Introduction  介绍

Natural enzymes are commonly used as catalysts in a variety of fields, such as environmental monitoring, agriculture, and the food industry.^1–3 Peroxidase is a natural enzyme that catalyses the transfer of electrons from an electron donor to peroxides, which are electron acceptors.^4–6 Convenient detection of hydrogen peroxide is of practical importance as it plays important roles in several fields including food, pharmaceutical and plastic industries, environmental chemistry, biological analysis, and clinical diagnostics.^7–10 The use of peroxidase enzymes in diagnostics is most prominent and forms the basis for majority of ELISA-based assays, where hydrogen peroxide acts as an electron acceptor in the oxidation of chromogenic substrates into coloured compounds.^1,11–13 However, the inherent disadvantages of natural enzymes, such as high cost, high pH sensitivity, and poor thermal stability challenge their practical applications.^14–18 Hence, artificial peroxidase-mimicking materials as an alternative to natural enzymes have attracted considerable attention in recent years.^14,19–22 To this end, various nanoparticles have emerged as natural enzyme-mimics with the ability to catalyse reactions in the absence of a natural enzyme. These nanoparticles, referred to as nanozymes, offer better stability, ease of synthesis and modification, comparable catalytic activity, and lower cost overcoming the limitations of the natural enzymes.
天然酶通常作为催化剂广泛应用于多个领域，如环境监测、农业和食品工业。^1–3 过氧化物酶是一种天然酶，催化电子从电子供体转移到过氧化物，后者是电子受体。^4–6 方便检测过氧化氢在多个领域中具有实际重要性，包括食品、制药和塑料工业、环境化学、生物分析和临床诊断。^7–10 过氧化物酶在诊断中的应用最为突出，构成了大多数基于 ELISA 的检测的基础，其中过氧化氢作为电子受体，在显色底物的氧化中转化为有色化合物。^1,11–13 然而，天然酶固有的缺点，如高成本、高 pH 敏感性和差的热稳定性，挑战了它们的实际应用。^14–18 因此，作为天然酶替代品的人工过氧化物酶模拟材料近年来引起了相当大的关注。^14,19–22 为此，各种纳米颗粒作为天然酶的模拟物应运而生，能够在没有天然酶的情况下催化反应。这些纳米颗粒被称为纳米酶，提供了更好的稳定性、合成和改性的便利、可比的催化活性以及更低的成本，克服了天然酶的局限性。

Various nanomaterials, such as metallic nanoparticles, metallic oxides and salts, conducting polymer nanoparticles, and quantum dots, have been studied for peroxidase-mimicking activity in applications such as immunoassays and the detection of biochemicals including glutathione, ascorbic acid, glucose, and cholesterol.^1,19,23–25 Iron oxide nanoparticles have attracted a great deal of attention as artificial non-proteinaceous nanozymes that mimic the catalytic characteristics of peroxidase enzymes.^26–28 These nanoparticles can oxidise the substrate 3,3′,5,5′-tetramethyl benzidine (TMB) in the presence of hydrogen peroxide, resulting in a colourimetric change from colourless to blue, which can be observed by the naked eye and analysed using ultraviolet-visible (UV-vis) spectrophotometry.^13,29 Unlike natural peroxidase enzyme, iron oxide nanoparticles are not susceptible to proteolytic degradation or affected by other environmental factors, such as temperature, pH, ionic strength, and heavy metals.³⁰
各种纳米材料，如金属纳米颗粒、金属氧化物和盐、导电聚合物纳米颗粒以及量子点，已被研究用于类过氧化物酶活性，应用于免疫测定和生化物质的检测，包括谷胱甘肽、抗坏血酸、葡萄糖和胆固醇。^1,19,23–25 氧化铁纳米颗粒作为模仿过氧化物酶催化特性的人工非蛋白质纳米酶，受到了极大的关注。^26–28 这些纳米颗粒可以在过氧化氢存在下氧化底物 3,3′,5,5′-四甲基苯胺（TMB），导致颜色从无色变为蓝色，这可以用肉眼观察并通过紫外-可见（UV-vis）分光光度法进行分析。^13,29 与天然过氧化物酶不同，氧化铁纳米颗粒不易受到蛋白水解降解或其他环境因素的影响，如温度、pH、离子强度和重金属。³⁰

Despite iron oxide nanoparticles being commonly studied as nanozymes, they undergo aggregation in particular when used in isolation in an aqueous environment, decreasing their total surface area, and thus significantly compromising their catalytic activity.³⁰ To this end, iron oxide nanoparticles have been immobilised on solid substrates including nanoparticles and polymer matrices. For example, iron oxide nanozyme particles were attached to cotton-based textiles for pollutant removal, exhibiting elimination of dye with nanozyme activity.³¹ Geleto et al. fabricated nanocellulose-based iron oxide–silver nanozymes for enhanced antibacterial and wound healing applications.³² Also, electrospun composites with iron oxide nanoparticles have been fabricated, showing high catalytic activity.³³ Another report by Satvekar et al. demonstrated the fabrication of a silica/chitosan organic–inorganic hybrid material, assimilated with iron oxide magnetic nanoparticles, for hydrogen peroxide biosensing, exhibiting high selectivity and sensitivity.³⁴ The focus of these approaches has been to stabilise iron oxide nanoparticles to overcome nanoparticle aggregation-induced activity loss. However, these substrate-based approaches do not provide any tuneability or on-demand catalytic response.
尽管氧化铁纳米颗粒作为纳米酶被广泛研究，但它们在水环境中单独使用时会发生聚集，降低其总表面积，从而显著影响其催化活性。³⁰ 为此，氧化铁纳米颗粒已被固定在包括纳米颗粒和聚合物基质的固体基材上。例如，氧化铁纳米酶颗粒被附着在基于棉花的纺织品上以去除污染物，展现出具有纳米酶活性的染料去除效果。³¹ Geleto 等 制造了基于纳米纤维素的氧化铁-银纳米酶，以增强抗菌和伤口愈合应用。³² 此外，已制造出含有氧化铁纳米颗粒的电纺复合材料，显示出高催化活性。³³ Satvekar 等 的另一项报告展示了制造一种与氧化铁磁性纳米颗粒相结合的二氧化硅/壳聚糖有机-无机混合材料，用于过氧化氢生物传感，展现出高选择性和灵敏度。³⁴ 这些方法的重点是稳定氧化铁纳米颗粒，以克服纳米颗粒聚集引起的活性损失。然而，这些基于基材的方法并不提供任何可调性或按需催化响应。

Herein, we report the development of polymer nanocomposite films with on-demand activatable catalytic nanozymatic response. We used in situ miniemulsion polymerisation to prepare colloidally stable poly(styrene-stat-n-butyl acrylate)/iron oxide-embedded silica nanoparticle (P(St-stat-nBA)/FeSiNP) nanocomposites with an innate ability to undergo film formation at ambient temperature virtually on any kind of substrate. The porous FeSiNPs were used as peroxide mimicking nanozyme sensing fillers. It is important to note that nanocomposite films with tuneable and activatable nanozymatic activity have to the best of our knowledge never been explored previously.
在此，我们报告了具有按需激活催化纳米酶反应的聚合物纳米复合膜的开发。我们使用原位微乳液聚合制备了胶体稳定的聚（苯乙烯-stat-n-丁基丙烯酸酯）/铁氧化物嵌入的二氧化硅纳米颗粒（P(St-stat-nBA)/FeSiNP）纳米复合材料，具有在几乎任何类型基材上在常温下进行膜形成的固有能力。多孔的 FeSiNP 被用作过氧化物模拟纳米酶传感填料。值得注意的是，具有可调和可激活纳米酶活性的纳米复合膜在我们所知的范围内之前从未被探索过。

