Polydopamine-Coated Co3O4 Nanoparticles as an Efficient Catalase Mimic for Fluorescent Detection of Sulfide Ion

Surface engineering of nanozymes has been recognized as a potent strategy to improve their catalytic activity and specificity. We synthesized polydopamine-coated Co3O4 nanoparticles (PDA@Co3O4 NPs) through simple dopamine-induced self-assembly and demonstrated that these NPs exhibit catalase-like activity by decomposing H2O2 into oxygen and water. The activity of PDA@Co3O4 NPs was approximately fourfold higher than that of Co3O4 NPs without PDA, possibly due to the additional radical scavenging activity of the PDA shell. In addition, PDA@Co3O4 NPs were more stable than natural catalase under a wide range of pH, temperature, and storage time conditions. Upon the addition of a sample containing sulfide ion, the activity of PDA@Co3O4 NPs was significantly inhibited, possibly because of increased mass transfer limitations via the absorption of the sulfide ion on the PDA@Co3O4 NP surface, along with NP aggregation which reduced their surface area. The reduced catalase-like activity was used to determine the levels of sulfide ion by measuring the increased fluorescence of the oxidized terephthalic acid, generated from the added H2O2. Using this strategy, the target sulfide ion was sensitively determined to a lower limit of 4.3 µM and dynamic linear range of up to 200 µM. The fluorescence-based sulfide ion assay based on PDA@Co3O4 NPs was highly precise when applied to real tap water samples, validating its potential for conveniently monitoring toxic elements in the environment.


Introduction
Sulfide ions (S 2− ), which are among the most harmful contaminants, are extensively released into aqueous environments through various agricultural and industrial processes. These ions can exist in the human body and take part in the antioxidant process in liver and lung, or act as vasodilators [1][2][3]. Importantly, an imbalance in S 2− levels has been implicated in various diseases such as Alzheimer's disease, Down's syndrome, hyperglycemia, and liver cirrhosis [4][5][6][7]. Abnormally high levels of S 2− can directly threaten both the ecological environment and human health. To date, many methods for detecting S 2− have been developed, such as gas chromatography [8,9], titration [10], extraction [11], colorimetric [12,13], electrochemical [14], and fluorometric assays [15]. However, these methods are often time-consuming because of the sample pre/post treatments required, involved complicated assay procedures, and need for qualified operators [16]. Thus, more rapid, convenient, selective, sensitive, and reliable analytical methods for S 2− detection are urgently needed.
Catalase, which is commonly found in most aerobic organisms, plays a crucial role in protecting cells against oxidative damage, by decomposing H 2 O 2 into non-harmful O 2 and H 2 O [17]. Similar to other natural enzymes, catalase is unstable under harsh conditions, is costly to produce and purify, and is difficult to recycle. To overcome these limitations, studies aimed at developing an appropriate catalase mimic from nanomaterials exhibiting enzyme-like activities (nanozymes) have gained attention. To date, several types of catalasemimicking nanozymes have been reported, such as cerium oxide nanoparticles [18], iron oxide NPs [19], and cobalt oxide (Co 3 O 4 ) NPs [20,21]. Among these, Co 3 O 4 NPs showed morphology-dependent catalase-like activity; however, few studies have reported their surface engineering, which is an efficient approach for engineering nanozymes with improved activity [22,23].
Herein, we developed polydopamine (PDA)-coated Co 3 O 4 NPs (PDA@Co 3 O 4 NPs) as highly active catalase-mimicking nanozymes and applied these NPs in fluorescent detection of S 2− . PDA can be produced through self-polymerization of dopamine (DA) without the involvement of any organic solvent, yielding unique adhesion properties based on its active catechol and alkane groups, to facilitate substrate adsorption and product desorption in diverse reactions [24][25][26]. The synthesized PDA@Co 3 O 4 NPs showed high catalaselike activity in degrading H 2 O 2 , which inhibited the formation of extremely fluorescent 2-hydroxy terephthalic acid, to decrease the fluorescent signal. Interestingly, S 2− in the sample may interact with PDA@Co 3 O 4 NPs, resulting in decreased catalase-like activity via increased substrate transfer limitations and a decreased available surface area for catalytic events. We detected the target S 2− in a PDA@Co 3 O 4 NPs-based fluorescent assay and investigated various analytical characteristics, such as selectivity, sensitivity, stability, and practical utility along with the detection precision.

