5.23 104.60 0.28 4.93
15.00 15.23 101.53 1.11 14.89
Apple – – – 1.12 –
5.00 5.45 109.00 0.50 5.12
15.00 14.77 98.47 1.11 15.03
Dragon fruit – – – 0.52 –
5.00 5.23 104.60 0.14 4.89
15.00 15.23 101.53 0.55 14.91
Onion – – – 1.33 –
5.00 5.23 104.60 0.64 5.13
15.00 13.87 92.47 0.86 14.81
Beer – – – 0.58 –
5.00 5.46 109.20 0.24 5.05
15.00 14.32 95.47 0.67 15.06
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4. Conclusion

In summary, a novel nano sensing system was constructed for improving the selectivity, sensitivity, and rapid detection of H2S based on the cascaded signal amplification strategy, the unique spatial structure of Try-AgAuNFs, and the synergistic effect of strong Au—S bonds and Ag—S bonds. This analytical system has four outstanding advantages. Firstly, the Try-AgAuNFs exhibit excellent stability and structural specificity, effectively preventing interference from biothiols and, thereby, enhancing detection selectivity. Secondly, the high Ag loading enables a 30-fold cascade signal amplification, substantially improving sensitivity. Thirdly, the dual Au—S and Ag—S bonding interactions enhance response, enabling real-time H2S monitoring. This work reports, for the first time, a detailed investigation of the H2S-triggered etching mechanism of Try-AgAuNFs based on a 30-fold cascaded signal amplification strategy using UV–Vis spectroscopy, TEM imaging, and XPS analysis. More importantly, this work establishes both a new approach for fabricating highly stable, protein-protected Ag—Au nanostructures and a mechanistic framework linking protein steric hindrance with cascade signal amplification for selective etching. Additionally, this method shows satisfactory performance for the assessment of the freshness of complex food samples, indicating its great potential in the field of food safety.

CRediT authorship contribution statement

Hongyu Chen: Writing – original draft, Methodology, Funding acquisition, Data curation, Conceptualization, Writing – review & editing. Yafang Wang: Visualization, Validation, Software, Methodology, Formal analysis. Xinrui Sun: Visualization, Validation, Software, Formal analysis. Nannan Wang: Visualization, Funding acquisition, Formal analysis. Wenping Zhu: Visualization, Funding acquisition, Formal analysis, Conceptualization. Manman Sun: Visualization, Funding acquisition, Formal analysis, Data curation. Qingfeng Li: Project administration, Investigation, Funding acquisition, Formal analysis. Yahong Chen: Visualization, Formal analysis, Data curation.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (12305076), Natural Science Foundation of Henan Province (232300420396, 252300421736), Henan Provincial Science and Technology Research and Development Joint Fund (Industrial) (245101610092), Key Scientific and Technological Project of Henan Province (262102321123), Key Research Projects of Henan Higher Education Institutions (25A150038) and Young and Middle-aged Backbone Teachers Plan of Zhoukou Normal University.

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.fochx.2026.103830.

Appendix A. Supplementary data

Supplementary material

mmc1.docx (1.1MB, docx)

Data availability

Data will be made available on request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary material

mmc1.docx (1.1MB, docx)

Data Availability Statement

Data will be made available on request.

Articles from Food Chemistry: X are provided here courtesy of Elsevier

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