The developed P(St-stat-nBA)/FeSiNP nanocomposite films have several advantages: (i) conjugation in silica nanoparticles limits the extent of iron oxide nanoparticle agglomeration; (ii) the hydrophilic nature of the silica shell enables their potential use in miniemulsion polymerisation as a surfactant stabilising polymer particle; (iii) the nanocomposite films can be stretched to different lengths exposing fresh FeSiNPs to tailor and enhance catalytic response on-demand. The present nanocomposite film strategy makes this approach highly versatile, thus challenging the status quo in functional composites for sensing and industrial applications, providing the channel to develop long-lasting sensors with tuneable responses.
所开发的 P(St-stat-nBA)/FeSiNP 纳米复合材料薄膜具有几个优点：(i) 硅纳米颗粒中的共轭限制了铁氧化物纳米颗粒的聚集程度；(ii) 硅壳的亲水特性使其在微乳液聚合中作为表面活性剂稳定聚合物颗粒的潜在应用成为可能；(iii) 纳米复合材料薄膜可以被拉伸到不同的长度，暴露出新鲜的 FeSiNP，以便根据需要定制和增强催化响应。目前的纳米复合材料薄膜策略使这种方法具有高度的多功能性，从而挑战了现状在传感和工业应用中的功能复合材料，提供了开发具有可调响应的持久传感器的渠道。

Materials and methods  材料与方法

Materials  材料

Iron(iii) chloride hexahydrate (FeCl₃·6H₂O, 97%), iron(ii) chloride tetrahydrate (FeCl₂·4H₂O, 98%), and anhydrous sodium hydroxide pellets (NaOH, 98%) were used in the synthesis of iron oxide nanoparticles and were purchased from Sigma-Aldrich (Australia). Hexadecyltrimethylammonium bromide (CTAB, 98%, powder), cetyltrimethylammonium chloride (CTAC, 25 wt% in H₂O), cyclohexane, triethanolamine (TEA), tetraethyl orthosilicate (TEOS), magnesium powder (99%), and hydrochloric acid (HCl, 37%) were purchased from Sigma-Aldrich (Australia) and were used in the synthesis of iron oxide-embedded silica nanoparticles. Acetic acid, sodium acetate (NaCH₃COO, ≥99%), hydrogen peroxide (H₂O₂, 30% w/w in H₂O), 3,3′,5,5′-tetramethylbenzidine (TMB, ≥98%), and l-ascorbic acid were purchased from Sigma-Aldrich (Australia). Styrene (St, Sigma-Aldrich, ≥99%) and n-butyl acrylate (nBA, Sigma-Aldrich, ≥99%) were purified through a filtration column filled with activated basic aluminium oxide powder (Sigma-Aldrich, Brockmann I) to remove the inhibitor. Azobisisobutyronitrile (AIBN, Sigma-Aldrich) was recrystallised in water from acetone. Hexadecane (HD, Sigma-Aldrich, 99%) and sodium dodecyl sulfate (SDS, Sigma-Aldrich, ≥99%) were used as received. Dimethyl sulfoxide (DMSO) and glycine were purchased from Chem-Supply (Australia). Sodium acetate buffer (0.2 M) was prepared by dissolving sodium acetate in Milli-Q water and adjusting the pH with acetic acid and NaOH. The water used in all experiments was Milli-Q (18.2 MΩ.cm at 25 °C) water.
氯化铁(iii)六水合物 (FeCl₃·6H₂O, 97%)、氯化铁(ii)四水合物 (FeCl₂·4H₂O, 98%) 和无水氢氧化钠颗粒 (NaOH, 98%) 用于合成铁氧化物纳米颗粒，均购自 Sigma-Aldrich（澳大利亚）。十六烷基三甲基氯化铵 (CTAB, 98%, 粉末)、十六烷基三甲基氯化铵 (CTAC, 25 wt% in H₂O)、环己烷、三乙醇胺 (TEA)、四乙氧基硅烷 (TEOS)、镁粉 (99%) 和盐酸 (HCl, 37%) 也购自 Sigma-Aldrich（澳大利亚），用于合成嵌入铁氧化物的二氧化硅纳米颗粒。醋酸、醋酸钠 (NaCH₃COO, ≥99%)、过氧化氢 (H₂O₂, 30% w/w in H₂O)、3,3′,5,5′-四甲基联苯胺 (TMB, ≥98%) 和l-抗坏血酸均购自 Sigma-Aldrich（澳大利亚）。 苯乙烯 (St, Sigma-Aldrich, ≥99%) 和 n-丁基丙烯酸酯 (nBA, Sigma-Aldrich, ≥99%) 通过填充有活性碱性铝氧化物粉末 (Sigma-Aldrich, Brockmann I) 的过滤柱进行纯化，以去除抑制剂。偶氮二异丁腈 (AIBN, Sigma-Aldrich) 在水中从丙酮重结晶。十六烷 (HD, Sigma-Aldrich, 99%) 和十二烷基硫酸钠 (SDS, Sigma-Aldrich, ≥99%) 直接使用。二甲基亚砜 (DMSO) 和甘氨酸购自 Chem-Supply (澳大利亚)。醋酸钠缓冲液 (0.2 M) 通过将醋酸钠溶解在 Milli-Q 水中并用醋酸和氢氧化钠调整 pH 制备。所有实验中使用的水为 Milli-Q (25 °C 时 18.2 MΩ.cm) 水。

Synthesis of FeSiNPs  FeSiNPs 的合成

Iron oxide (γ-Fe₂O₃) nanoparticles were synthesised via co-precipitation according to the method reported previously.³⁵ Iron(iii) chloride hexahydrate (2.703 g) and iron(ii) chloride tetrahydrate (0.994 g) were combined in a 150 mL beaker, maintaining a 2 : 1 ferric to ferrous ratio. NaOH (1.6 g) dissolved in 40 mL of deionised water was introduced into the iron mixture under stirring at 400 rpm for 1 min. The solution was heated to 50 °C and stirred for 30 min. Upon observing an inadequate pH level, an additional 0.4 g of NaOH was added in 10 mL of water, and the solution was stirred at the same speed for another 30 min at the same temperature. The nanoparticles formed were magnetically separated and washed thrice with Milli-Q water, followed by centrifugation (8000 rpm). The resultant paste containing the nanoparticles was oven-dried at 100 °C for 12 h, yielding 1.211 g of dry product.
氧化铁 (γ-Fe₂O₃) 纳米颗粒是通过共沉淀法合成的，具体方法参考之前的报道。³⁵ 六水合氯化铁(三价) (2.703 g) 和四水合氯化铁(二价) (0.994 g) 在一个 150 mL 的烧杯中混合，保持 2 : 1 的三价铁与二价铁的比例。将溶解在 40 mL 去离子水中的氢氧化钠 (1.6 g) 在 400 rpm 搅拌下加入铁混合物中，搅拌 1 分钟。将溶液加热至 50 °C 并搅拌 30 分钟。观察到 pH 值不足后，额外加入 0.4 g 氢氧化钠溶解在 10 mL 水中，并在相同温度下以相同速度再搅拌 30 分钟。形成的纳米颗粒通过磁力分离并用 Milli-Q 水洗涤三次，随后进行离心（8000 rpm）。含有纳米颗粒的浆料在 100 °C 下烘干 12 小时，得到 1.211 g 的干产品。

The synthesis of FeSiNPs was conducted based on the method described previously (Fig. S1, ESI†).³⁶ The procedure commenced with the ultrasonication of iron oxide nanoparticles in 3 mL of deionised water until achieving a uniform dispersion. The dispersed nanoparticles were then transferred to a 150 mL conical flask containing 8 mL of CTAC, 360 mg of TEA, and 72 mL of deionised water. The mixture was stirred at 400 rpm for 1 h, maintaining a temperature of 60 °C. Subsequently, 32 mL of cyclohexane and 8 mL of TEOS were added, and the solution was aged for 14 h under the same conditions. The sample was then washed with acetone and centrifuged at 10 000 rpm until the supernatant became clear. The particles were air-dried in a fume cupboard for 72 h. Magnetic-responsive particles were separated using a strong magnet bar for further analysis.
FeSiNPs 的合成是基于之前描述的方法进行的（图 S1，ESI†）。³⁶ 该过程首先在 3 mL 去离子水中超声处理氧化铁纳米颗粒，直到获得均匀分散。然后将分散的纳米颗粒转移到一个 150 mL 的锥形瓶中，瓶内含有 8 mL 的 CTAC、360 mg 的 TEA 和 72 mL 的去离子水。混合物在 60 °C 下以 400 rpm 搅拌 1 小时。随后，加入 32 mL 的环己烷和 8 mL 的 TEOS，并在相同条件下老化 14 小时。然后用丙酮洗涤样品，并以 10,000 rpm 离心，直到上清液变清澈。颗粒在通风柜中空气干燥 72 小时。使用强磁铁棒分离磁响应颗粒以进行进一步分析。