Synthesis and Characterization of PDA@Co 3 O 4 NPs
PDA@Co 3 O 4 NPs were synthesized following a previously reported method for DAinduced self-assembly with some modifications [25]. First, 250 mg CoSO 4 ·7H 2 O was dissolved in 50 mL of Tris-HCl buffer (pH 8.5) at 25 • C. DA (1 mg/mL) was added to the solution, followed by sonication at a frequency of 40 kHz and power of 160 W for 20 min. Next, 2 mL of 50 mM NaBH 4 was added, and the mixture was incubated at 60 • C for 4 h. The pellet was collected by a centrifugation (8000× g, 8 min), followed by freeze-drying to obtain the resulting black powders. Bare Co 3 O 4 NPs without a PDA shell were synthesized using the same procedures except that H 2 O was added instead of DA.
The synthesized materials were analyzed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM), using a field emission scanning electron microscope (Hitachi S-4700, Tokyo, Japan) and transmission electron microscope (FEI Tecnai, OR), respectively. Elemental composition was analyzed using an energy-dispersive spectrometer (EDX) (Bruker, Billerica, MA, U.S.A.). For SEM analyses, the suspension of sonicated NPs was dried on a silicon wafer. For TEM analyses, 5 µL of the suspension of sonicated NPs was dropped onto a carbon-coated copper TEM grid (Electron Microscopy Sciences, Hatfield, U.K.) followed by drying overnight at room temperature (RT). Fourier transform infrared (FT-IR) spectra of the NPs were obtained using an FT-IR spectrophotometer (FT/IR-4600, JASCO, Tokyo, Japan). X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) were conducted using an X-ray diffractometer (D/MAX-2500, Rigaku Corporation, Tokyo, Japan) and XPS reader (Gemini, Molecular Devices, Sunnyvale, CA, U.S.A.), respectively. The size distribution of the NPs was determined using dynamic light scattering (DLS) (Zetasizer, Malvern Instruments, Malvern, U.K.).

Evaluation of Enzyme-Like Activity of PDA@Co 3 O 4 NPs
Catalase-like activity of PDA@Co 3 O 4 NPs was examined by using TA as a fluorescent probe in the presence of H 2 O 2 . In a standard assay, PDA@Co 3 O 4 NPs or bare Co 3 O 4 NPs (both at 100 µg/mL), H 2 O 2 (20 mM), and TA (0.625 mM) were incubated in NaAc buffer solution (0.1 M, pH 6.0) for 20 min at RT under UV irradiation at 254 nm using a UV transilluminator (Core-Bio System, Seoul, Korea). The fluorescent signal was monitored using a microplate reader (Synergy H1, BioTek, Winooski, VT, U.S.A.) at excitation and emission wavelengths of 315 and 420 nm, respectively. The effects of the PDA@Co 3 O 4 NP concentration on their catalytic activity were examined following the same procedures but with varying concentrations of PDA@Co 3 O 4 NPs (0, 6.25, 12.5, 25, 50, 100, 150, 200, 250, and 300 µg/mL). The effects of the reaction pH and temperature on the catalytic activity of PDA@Co 3 O 4 NPs and natural free catalase were evaluated over wide pH (3.0-9.0) and temperature (4-80 • C) ranges. Stabilities of PDA@Co 3 O 4 NPs and free catalase were investigated by incubating them for 8 h in NaAc buffer at various pH values (3.0-10.0) at RT or temperatures (4-80 • C) at pH 6.0. The long-term stabilities of PDA@Co 3 O 4 NPs and free catalase were also evaluated by incubating them in NaAc buffer (pH 6.0) under static conditions at RT. The initial activities were determined by measuring the fluorescence intensity under standard assay conditions, and relative activity (%) was determined by calculating the ratio of residual to initial activity. Fluorescent images were acquired using a fluorescence imaging system (Kodak, Tokyo, Japan).