Preparation of P(St-stat-nBA)/FeSiNP nanocomposite latexes using miniemulsion polymerisation
使用微乳液聚合制备 P(St-stat-nBA)/FeSiNP 纳米复合乳胶

An aqueous dispersion of FeSiNPs was prepared by mixing FeSiNPs (concentrations of 5, 10, and 20 wt% relative to monomers) with 15 mL of water for 15 min, followed by ultrasonication (Branson digital sonifier) at 20% amplitude on ice for 5 min. The organic phase comprised St and nBA as monomers with a weight ratio of 1 : 1 (total of 7 wt% relative to water), HD as a co-stabiliser (5 wt% relative to monomers), AIBN as an initiator (0.25 M relative to HD and monomers), and SDS as a surfactant (1 wt% relative to the organic phase – monomer, HD and AIBN). The FeSiNP dispersion was subsequently mixed with the organic phase in a 20 mL glass bottle for 15 min on ice, followed by 10 min ultrasonication at 20% amplitude on ice to obtain monomer droplets decorated with FeSiNP. After that, the acquired miniemulsion underwent degassing for 20 min on ice. Finally, polymerisation was conducted at 70 °C for 24 h (Scheme 1).
FeSiNPs 的水相分散液是通过将 FeSiNPs（相对于单体的浓度为 5%、10%和 20%）与 15 毫升水混合 15 分钟制备的，随后在冰上以 20%的振幅进行超声处理（Branson 数字声波仪）5 分钟。有机相由 St 和nBA 作为单体，重量比为 1:1（相对于水总共为 7 wt%），HD 作为共稳定剂（相对于单体为 5 wt%），AIBN 作为引发剂（相对于 HD 和单体为 0.25 M），SDS 作为表面活性剂（相对于有机相——单体、HD 和 AIBN 为 1 wt%）。随后，FeSiNP 分散液与有机相在一个 20 毫升的玻璃瓶中混合 15 分钟，冰上以 20%的振幅进行超声处理 10 分钟，以获得装饰有 FeSiNP 的单体液滴。之后，获得的微乳液在冰上进行脱气 20 分钟。最后，在 70°C 下进行聚合反应 24 小时（方案 1）。

Nanocomposite film preparation
纳米复合膜制备

To fabricate P(St-stat-nBA)/FeSiNP nanocomposite films, the as-synthesised nanocomposite latex was initially degassed in a vacuum chamber for 30 min to remove microbubbles. Subsequently, 10 mL of latex was drop-casted into a 25 × 25 mm silicone mould and then left to undergo film formation under ambient conditions (∼20 °C and 1 atm) for approximately 2 weeks to obtain robust standalone films (Scheme 1).
为了制造 P(St-stat-nBA)/FeSiNP 纳米复合薄膜，初步合成的纳米复合乳液在真空室中脱气 30 分钟，以去除微气泡。随后，将 10 毫升乳液滴铸到 25 × 25 毫米的硅胶模具中，然后在环境条件下（约 20°C 和 1 atm）静置约 2 周，以获得坚固的独立薄膜（方案 1）。

Characterisation  特征描述

Gravimetry  重力测量

After polymerisation, monomer conversion was calculated by gravimetric analysis. 1.5 g of the P(St-stat-nBA)/FeSiNP latex was weighed in a pre-weighed aluminium pan and covered with perforated aluminium foil and dried in a vacuum oven at 30 °C for 24 h to allow the evaporation of unreacted monomers and water. The weight difference between the initial and the vacuum-dried latex yielded the monomer conversion.
聚合后，通过重力分析计算单体转化率。将 1.5 克 P(St-stat-nBA)/FeSiNP 乳胶称量在一个预先称重的铝盘中，覆盖上打孔的铝箔，并在 30°C 的真空烘箱中干燥 24 小时，以便蒸发未反应的单体和水。初始乳胶与真空干燥乳胶之间的重量差异得出了单体转化率。

Dynamic light scattering (DLS)
动态光散射 (DLS)

The hydrodynamic diameters of monomer droplets and polymer particles were measured using dynamic light scattering (DLS, Malvern Zetasizer Ultra). The DLS sample was prepared by diluting 1–2 drops of the miniemulsion or nanocomposite latex in water and subsequently, subjecting it to three runs with 5 sub-measurements per run. Average intensity data of the three runs are presented along with the polydispersity.
单体液滴和聚合物颗粒的水动力直径通过动态光散射（DLS，Malvern Zetasizer Ultra）进行测量。DLS 样品是通过将 1-2 滴微乳液或纳米复合乳胶稀释在水中制备的，随后进行三次实验，每次实验进行 5 次子测量。三次实验的平均强度数据与多分散性一起呈现。

Gel permeation chromatography (GPC)
凝胶渗透色谱 (GPC)

Gel permeation chromatography was used to determine the number-average (M_n) and weight-average (M_w) molecular weights of the polymer and the molecular weight distributions. The instrument comprised an LC-20AT pump (Shimadzu) and a SIL-20A HT autosampler (Shimadzu). Tetrahydrofuran (THF, HPLC Grade, RCI Labscan Ltd.) was used as an eluent at 40 °C, and a flow rate of 1.0 mL min⁻¹ was applied with an injection volume of 50 μL. The system was calibrated against linear polystyrene standards. GPC samples were prepared by dissolving the dry nanocomposite from gravimetric analysis in THF with the ratio of 1 mg : 2 mL, followed by filtration using a syringe filter (13 mm-Ø, PTFE membrane, 0.45 μm, Adelab Scientific) to remove the FeSiNP.
凝胶渗透色谱法用于测定聚合物的数均分子量（M_n）和重均分子量（M_w）以及分子量分布。仪器包括一台 LC-20AT 泵（岛津）和一台 SIL-20A HT 自动进样器（岛津）。四氢呋喃（THF，HPLC 级，RCI Labscan Ltd.）作为流动相，在 40°C 下使用，流速为 1.0 mL min⁻¹，注射体积为 50 μL。该系统以线性聚苯乙烯标准进行校准。GPC 样品通过将干燥的纳米复合材料（来自称重分析）溶解在 THF 中，比例为 1 mg : 2 mL，随后使用注射器过滤器（直径 13 mm，PTFE 膜，0.45 μm，Adelab Scientific）过滤以去除 FeSiNP。

Tensile testing  拉伸测试

Tensile testing of the P(St-stat-nBA)/FeSiNP films was conducted using a Mark-10 ESM303 instrument. The as-made films in silicone moulds were trimmed to approximately 5 mm in width and 20 mm in length. Each film was clamped at two ends and stretched at a rate of 20 mm min⁻¹ until fracture. The width and thickness of the films, as well as the initial distance between two clamps or gauge length, were measured using a digital caliper. These parameters were used to convert the raw data, expressed in load (N) versus travel (mm), to stress–strain plots. A minimum of three samples from each film were measured and data are presented as average ± standard deviation.
对 P(St-stat-nBA)/FeSiNP 薄膜的拉伸测试使用 Mark-10 ESM303 仪器进行。制备好的薄膜在硅胶模具中修剪至约 5 毫米宽和 20 毫米长。每个薄膜在两端夹紧，并以 20 毫米每分钟⁻¹的速度拉伸直至断裂。薄膜的宽度和厚度，以及两个夹具之间的初始距离或标距，使用数字卡尺进行测量。这些参数用于将以载荷（N）与位移（mm）表示的原始数据转换为应力-应变图。每个薄膜至少测量三个样本，数据以平均值±标准差的形式呈现。

Scanning electron microscopy (SEM)
扫描电子显微镜 (SEM)