Steady-state kinetic parameters of the catalase-like activity of PDA@Co 3 O 4 NPs were determined based on oxygen production measured using a dissolved oxygen meter (Eutech DO 6+, Thermo Fisher Scientific, Waltham, MA, U.S.A.). Various concentrations of H 2 O 2 solution were added to NaAc buffer and continually stirred until the dissolved oxygen value was stable, followed by addition of PDA@Co 3 O 4 NPs (100 µg/mL).  O 4 NPs (both at 100 µg/mL) were added to NaAc buffer (0.1 M, pH 4.0) containing TMB (0.5 mM), followed by incubation for 5 min at RT. The blue color intensity was recorded at 652 nm using a microplate reader (Synergy H1). Oxidase (OXD)-like activities were measured following the same procedure as in the POD-assay but in the absence of H 2 O 2 . Superoxide dismutase (SOD)-like activities were evaluated by measuring the reduction in cytochrome C at 550 nm in the presence of superoxide radicals (O 2 •− ). Xanthine and xanthine oxidase (XO) were used as the source to generate O 2 •− . Briefly, PDA@Co 3 O 4 NPs (100 µg/mL) was added to phosphate buffer (0.05 m, pH 7.4) containing xanthine (0.05 mM), XO (0.15 mU/mL), and cytochrome C (0.01 mM), followed by incubation for 10 min at RT in the dark. The resultant solutions were centrifuged, and used to monitor the absorbance intensities at 550 nm using a microplate reader (Synergy H1).

Detection of S 2− Using PDA@Co 3 O 4 NPs
Detection of S 2− using the catalase-like activity of PDA@Co 3 O 4 NPs was performed as follows. First, aqueous solutions containing various concentrations of sodium sulfide were prepared as the S 2− source (0-600 µM). The S 2− sample solutions were added to an assay mixture containing PDA@Co 3 O 4 NPs (100 µg/mL), H 2 O 2 (20 mM), and TA (0.625 mM) in NaAc buffer solution (0.1 M, pH 6.0). The reaction mixture was incubated for 20 min at RT under UV irradiation (254 nm), and the resulting fluorescent signals were recorded as aforementioned. The limit of detection (LOD) values were calculated as 3 times of standard deviation (SD) of blank value over the slope of the calibration curve.
To demonstrate the practical utility of the proposed assay, real tap water samples were spiked with S 2− . For this assay, tap water samples were first collected from the laboratory and filtered through a syringe membrane with a pore size of 0.45 µm to eliminate undesirable molecules. Then, predetermined amounts of S 2− were added to the collected tap water samples to prepare spiked samples containing final S 2− concentrations of 50, 100, and 200 µM. Finally, the concentrations of S 2− in the spiked tap water samples were determined as described above. To measure the accuracy and reproducibility of the assay, we calculated the recovery rate (recovery rate (%) = measured value/actual value × 100) and the coefficient of variation (CV (%) = SD/average × 100), from the six independent assay results.

Synthesis of PDA@Co 3 O 4 NPs as an Efficient Catalase Mimic to Detect S 2−
The procedure used for DA-u of DA to form a PDS shell on the Co 3 O 4 NPs, would have enhanced catalase-like activity with help of additional radical scavenging activity of the PDA shell. Based on this enhanced activity, a highly sensitive system for S 2− detection was developed. The high catalase-like activity of PDA@Co 3  min at RT under UV irradiation (254 nm), and the resulting fluorescent signals were recorded as aforementioned. The limit of detection (LOD) values were calculated as 3 times of standard deviation (SD) of blank value over the slope of the calibration curve.
To demonstrate the practical utility of the proposed assay, real tap water samples were spiked with S 2− . For this assay, tap water samples were first collected from the laboratory and filtered through a syringe membrane with a pore size of 0.45 μm to eliminate undesirable molecules. Then, predetermined amounts of S 2− were added to the collected tap water samples to prepare spiked samples containing final S 2− concentrations of 50, 100, and 200 µ M. Finally, the concentrations of S 2− in the spiked tap water samples were determined as described above. To measure the accuracy and reproducibility of the assay, we calculated the recovery rate (recovery rate (%) = measured value/actual value × 100) and the coefficient of variation (CV (%) = SD/average × 100), from the six independent assay results.

Synthesis of PDA@Co3O4 NPs as an Efficient Catalase Mimic to Detect S 2−
The procedure used for DA-u of DA to form a PDS shell on the Co3O4 NPs, would have enhanced catalase-like activity with help of additional radical scavenging activity of the PDA shell. Based on this enhanced activity, a highly sensitive system for S 2− detection was developed. The high catalase-like activity of PDA@Co3O4 NPs induced inhibition of the formation of fluorescent 2-hydroxyl TA in the presence of H2O2, yielding a very low fluorescence background. Importantly, S 2− in the sample solution selectively interacted with the surface of PDA@Co3O4 NPs, resulting in a significant reduction in their catalaselike activity and concomitant increase in fluorescence via the facilitated formation of 2hydroxyl TA. Specifically, S 2− was predicted to be adsorbed on the surface of PDA@Co3O4 NPs, causing them to aggregate and leading to mass transfer limitations. These factors significantly reduced the catalase-like activity of PDA@Co3O4 NPs and increased the fluorescence signal proportionally to the amount of target S 2− (Figure 1).

Characterization of PDA@Co3O4 NPs
Structural characteristics of PDA@Co3O4 NPs were analyzed and compared with those of bare Co3O4 NPs without a PDA shell by TEM and SEM images. Bare Co3O4 NPs had spherical shape with 20.03 ± 2.82 nm diameter, calculated from their TEM images, and

Characterization of PDA@Co 3 O 4 NPs
Structural characteristics of PDA@Co 3 O 4 NPs were analyzed and compared with those of bare Co 3 O 4 NPs without a PDA shell by TEM and SEM images. Bare Co 3 O 4 NPs had spherical shape with 20.03 ± 2.82 nm diameter, calculated from their TEM images, and importantly, relatively thick (~15 nm) shells were clearly observed outside the core NPs from the PDA@Co 3 O 4 NPs (Figure 2a,b). In basic conditions, DA is known to easily interact with the surface of the NPs by a variety of interactions, including electrostatic interaction, metal coordination, and hydrogen bonding, and induce its polymerization among another DA monomers [25][26][27][28][29][30]. Thus, it was believed that the layer around the Co 3 O 4 NPs was PDA shell, as also clearly observed in the SEM images ( Figure S1 in Supplementary Materials). As the concentrations of DA increased, the thickness of polymeric shell concomitantly increased, which was similar to the previous studies ( Figure S2) [25,31]. High-resolution TEM (HRTEM) imaging and selected area electron diffraction imaging (SAED) demonstrated the presence of crystalline Co 3 O 4 in PDA@Co 3 O 4 NPs, which fit well with the reported data (JCPDS no. 76-1802) (Figure 2c,d). EDX images also proved the presence of Co, N, and O, which were well distributed throughout the material (Figure 2e). The elemental composition ratios within the PDA@Co 3 O 4 NPs are provided in Table S1. importantly, relatively thick (~15 nm) shells were clearly observed outside the core NPs from the PDA@Co3O4 NPs (Figure 2a,b). In basic conditions, DA is known to easily interact with the surface of the NPs by a variety of interactions, including electrostatic interaction, metal coordination, and hydrogen bonding, and induce its polymerization among another DA monomers [25][26][27][28][29][30]. Thus, it was believed that the layer around the Co3O4 NPs was PDA shell, as also clearly observed in the SEM images ( Figure S1 in Supplementary Materials). As the concentrations of DA increased, the thickness of polymeric shell concomitantly increased, which was similar to the previous studies ( Figure S2) [25,31]. High-resolution TEM (HRTEM) imaging and selected area electron diffraction imaging (SAED) demonstrated the presence of crystalline Co3O4 in PDA@Co3O4 NPs, which fit well with the reported data (JCPDS no. 76-1802) (Figure 2c,d). EDX images also proved the presence of Co, N, and O, which were well distributed throughout the material (Figure 2e). The elemental composition ratios within the PDA@Co3O4 NPs are provided in Table S1. XRD, FT-IR, and XPS analyses were additionally performed to characterize the synthesized PDA@Co3O4 NPs in detail. The XRD patterns clearly confirmed the presence of crystalline Co3O4, and the peaks of PDA@Co3O4 NPs kept nearly the same intensity compared