SEM was conducted on the fabricated films in unstretched and stretched conditions. The film pieces were coated with Pt (10 nm) for SEM imaging and 20 nm carbon for EDS mapping. Images were taken using an FEI Nova NanoSEM 230 FE-SEM system operating at an accelerating voltage of 5 kV. For stretched imaging, the films were stretched by ∼100% prior to coatings.
对制造的薄膜在未拉伸和拉伸条件下进行了扫描电子显微镜（SEM）观察。薄膜片被涂覆了 10 纳米的铂用于 SEM 成像，以及 20 纳米的碳用于能量色散光谱（EDS）映射。使用 FEI Nova NanoSEM 230 FE-SEM 系统在 5 kV 的加速电压下拍摄了图像。对于拉伸成像，薄膜在涂层之前被拉伸了约 100%。

Scanning transmission electron microscopy (STEM) and electron energy loss spectroscopy (EELS)
扫描传输电子显微镜（STEM）和电子能量损失光谱（EELS）

Annular dark-field STEM (ADF-STEM) imaging and EELS spectroscopy were carried out using the JEOL GrandArm2 equipped with a Gatan Continuum energy filter with a K3 detector, operated at 300 kV. The TEM samples of FeSiNP were prepared by drop casting the sonicated FeSiNP suspensions directly onto lacy carbon film-coated TEM Cu grids and dried in a vacuum for 20 minutes.
环形暗场扫描透射电子显微镜（ADF-STEM）成像和电子能量损失谱（EELS）实验是在配备有 Gatan Continuum 能量滤波器和 K3 探测器的 JEOL GrandArm2 上进行的，操作电压为 300 kV。FeSiNP 的透射电子显微镜样品是通过将超声处理的 FeSiNP 悬浮液直接滴加到涂有网状碳膜的铜网格上，然后在真空中干燥 20 分钟制备的。

Optimisation of conditions for peroxidase-mimicking activity of bare FeSiNPs
裸 FeSiNPs 的过氧化物酶模拟活性条件优化

A series of experiments were carried out to optimise the conditions for peroxidase-like activity of FeSiNPs. The impact of pH on the reaction was determined through alterations of the pH of sodium acetate buffer from 2.5 to 7.5. Also, the effect of temperature was studied at a range of 25 °C to 45 °C. The experimental procedure involved exposing the bare FeSiNPs to various temperatures (25 to 45 °C) in the presence of TMB (1000 μM from 10 mM stock dissolved in DMSO) and H₂O₂ (700 mM) in sodium acetate buffer (pH 3.5) for a duration of 20 min in the dark. The absorbance was subsequently measured at 652 nm using a plate reader (Multiskan Sky, Thermo Fisher Scientific). The effect of pH on nanozyme activity of FeSiNPs was similarly assessed over a pH range from 2.5 to 7.5 at room temperature in the dark.
一系列实验被开展以优化 FeSiNPs 的类过氧化物酶活性的条件。通过将醋酸钠缓冲液的 pH 从 2.5 调整到 7.5，确定了 pH 对反应的影响。此外，温度的影响在 25°C 到 45°C 的范围内进行了研究。实验程序涉及在黑暗中将裸露的 FeSiNPs 暴露于不同温度（25 到 45°C）下，存在 TMB（1000 μM，来自 10 mM 的 DMSO 溶液）和 H₂O₂（700 mM）在醋酸钠缓冲液（pH 3.5）中，持续 20 分钟。随后使用板式读数仪（Multiskan Sky，Thermo Fisher Scientific）在 652 nm 处测量吸光度。FeSiNPs 的纳米酶活性在室温下的黑暗中也在 pH 2.5 到 7.5 的范围内进行了类似评估。

Determination of catalytic parameters
催化参数的确定

Catalytic parameters were determined by altering the concentration of H₂O₂ (100–1000 mM) while keeping the concentration of TMB fixed at 1000 μM and also for TMB concentration (100–1000 μM) while maintaining a fixed H₂O₂ concentration of 700 mM. The absorbance of the reaction mixture was measured at 652 nm after 20 min of incubation in the dark, and catalytic parameters were determined by fitting the absorbance data to the Michaelis–Menten equation as follows:
催化参数通过改变 H₂O₂的浓度（100–1000 mM）来确定，同时保持 TMB 的浓度固定在 1000 μM，并且在保持 H₂O₂浓度为 700 mM 的情况下，改变 TMB 浓度（100–1000 μM）。在黑暗中孵育 20 分钟后，测量反应混合物在 652 nm 处的吸光度，并通过将吸光度数据拟合到米氏-门腾方程来确定催化参数，如下所示： (1) where V₀ is the rate at which the substrate (TMB) is converted to a product, V_max denotes the maximum rate of conversion (obtained when substrates saturate the active sites of the enzyme), [S] is the concentration of the substrate, and K_m represents the Michaelis–Menten constant.
其中 V₀ 是底物（TMB）转化为产物的速率，V_max 表示最大转化速率（当底物饱和酶的活性位点时获得），[S] 是底物的浓度，K_m 代表米哈利斯-门腾常数。

Mechanism of bare FeSiNPs activity
裸 FeSiNPs 活性的机制

A hydroxyl radical scavenger, ascorbic acid, was used to study the mechanism of enzyme-mimicking activity of bare FeSiNPs. A certain amount of ascorbic acid (50 μg) was added to the FeSiNP solution containing 1000 μM TMB and 700 mM H₂O₂ in sodium acetate buffer at pH 3.5. The absorbance was measured at 652 nm after 20 min of incubation in the dark.
一种羟基自由基清除剂——抗坏血酸，被用来研究裸 FeSiNPs 的酶模拟活性机制。在 pH 3.5 的醋酸钠缓冲液中，向含有 1000 μM TMB 和 700 mM H₂O₂的 FeSiNP 溶液中添加了一定量的抗坏血酸（50 μg）。在黑暗中孵育 20 分钟后，在 652 nm 处测量吸光度。

Calibration curve with bare FeSiNPs
裸 FeSiNPs 的校准曲线

A calibration curve was obtained by analysing the catalytic activity of bare FeSiNPs using TMB and H₂O₂. The FeSiNPs were dispersed in sodium acetate buffer (pH 3.5) and the dispersion was serially diluted using the acetate buffer. 556 μL of 0.2 M acetate buffer was added per well in a 48-well tissue culture plate, followed by the addition of 100 μL of FeSiNP dispersion. Finally, 64 μL of H₂O₂ (700 mM, from 30% w/w stock) and 80 μL of TMB (1000 μM, 10 mM stock solution in DMSO) were added. The final concentration range of the nanoparticles was 0–1.25 mg/800 μL in each well. The plate was incubated in the dark for 20 min. 150 μL of the reaction mixture from each well was aliquoted and absorbance was measured immediately at 652 nm using a plate reader (Multiskan Sky, Thermo Fisher Scientific).
通过使用 TMB 和 H₂O₂分析裸 FeSiNPs 的催化活性获得了校准曲线。将 FeSiNPs 分散在醋酸钠缓冲液（pH 3.5）中，并使用醋酸盐缓冲液进行系列稀释。在 48 孔组织培养板的每个孔中添加 556 μL 的 0.2 M 醋酸盐缓冲液，然后添加 100 μL 的 FeSiNP 分散液。最后，添加 64 μL 的 H₂O₂（700 mM，来自 30% w/w 的储备液）和 80 μL 的 TMB（1000 μM，DMSO 中的 10 mM 储备溶液）。每个孔中纳米颗粒的最终浓度范围为 0–1.25 mg/800 μL。将板在黑暗中孵育 20 分钟。每个孔中取 150 μL 反应混合物，并立即使用酶标仪（Multiskan Sky，Thermo Fisher Scientific）在 652 nm 处测量吸光度。

Catalytic activity of P(St-stat-nBA)/FeSiNP nanocomposite films
P(St-stat-nBA)/FeSiNP 纳米复合薄膜的催化活性

Catalytic activities of P(St-stat-n-BA)/FeSiNP films were evaluated both in stretched and unstretched conditions. The nanocomposite films were cut into 5 × 5 mm pieces and three pieces per group were analysed. For the stretched experiments, films were hand-stretched to the desired lengths (50% and 100% extension) and attached to two needle sticks firmly set at predetermined lengths in a PDMS block. All samples were immersed in 656 μL of 0.2 M acetate buffer in a 48-well tissue culture plate, followed by the addition of 64 μL of H₂O₂ (from 30% w/w stock) and 80 μL of TMB (from 10 mM stock in DMSO). The samples were incubated in the dark for 20 min. Subsequently, 150 μL of the reaction mixture from each well was transferred to a 96-well tissue culture plate and absorbance was recorded at 652 nm using a plate reader.
P(St-stat-n-BA)/FeSiNP 薄膜的催化活性在拉伸和未拉伸条件下进行了评估。纳米复合薄膜被切割成 5 × 5 mm 的小块，每组分析三块。对于拉伸实验，薄膜被手动拉伸到所需长度（50% 和 100% 延伸），并固定在 PDMS 块中预定长度的两个针棒上。所有样品浸泡在 656 μL 的 0.2 M 醋酸盐缓冲液中，放置在 48 孔组织培养板中，然后加入 64 μL 的 H₂O₂（来自 30% w/w 储备液）和 80 μL 的 TMB（来自 10 mM 储备液在 DMSO 中）。样品在黑暗中孵育 20 分钟。随后，从每个孔中转移 150 μL 反应混合物到 96 孔组织培养板，并使用酶标仪在 652 nm 处记录吸光度。

Sustained nanozyme activity
持续的纳米酶活性

A time-based study was conducted with the P(St-stat-nBA)/10 wt% FeSiNP films. The film pieces (5 × 5 mm) were stretched 100% over PDMS blocks as described above and immersed in a 48-well tissue culture plate containing 656 μL of 0.2 M acetate buffer (pH 3.5), 64 μL of H₂O₂ (from 30% w/w stock) and 80 μL of TMB (from 10 mM stock in DMSO). The reaction was conducted for 2 h and the absorbance was recorded live using an Ocean Optics HR 4000 spectrometer at normal incidence with a focused spot size of 1 mm².
进行了一项基于时间的研究，使用了 P(St-stat-nBA)/10 wt% FeSiNP 薄膜。薄膜片（5 × 5 mm）在上述描述的 PDMS 块上拉伸 100%，并浸入含有 656 μL 0.2 M 醋酸盐缓冲液（pH 3.5）、64 μL H₂O₂（来自 30% w/w 库存）和 80 μL TMB（来自 10 mM DMSO 库存）的 48 孔组织培养板中。反应进行 2 小时，使用 Ocean Optics HR 4000 光谱仪在正常入射下实时记录吸光度，聚焦点大小为 1 mm²。

On-demand catalytic response
按需催化反应

The nanocomposite films (4 pieces, 5 × 5 mm) were immersed in 656 μL of 0.2 M acetate buffer in a 48-well tissue culture plate, followed by the addition of 64 μL of H₂O₂ (from 30% w/w stock) and 80 μL of TMB (from 10 mM stock in DMSO). After 18 h of incubation at room temperature in the dark, a 150 μL volume was withdrawn from each well and read at 652 nm in duplicate. The first film piece was hand-stretched using two tweezers under the reagent for 20 min, while the remaining three pieces were left unstretched. After 20 min, absorbance was read again for all of the films. Then the second film piece was stretched similarly for 20 min and absorbance was read. After two more measurements (at 20 min interval), the third piece was stretched similarly. The readings were recorded in total up to 200 min. The fourth film piece remained unstretched from the beginning to the end of the experiment.
纳米复合薄膜（4 片，5 × 5 mm）浸泡在 656 μL 的 0.2 M 醋酸盐缓冲液中，放置在 48 孔组织培养板中，随后加入 64 μL 的 H₂O₂（来自 30% w/w 的储备液）和 80 μL 的 TMB（来自 10 mM 的 DMSO 储备液）。在黑暗中室温孵育 18 小时后，从每个孔中取出 150 μL 的液体，并在 652 nm 处进行重复测量。第一片薄膜在试剂下用两把镊子手动拉伸 20 分钟，而其余三片保持未拉伸状态。20 分钟后，所有薄膜的吸光度再次测量。然后第二片薄膜以类似方式拉伸 20 分钟并测量吸光度。经过两次测量（间隔 20 分钟），第三片薄膜也以类似方式拉伸。总共记录的测量时间长达 200 分钟。第四片薄膜从实验开始到结束始终保持未拉伸状态。

Results and discussion  结果与讨论

Characterisation of FeSiNP
FeSiNP 的表征

Fig. 1a shows the morphology of FeSiNPs as imaged using annular dark field scanning transmission electron microscopy (ADF-STEM). We observed a dendritic porous silica nanoparticle structure embedded with iron oxide nanoparticles. The iron oxide nanoparticles used for the FeSiNPs formulation were prepared using a precipitation process, which resulted in iron oxide nanoparticles of approximately 100 nm size as evident in the DLS and STEM data (Table S1 and Fig. S2, ESI†). The average size of the FeSiNPs was approximately 100 nm in diameter. The oxidation states of iron and silicon as well as the distribution of iron oxide particles in FeSiNP nanoparticle were further confirmed using EELS analysis (Fig. 1b) and STEM-EELS mapping (Fig. 1c). EELS spectra of Si L_2,3 and Fe L_2,3 edges of FeSiNP revealed characteristic spectral features corresponding to silica and iron oxide. The presence and morphology of iron oxide particles embedded in the silica of FeSiNPs were further confirmed by STEM-EELS maps.
图 1a 显示了使用环形暗场扫描透射电子显微镜 (ADF-STEM) 成像的 FeSiNPs 的形态。我们观察到一种树枝状多孔二氧化硅纳米颗粒结构，嵌入了氧化铁纳米颗粒。用于 FeSiNPs 配方的氧化铁纳米颗粒是通过沉淀过程制备的，结果得到的氧化铁纳米颗粒大小约为 100 nm，这在 DLS 和 STEM 数据中得到了证实（表 S1 和图 S2，ESI†）。FeSiNPs 的平均直径约为 100 nm。铁和硅的氧化态以及 FeSiNP 纳米颗粒中氧化铁颗粒的分布通过 EELS 分析进一步确认（图 1b）和 STEM-EELS 映射（图 1c）。FeSiNP 的 Si L_2,3 和 Fe L_2,3 边缘的 EELS 光谱显示出与二氧化硅和氧化铁相对应的特征光谱特征。通过 STEM-EELS 图进一步确认了嵌入 FeSiNPs 二氧化硅中的氧化铁颗粒的存在和形态。

Characterisation of polymer latex
聚合物乳胶的表征

Colloidally stable poly(styrene-stat-n-butyl acrylate) (P(St-stat-nBA))/FeSiNP nanocomposite latexes were synthesised using miniemulsion polymerisation. Miniemulsion polymerisation is a one-pot synthesis strategy to prepare polymer/filler nanocomposite colloids. In miniemulsion, polymerisation proceeds by droplet nucleation within submicron-sized monomer droplets, produced using high energy mixing, ideally resulting in one-to-one conversion of monomer droplets into the polymer particles.³⁷ Miniemulsion polymerisation typically forms filler-decorated polymer particles potentially at the interface in the case of nanocomposites when using fillers such as graphene oxide.^38–43
胶体稳定的聚（苯乙烯-stat-n-丁基丙烯酸酯）(P(St-stat-nBA))/FeSiNP 纳米复合乳液通过微乳液聚合合成。微乳液聚合是一种一锅合成策略，用于制备聚合物/填料纳米复合胶体。在微乳液中，聚合通过在亚微米级单体液滴内的液滴成核进行，液滴是通过高能混合产生的，理想情况下实现单体液滴向聚合物颗粒的一对一转化。³⁷ 微乳液聚合通常在使用如氧化石墨烯等填料的情况下形成填料装饰的聚合物颗粒，可能位于纳米复合材料的界面。^38–43