with those of bare Co3O4 NPs, proving that the PDA layer does not negatively affect the crystalline structure of core Co3O4 NPs (Figure 3a). The FT-IR spectra confirmed the chemical structure of PDA@Co3O4 NPs, with the peaks corresponding to C-O stretch (1295 cm −1 ), N-H stretch (1510 cm −1 ), C-H stretch (around 3000 cm −1 ), and aromatic ring (1605 cm −1 ), which demonstrated the presence of the PDA layer on the surface of Co3O4 NPs (Figure 3b). The PDA peaks around 3400 cm −1 , which corresponded to the hydrogen bonds of O-H and N-H, were shifted in PDA@Co3O4 NPs, proving the interaction between Co3O4 NPs and catechol hydroxyl group of PDA [24]. Moreover, XPS analysis revealed peaks corresponding to C, N, O, and Co at 283.37, 398.23, 530.34, and 779.3 eV, respectively (Figure S3). The appearance of C, N, and O elements was attributed to the presence of PDA on the Co3O4 NP surface. In addition, the electronic configurations of the O and Co peaks supported the presence of Co3O4 NPs (Figure 3c,d) [31]. All these characterizations confirm that PDA@Co3O4 NPs were successfully formed by incorporation of a PDA layer on crystalline Co3O4 NPs.

Evaluation of the Catalase-Like Activity of PDA@Co3O4 NPs
Catalase-like activities of PDA@Co3O4 NPs and control Co3O4 NPs were evaluated via the decomposition of H2O2 by monitoring the changes in the fluorescent intensities of TA. In the absence of catalase mimics, H2O2 under UV irradiation produced hydroxyl radicals which further reacted with TA, generating highly fluorescent 2-hydroxy TA. PDA@Co3O4 NPs or bare Co3O4 NPs catalyzed the decomposition of H2O2 to H2O and O2, resulting in a decrease in the fluorescent signal, and importantly, PDA@Co3O4 NPs exhibited much higher activity, which was up to approximately fourfold higher than that of bare Co3O4 NPs (Figure 4a,b). The thickness of the PDA layer significantly affected the catalase-like activity of the materials (Figure 4b). PDA@Co3O4 NPs with 2 mg/mL DA (2-PDA@Co3O4 NPs) and PDA@Co3O4 NPs with 1 mg/mL DA (1-PDA@Co3O4 NPs) exhibited higher activity than that of PDA@Co3O4 NPs with 0.5 mg/mL DA (0.5-PDA@Co3O4 NPs), and the activity difference between 2-PDA@Co3O4 NPs and 1-PDA@Co3O4 NPs was not significant. Thus, 1-PDA@Co3O4 NPs were chosen and used for further studies. We also investigated the other oxidoreductases (POD, OXD, and SOD)-like activities of PDA@Co3O4 NPs and bare Co3O4 NPs ( Figure S4). Both PDA@Co3O4 NPs and bare Co3O4 NPs were unable to remove the O2 •− produced by the xanthine and XO reaction. In terms of POD-and OXD-like activity, the PDA@Co3O4 NPs, unlike Co3O4 NPs, could not oxidize TMB to produce blue-color product (oxidized TMB), which can be measured at 652 mm. It indicated that the developed PDA@Co3O4 NPs almost solely exhibited high catalase-like activity, which is beneficial for their utilization in catalase-mediated applications.  •− produced by the xanthine and XO reaction. In terms of POD-and OXD-like activity, the PDA@Co 3 O 4 NPs, unlike Co 3 O 4 NPs, could not oxidize TMB to produce blue-color product (oxidized TMB), which can be measured at 652 mm. It indicated that the developed PDA@Co 3 O 4 NPs almost solely exhibited high catalase-like activity, which is beneficial for their utilization in catalase-mediated applications. Several parameters affecting the activity, such as the concentrations of PDA@Co3O4 NPs, reaction pH, and temperature were examined to obtain the optimal reaction conditions ( Figure S5). With increasing concentrations of PDA@Co3O4 NPs, the fluorescence intensity gradually decreased, and 100 μg/mL of PDA@Co3O4 NPs was selected for further experiments ( Figure S5a). Similar to natural catalase, the activity of PDA@Co3O4 NPs was dependent on the reaction pH and temperature, and pH 6 and RT were found to be the optimal assay conditions ( Figure S5b,c). PDA@Co3O4 NPs showed high activity (over 60%) over broad pH and temperature ranges, whereas natural catalase did not show considerable activity (below 40%) under harsh conditions (acidic or basic pH, and high temperature over 60 °C). This difference may have resulted from the coated PDA layer, which shows additional catalase-like activity even under harsh reaction environments [32].