In this work, we employed a statistical copolymer of styrene (St) and n-butyl acrylate (nBA) due to the ability of the resulting polymer to undergo film formation at ambient temperature as previously reported by us and others.^44–49 The weight ratio of St and nBA was maintained at 1 : 1 to obtain a theoretical glass transition temperature of ∼3 °C (based on the Fox equation).⁵⁰ The polymerisation was conducted at 70 °C for 24 h using AIBN as an initiator, FeSiNP (5, 10, and 20 wt%; relative to monomers) and 1 wt% SDS (relative to organic phase) for 24 h. We obtained highly colloidally stable latexes at all filler loadings with monomer conversions of >85% (Fig. 2a and Table S2, ESI†). The polymer molecular weights were in the typical range for such nanocomposites.^51,52 GPC analysis revealed a unimodal distribution with the M_n of ∼100 to 110 kg mol⁻¹ (Fig. 2b, c and Table S3, ESI†). Polymer particle size determined using DLS was also in the typical range (80–150 nm) reported for polymer nanocomposites prepared using miniemulsion polymerisation (Table S2, ESI†).^47,53
在这项工作中，我们采用了苯乙烯（St）和n-丁基丙烯酸酯（nBA）的统计共聚物，因为所得到的聚合物能够在室温下形成薄膜，正如我们和其他人之前所报道的那样。^44–49 St 和nBA 的重量比保持在 1 : 1，以获得约 3 °C 的理论玻璃转变温度（基于 Fox 方程）。⁵⁰ 聚合反应在 70 °C 下进行 24 小时，使用 AIBN 作为引发剂，FeSiNP（5%、10%和 20% wt%；相对于单体）和 1 wt% SDS（相对于有机相）进行 24 小时。我们在所有填料负载下获得了高度胶体稳定的乳胶，单体转化率超过 85%（图 2a和表 S2，ESI†）。聚合物的分子量在此类纳米复合材料的典型范围内。^51,52 GPC 分析显示出单峰分布，M_n约为 100 到 110 kg mol⁻¹（图 2b, c和表 S3，ESI†）。 使用动态光散射（DLS）确定的聚合物颗粒大小也在使用微乳液聚合制备的聚合物纳米复合材料的典型范围（80–150 nm）内（表 S2，ESI†）。^47,53

Next, the obtained P(St-stat-nBA)/FeSiNP nanocomposite latexes comprising different concentrations of FeSiNPs were drop-cast in silicone moulds to prepare nanocomposite films at ambient temperature (Scheme 1). SEM imaging was conducted to characterise the surface profile of the obtained films under both unstretched and stretched conditions to visualise any change to the surface profile as a result of stretching. In the case of unstretched films, SEM images revealed rough surfaces in all films regardless of FeSiNP loading (Fig. 3). In all films, we observed features akin to crevices on the film surface exposing embedded FeSiNPs. Higher magnification imaging revealed the presence of more aggregated spherical nanoparticles within these crevices, indicating potential interference during film formation and leading to their incomplete coverage, which could explain the emergence of crevices. These crevices do not result from film cracking, instead we hypothesise that during the film formation, decorated polymer particles start to coalesce, enhancing inter-FeSiNP interactions and resulting in FeSiNP aggregation. Such FeSiNP aggregation will in turn interfere with polymer particle coalescence to some extent, resulting in incomplete coverage on the film surface. These crevice-like features increased in films with increasing FeSiNP loading as anticipated (Fig. S3, ESI†). When the films were stretched, crevices became more evident leading to the exposure of FeSiNPs embedded within the films under all conditions. As shown in Fig. 3, FeSiNPs became clearly visible on all films, with an increasing number of FeSiNPs becoming exposed with increasing filler loading. SEM-EDS analysis of unstretched and stretched P(St-stat-nBA)/FeSiNP films confirmed the presence of silicon and iron from the FeSiNPs on the film surface (Fig. S4 and S5, ESI†). EDS mapping indicated potentially higher concentrations of silicon inside these crevices (Fig. S6, ESI†).
接下来，获得的 P(St-stat-nBA)/FeSiNP 纳米复合乳液中含有不同浓度的 FeSiNP，采用滴铸法在硅胶模具中制备纳米复合薄膜，温度为常温(方案 1)。进行了 SEM 成像，以表征获得薄膜在未拉伸和拉伸条件下的表面特征，以可视化拉伸导致的表面特征变化。在未拉伸薄膜的情况下，SEM 图像显示所有薄膜的表面均粗糙，无论 FeSiNP 的负载量如何(图 3)。在所有薄膜中，我们观察到类似于薄膜表面裂缝的特征，暴露出嵌入的 FeSiNP。更高倍数的成像揭示了这些裂缝内存在更多聚集的球形纳米颗粒，表明在薄膜形成过程中可能存在干扰，导致其覆盖不完全，这可能解释了裂缝的出现。这些裂缝并不是由于薄膜开裂造成的，我们假设在薄膜形成过程中，装饰的聚合物颗粒开始聚集，增强了 FeSiNP 之间的相互作用，导致 FeSiNP 聚集。 这种 FeSiNP 聚集反过来会在一定程度上干扰聚合物颗粒的合并，导致薄膜表面覆盖不完全。这些裂缝状特征在 FeSiNP 负载量增加的薄膜中如预期般增加（图 S3，ESI†）。当薄膜被拉伸时，裂缝变得更加明显，导致在所有条件下嵌入薄膜中的 FeSiNP 暴露出来。如图 3所示，FeSiNP 在所有薄膜上变得清晰可见，随着填料负载量的增加，暴露的 FeSiNP 数量也在增加。对未拉伸和拉伸的 P(St-stat-nBA)/FeSiNP 薄膜的 SEM-EDS 分析确认了薄膜表面存在来自 FeSiNP 的硅和铁（图 S4 和 S5，ESI†）。EDS 映射表明这些裂缝内部可能存在更高浓度的硅（图 S6，ESI†）。

The effect of FeSiNP on mechanical properties in standalone nanocomposite films was studied using uniaxial tensile testing. The tensile strength increased with increasing FeSiNP concentration from 5 wt% (0.78 ± 0.08 MPa) to 10 wt% (1.71 ± 0.21 MPa) (Table S4, ESI†). However, a further increase to 20 wt% caused no further increase in tensile strength (1.15 ± 0.09 MPa). On the contrary, elongation at break decreased with increasing FeSiNP concentration from 5 wt% (∼1448%) to 10 wt% (847 ± 27%). However, no further change in tensile strength was observed when FeSiNP concentration was increased to 20 wt% (860 ± 46%) (Table S4, ESI†). We postulate that increasing the filler concentration from 10 wt% to 20 wt% may have induced a greater extent of FeSiNPs agglomeration during the formation of the film. The evidence of a greater extent of FeSiNP agglomeration at 20 wt% loading compared to 10 wt% is supported by the SEM data, where the presence of nanoparticle agglomeration became more clearly visible within the crevices under stretched film conditions. Such agglomeration compromises the ability of nanocomposite films to dissipate stress with increasing strain, thus compromising both tensile strength and elongation at break.
FeSiNP 对独立纳米复合薄膜机械性能的影响通过单轴拉伸测试进行了研究。随着 FeSiNP 浓度从 5 wt%（0.78 ± 0.08 MPa）增加到 10 wt%（1.71 ± 0.21 MPa），拉伸强度增加（表 S4，ESI†）。然而，进一步增加到 20 wt%并未导致拉伸强度的进一步增加（1.15 ± 0.09 MPa）。相反，断裂伸长率随着 FeSiNP 浓度从 5 wt%（∼1448%）增加到 10 wt%（847 ± 27%）而减少。然而，当 FeSiNP 浓度增加到 20 wt%时，拉伸强度没有进一步变化（860 ± 46%）（表 S4，ESI†）。我们推测，将填料浓度从 10 wt%增加到 20 wt%可能在薄膜形成过程中引起了 FeSiNPs 的更大程度的聚集。SEM 数据支持了在 20 wt%负载下与 10 wt%相比，FeSiNP 聚集程度更大的证据，在拉伸薄膜条件下，纳米颗粒聚集的存在变得更加明显。 这种聚集妨碍了纳米复合材料薄膜在应变增加时耗散应力的能力，从而影响了抗拉强度和断裂伸长率。