Under the optimized conditions, stabilities of PDA@Co3O4 NPs, depending on the pH, temperature, and storage time at RT, were assessed and compared with those of natural catalase. As expected, under all conditions, PDA@Co3O4 NPs clearly showed improved stabilities, maintaining over 90% of their initial activity, while natural catalase lost over half of its activity under harsh conditions (acidic pH below 4, temperature over 50 °C, and storage over 15 days) (Figure 4c-e). The clear improvement in the stability of PDA@Co3O4 NPs is beneficial for their practical applications.
Steady-state kinetic assays of PDA@Co3O4 NPs were performed to determine the Michaelis constant (Km) and maximal reaction velocity (Vmax), which are important to elucidate reaction mechanism [33]. According to the Michaelis-Menten curve obtained using different H2O2 concentrations and the corresponding Lineweaver-Burk plot, the kinetic parameters were calculated and compared with those of previously reported values from other Co3O4-based catalase mimics and natural catalase ( Figure S6 and Table S2). The Km value of PDA@Co3O4 NPs was 22.1 mM, which was over twofold lower than that of natural catalase and among the lowest values reported for Co3O4-based catalase mimics. These Several parameters affecting the activity, such as the concentrations of PDA@Co 3 O 4 NPs, reaction pH, and temperature were examined to obtain the optimal reaction conditions ( Figure S5). With increasing concentrations of PDA@Co 3 O 4 NPs, the fluorescence intensity gradually decreased, and 100 µg/mL of PDA@Co 3 O 4 NPs was selected for further experiments ( Figure S5a). Similar to natural catalase, the activity of PDA@Co 3 O 4 NPs was dependent on the reaction pH and temperature, and pH 6 and RT were found to be the optimal assay conditions (Figure S5b,c). PDA@Co 3 O 4 NPs showed high activity (over 60%) over broad pH and temperature ranges, whereas natural catalase did not show considerable activity (below 40%) under harsh conditions (acidic or basic pH, and high temperature over 60 • C). This difference may have resulted from the coated PDA layer, which shows additional catalase-like activity even under harsh reaction environments [32].
Under the optimized conditions, stabilities of PDA@Co 3 O 4 NPs, depending on the pH, temperature, and storage time at RT, were assessed and compared with those of natural catalase. As expected, under all conditions, PDA@Co 3 O 4 NPs clearly showed improved stabilities, maintaining over 90% of their initial activity, while natural catalase lost over half of its activity under harsh conditions (acidic pH below 4, temperature over 50 • C, and storage over 15 days) (Figure 4c-e). The clear improvement in the stability of PDA@Co 3 O 4 NPs is beneficial for their practical applications.
Steady-state kinetic assays of PDA@Co 3 O 4 NPs were performed to determine the Michaelis constant (K m ) and maximal reaction velocity (V max ), which are important to elucidate reaction mechanism [33]. According to the Michaelis-Menten curve obtained using different H 2 O 2 concentrations and the corresponding Lineweaver-Burk plot, the kinetic parameters were calculated and compared with those of previously reported values from other Co 3 O 4 -based catalase mimics and natural catalase ( Figure S6 and Table S2

Analytical Capabilities of PDA@Co 3 O 4 NPs for the Detection of Sulfide Ion
PDA@Co 3 O 4 NPs with enhanced catalase-like performances were utilized to fluorescently detect environmentally harmful S 2− . In the absence of S 2− , TA-mediated fluorescence was significantly decreased because of the high catalase-like activity of PDA@Co 3 O 4 NPs (Figure 5a). In the presence of S 2− , the fluorescence was clearly restored due to the significant reduction in the activity of PDA@Co 3 O 4 NPs. The S 2− -mediated reduction in activity may have occurred because of the interaction of S 2− on the PDA surface and subsequent inhibition of H 2 O 2 decomposition, resulting in an increased TA-mediated fluorescence signal. The PDA@Co 3 O 4 NPs-based S 2− biosensing system showed high selectivity for S 2− (50 µM), while diverse interfering compounds such as small molecules (glucose, urea), biothiols (glutathione, cysteine), and common ions (Mg 2+ , NH 4 + , Ca 2+ , Cl − ) did not have any considerable signal (below the threshold line), even at tenfold higher concentrations (Figure 5b), confirming that the biosensing strategy selectively detected target S 2− . On increasing the concentrations of S 2− , fluorescence intensity gradually increased ( Figure S7). From the analysis of dose-response curves, the LOD was calculated as low as 4.3 µM with the linear range up to 200 µM (Figure 5c,d), which is sufficient for practical S 2− biosensing in the field [34,35].
We speculated that the possible mechanism of the PDA@Co 3 O 4 NP-mediated S 2− biosensing system was that the target S 2− aggressively adsorbed on PDA@Co 3 O 4 NPs and caused substrate transfer limitations via their aggregation, yielding reduced catalase-like activity. To confirm this prediction, the size of the PDA@Co 3 O 4 NPs was determined in the presence and absence of S 2− ( Figure S8). The experiments clearly showed that the PDA@Co 3 O 4 NPs were aggregated in the presence of S 2− , leading to larger particle sizes (around 800-1000 nm), whereas non-aggregated PDA@Co 3 O 4 NPs were less than 500 nm in size. This aggregation may reduce the surface area of PDA@Co 3 O 4 NPs and, thus, reduce the response towards H 2 O 2 [36].
Finally, to investigate the practical biosensing capability of the developed system, the PDA@Co 3 O 4 NP-based assay was used to determine S 2− in spiked tap water samples, prepared at three concentrations of S 2− (50, 100, 200 µM). The biosensor quantified S 2− in tap water with good precision and accuracy, with CVs from 3.56 to 6.67% and recovery from 99.75 to 102.43% (Table 1), validating the excellent reproducibility and reliability. These results suggest that the PDA@Co 3 O 4 NP-based fluorometric biosensor can be used as an analytical system for the determination of S 2− in real aqueous environments. thiols (glutathione, cysteine), and common ions (Mg 2+ , NH4 + , Ca 2+ , Cl − ) did not have any considerable signal (below the threshold line), even at tenfold higher concentrations (Figure 5b), confirming that the biosensing strategy selectively detected target S 2− . On increasing the concentrations of S 2− , fluorescence intensity gradually increased ( Figure S7). From the analysis of dose-response curves, the LOD was calculated as low as 4.3 µ M with the linear range up to 200 µ M (Figure 5c,d), which is sufficient for practical S 2− biosensing in the field [34,35].

Conclusions
We demonstrated that PDA@Co 3 O 4 NPs are efficient catalase-like nanozymes, with competitive catalytic activity and stability compared with natural catalase and recently reported catalase-like nanozymes. We also proved that S 2− induced highly selective inhibition of the catalase-like activity of the PDA@Co 3 O 4 NPs, presumably due to the increased mass transfer limitation through aggregation. Based on the phenomena, S 2− was determined with high selectivity and sensitivity, and was quantified in real tap water with sufficient detection precision. This study provides an efficient approach for developing highly efficient nanozymes using simple surface engineering and nanozyme-mediated biosensors. These nanozymes show significant potential for use in diverse biotechnological applications.