Catalytic nanozyme activity
催化纳米酶活性

The peroxidase-mimicking catalytic nanozyme activity of the nanocomposite films was subsequently investigated using TMB as a substrate. TMB is commonly used in ELISA assays to determine the catalytic activity of natural enzymes. Typically, natural peroxidase enzymes in the presence of H₂O₂ generate hydroxyl radicals (˙OH), which react with TMB to produce a blue coloured oxidised product (TMB_ox) that absorbs light at 652 nm and can be utilised for both qualitative and quantitative analysis (Fig. 4a).⁵⁴ Before testing the catalytic activity, we optimised reaction conditions for the as-synthesised FeSiNPs. First, the effects of buffer pH and reaction temperature were investigated by changing the pH from 2.5 to 7.5 and the reaction temperature from room temperature (25 °C) to 45 °C. The catalytic activity increased initially, reaching the maximum activity at pH 3.5 (Fig. 4b). However, further increase in pH caused a continuous and significant reduction in the catalytic activity. The catalytic activity decreased systematically with an increase in the reaction temperature, exhibiting the best performance at room temperature (25 °C) and the lowest performance at 45 °C (Fig. 4c). These conditions were selected and maintained in all subsequent experiments unless stated otherwise. Next, the peroxidase-mimicking activity of the as-synthesised neat FeSiNPs was investigated. As anticipated, a linear correlation between FeSiNPs concentration and catalytic performance was observed with a regression coefficient (R²) of 0.973 (Fig. 4d). To determine the mechanism of catalytic activity of FeSiNPs, ascorbic acid was used as an oxygen free radical (˙OH and O₂˙⁻) scavenger.⁵⁵ The catalytic reactions were conducted similarly as described above with the incorporation of ascorbic acid, and the resulting absorbance of the product was quantified to determine the change in the catalytic activity. The absorbance decreased significantly with the incorporation of ascorbic acid (Fig. 4e). These results indicate that ascorbic acid reacted with ˙OH, inhibiting the oxidation of TMB, and thus causing a reduction in the formation of a blue colour product, confirming that the mechanism of action is indeed mediated by the ˙OH radicals. Based on the results, the catalytic mechanism is proposed to follow a previously published report as described in Fig. S7 and eqn (S1)–(S4) (ESI†).⁵⁶ In summary, iron (Fe²⁺) in FeSiNP catalyses the reduction of H₂O₂ to generate hydroxyl radicals (˙OH) and oxidised Fe³⁺ in the first step. This reaction is the rate limiting step. Next, the generated ˙OH radicals react with TMB to form a blue coloured oxidised product (TMB_ox). In the third step, ˙OH radicals react with H₂O₂ to form HO₂˙ radicals, which then reduce Fe³⁺ to generate the catalyst Fe²⁺ and a molecule of oxygen.
纳米复合膜的过氧化物酶模拟催化纳米酶活性随后使用 TMB 作为底物进行了研究。TMB 通常用于 ELISA 检测，以确定天然酶的催化活性。通常，天然过氧化物酶在 H₂O₂存在的情况下生成羟基自由基（˙OH），这些自由基与 TMB 反应生成一种蓝色的氧化产物（TMB_ox），该产物在 652 nm 处吸收光，可以用于定性和定量分析（图 4a）。⁵⁴ 在测试催化活性之前，我们优化了合成的 FeSiNPs 的反应条件。首先，通过将 pH 从 2.5 调整到 7.5，以及将反应温度从室温（25 °C）调整到 45 °C，研究了缓冲液 pH 和反应温度的影响。催化活性最初增加，在 pH 3.5 时达到最大活性（图 4b）。然而，pH 的进一步增加导致催化活性持续显著下降。 催化活性随着反应温度的升高而系统性下降，在室温（25 °C）时表现最佳，在 45 °C 时表现最低（图 4c）。除非另有说明，这些条件在所有后续实验中均被选择并保持。接下来，研究了合成的纯 FeSiNPs 的过氧化物酶模拟活性。正如预期的那样，观察到 FeSiNPs 浓度与催化性能之间存在线性相关，回归系数（R²）为 0.973（图 4d）。为了确定 FeSiNPs 的催化活性机制，使用抗坏血酸作为氧自由基（˙OH 和 O₂˙⁻）清除剂。⁵⁵ 催化反应的进行方式与上述相似，加入抗坏血酸后，量化产物的吸光度以确定催化活性的变化。随着抗坏血酸的加入，吸光度显著下降（图 4e）。 这些结果表明，抗坏血酸与˙OH 反应，抑制了 TMB 的氧化，从而导致蓝色产物的形成减少，确认了作用机制确实是由˙OH 自由基介导的。根据结果，催化机制被提议遵循之前发布的报告，如图 S7 和方程(S1)–(S4)所述(ESI†)。⁵⁶ 总之，FeSiNP 中的铁(Fe²⁺)催化 H₂O₂的还原，生成羟基自由基(˙OH)和氧化的 Fe³⁺，这是第一步。这一反应是速率限制步骤。接下来，生成的˙OH 自由基与 TMB 反应，形成蓝色氧化产物(TMB_ox)。在第三步，˙OH 自由基与 H₂O₂反应，形成 HO₂˙自由基，然后还原 Fe³⁺，生成催化剂 Fe²⁺和一分子氧。

A steady-state kinetics study was subsequently carried out to investigate the peroxidase-like catalytic activity of neat FeSiNPs by varying either the concentration of H₂O₂ or TMB while keeping the other component constant. The kinetic parameters (K_m and V_max) were calculated using the molar absorption coefficient (ε) of 39 000 M⁻¹ cm⁻¹ (at 652 nm) for TMB_ox.⁵⁷ Since natural peroxidase enzyme activity usually follows the Michaelis–Menten eqn (1), the data obtained in this study were fitted to a typical Michaelis–Menten curve (Fig. 4f and g) within the relevant concentration range using a nonlinear least-squares fitting method to determine the catalytic parameters (K_m and V_max). K_m is an indication of the affinity of an enzyme for its substrate; a lower K_m value suggests a higher affinity between the two. V_max is the maximum rate of conversion into the product. As the concentrations of H₂O₂ and TMB increased, the absorbance values at 652 nm also increased (represented as velocity, Fig. 4f and g, respectively). At higher concentrations of TMB and H₂O₂, there was no inhibition in the catalysis process. Additionally, Lineweaver–Burk double-reciprocal plots were generated (Fig. 4h and i) to estimate the affinity of the nanozyme (FeSiNPs) to the substrate (TMB). The K_m and V_max values were calculated to be 0.060 mM and 0.672 × 10⁻⁸ M s⁻¹ for H₂O₂, and 7.143 mM and 1.075 × 10⁻⁸ M s⁻¹ for TMB, respectively. Based on the obtained results, it can be deduced that FeSiNPs have a higher affinity towards H₂O₂ (lower K_m) than TMB. In comparison with the other previously reported nanozymes, the K_m value for H₂O₂ obtained for FeSiNPs in this work was amongst the lowest observed, whereas the calculated V_max value was comparable to the other nanozymes (Table S5, ESI†). A lower K_m value indicates a higher affinity of a nanozyme towards its substrate (H₂O₂ in this case). Therefore, in comparison to the other nanozyme listed in Table S5 (ESI†), FeSiNPs used in this work exhibited a higher affinity towards the H₂O₂ substrate. The catalytic efficiency of FeSiNPs, taking only the Fe component as the catalytic species into consideration, was calculated to be 6.35 × 10⁻⁴ s⁻¹ for H₂O₂ and 1.02 × 10⁻³ s⁻¹ for TMB.
随后进行了一项稳态动力学研究，以通过改变 H₂O₂或 TMB 的浓度，同时保持另一组分不变，来研究纯 FeSiNPs 的类过氧化物酶催化活性。动力学参数（K_m和V_max）是使用 TMB_ox的摩尔吸收系数（ε）39 000 M⁻¹ cm⁻¹（在 652 nm 处）计算得出的。⁵⁷由于天然过氧化物酶的活性通常遵循迈克利斯-门腾方程 (1)，本研究获得的数据在相关浓度范围内使用非线性最小二乘拟合方法拟合到典型的迈克利斯-门腾曲线（图 4f 和 g），以确定催化参数（K_m和V_max）。K_m是酶对其底物亲和力的指示；较低的K_m值表明两者之间的亲和力较高。 V_max 是转化为产物的最大速率。随着 H₂O₂ 和 TMB 的浓度增加，652 nm 处的吸光度值也增加（分别表示为速度，图 4f 和 g）。在较高浓度的 TMB 和 H₂O₂ 下，催化过程没有抑制。此外，生成了 Lineweaver–Burk 双倒数图（图 4h 和 i）以估计纳米酶（FeSiNPs）对底物（TMB）的亲和力。K_m 和 V_max 的值分别计算为 H₂O₂ 的 0.060 mM 和 0.672 × 10⁻⁸ M s⁻¹，以及 TMB 的 7.143 mM 和 1.075 × 10⁻⁸ M s⁻¹。根据获得的结果，可以推断 FeSiNPs 对 H₂O₂ 的亲和力（较低的 K_m）高于 TMB。 与其他已报道的纳米酶相比，本研究中 FeSiNPs 获得的 H₂O₂的K_m值是观察到的最低值之一，而计算得出的V_max值与其他纳米酶相当（表 S5，ESI†）。较低的K_m值表明纳米酶对其底物（在此情况下为 H₂O₂）的亲和力更高。因此，与表 S5 中列出的其他纳米酶（ESI†）相比，本研究中使用的 FeSiNPs 对 H₂O₂底物表现出更高的亲和力。仅考虑 Fe 成分作为催化物种，FeSiNPs 的催化效率被计算为 H₂O₂为 6.35 × 10⁻⁴ s⁻¹，TMB 为 1.02 × 10⁻³ s⁻¹。

Next, we systematically investigated the peroxidase-mimicking activities of the P(St-stat-nBA)/FeSiNP nanocomposite films with different FeSiNP loadings. Films were prepared with similar thickness to eliminate the potential impact of thickness on the catalytic performance (Fig. 5a). First, we investigated unstretched films to obtain their ‘baseline’ performance and determine the impact of different FeSiNP loadings. As shown in Fig. 5b–d, there was a direct correlation between FeSiNP loading and catalytic performance (absorbance values). As expected, increasing the amount of FeSiNPs led to a dose-dependent increase in the catalytic performance, which can potentially be explained by the presence of FeSiNPs in the crevices on the film surface as observed in the SEM analysis (Fig. 3). The control neat P(St-stat-nBA) without FeSiNPs under the same experimental conditions exhibited absorbance values comparable to the buffer control (which was the baseline value ≡ 0), indicating that FeSiNPs present in the composite films were indeed functioning as nanozymes (Fig. S8, ESI†). Therefore, it can be deduced that P(St-stat-nBA)/FeSiNP films have potential to function as a nanozyme biosensor by making FeSiNPs available for catalysis. Considering the high stretchability of these P(St-stat-nBA)/FeSiNP films, we subsequently explored the possibility of tuning the catalytic performance by simply stretching them to different extents (50% and 100%). With increased stretching from 0 to 100%, a gradual increase in absorbance of the blue product (TMB_ox; a quantitative measure of catalytic performance) was observed for P(St-stat-nBA)/FeSiNP (5 wt%) and P(St-stat-nBA)/FeSiNP (20 wt%) films, whereas a sharp and more linear increase in absorbance was obtained for P(St-stat-nBA)/FeSiNP (10 wt%).
接下来，我们系统地研究了不同 FeSiNP 负载量的 P(St-stat-nBA)/FeSiNP 纳米复合膜的过氧化物酶模拟活性。膜的厚度相似，以消除厚度对催化性能的潜在影响（图 5a）。首先，我们研究了未拉伸的膜，以获得其“基线”性能并确定不同 FeSiNP 负载量的影响。如图 5b–d所示，FeSiNP 负载量与催化性能（吸光度值）之间存在直接相关性。正如预期的那样，增加 FeSiNP 的数量导致催化性能的剂量依赖性增加，这可以通过 SEM 分析中观察到的膜表面裂缝中的 FeSiNP 的存在来解释（图 3）。在相同实验条件下，未添加 FeSiNP 的纯 P(St-stat-nBA)的吸光度值与缓冲液对照（基线值≡0）相当，表明复合膜中存在的 FeSiNP 确实作为纳米酶发挥作用（图 S8，ESI†）。 因此，可以推断出 P(St-stat-nBA)/FeSiNP 薄膜有潜力作为纳米酶生物传感器，通过使 FeSiNPs 可用于催化。考虑到这些 P(St-stat-nBA)/FeSiNP 薄膜的高拉伸性，我们随后探索了通过简单地将其拉伸到不同程度（50%和 100%）来调节催化性能的可能性。从 0%到 100%的拉伸增加中，观察到 P(St-stat-nBA)/FeSiNP (5 wt%)和 P(St-stat-nBA)/FeSiNP (20 wt%)薄膜的蓝色产物（TMB_ox; 催化性能的定量测量）吸光度逐渐增加，而 P(St-stat-nBA)/FeSiNP (10 wt%)的吸光度则呈现出急剧且更线性的增加。

In the case of 5 wt% FeSiNP, almost all FeSiNPs present on the film surface (and crevices) participate in catalysis under unstretched conditions, and therefore, stretching induced only a marginal increase in the catalytic activity (Fig. 5b). It is hypothesised that 5 wt% FeSiNPs loading is quite low in nanocomposite films, and despite stretching, a very small amount of new surface or embedded FeSiNPs becomes available for catalysis. If this is the case, one would expect the absorbance values to improve with increasing amounts of FeSiNPs loading, which is what is observed under all conditions (0, 50 and 100% stretching). In the case of 20 wt%, the amount of FeSiNPs is too high, resulting in a stronger catalytic response compared to lower NP loadings but also causing greater NP agglomeration throughout the film. This agglomeration may limit the number of previously unexposed NPs embedded within the film from becoming available under stretching conditions, resulting in only a marginal increase in catalytic performance (Fig. 5d). In the case of 10 wt% FeSiNP-loaded films, we believe that the NP concentration is optimal in these films such that despite some level of NP agglomeration, previously unexposed NPs continuously become available for catalysis when stretched from 0, 50 to 100% (Fig. 5c). Overall, these results highlight that the developed nanocomposite films provide a platform with unprecedented tuneability in catalytic performance by simple stretching (Fig. 5e).
在 5 wt% FeSiNP 的情况下，几乎所有在薄膜表面（和缝隙）上的 FeSiNP 都在未拉伸条件下参与催化，因此，拉伸仅导致催化活性略微增加（图 5b）。假设 5 wt% FeSiNP 的负载量在纳米复合薄膜中相当低，尽管拉伸，只有极少量的新表面或嵌入的 FeSiNP 可用于催化。如果是这种情况，人们会期望随着 FeSiNP 负载量的增加，吸光度值会改善，这在所有条件下（0、50 和 100%拉伸）都是观察到的。在 20 wt%的情况下，FeSiNP 的数量过高，导致与较低的 NP 负载相比，催化反应更强，但也导致薄膜中 NP 的聚集更严重。这种聚集可能限制了在拉伸条件下薄膜中先前未暴露的 NP 变得可用的数量，导致催化性能仅略微提高（图 5d）。 在 10 wt% FeSiNP 负载薄膜的情况下，我们认为这些薄膜中的 NP 浓度是最佳的，因此尽管存在一定程度的 NP 聚集，之前未暴露的 NP 在从 0、50 到 100%的拉伸过程中持续可用于催化（图 5c）。总体而言，这些结果突显出所开发的纳米复合薄膜通过简单的拉伸提供了前所未有的催化性能可调性的平台（图 5e）。

To investigate the durability in terms of long-term performance, we selected the 10 wt% FeSiNP-loaded nanocomposite film under a 100% stretched condition and performed the catalytic response experiment for 2 h. As shown in (Fig. 5f), a linear catalytic response was observed (R² = 0.994) with time. A plausible explanation for this observation could be that the FeSiNPs that are present deeper inside the films increasingly get exposed to the reagents with time to show the catalytic activity. If there are enough FeSiNPs available on the film surface to continually drive catalysis with time, one would expect the absorbance to plateau and not continue to increase with time. These data highlight that the developed films can function as a long-lasting continuous monitoring platform.
为了研究长期性能方面的耐久性，我们选择了在 100%拉伸条件下的 10 wt% FeSiNP 负载纳米复合膜，并进行了 2 小时的催化反应实验。如(图 5f)所示，随着时间的推移，观察到了线性催化反应（R² = 0.994）。对此观察的一个合理解释可能是，随着时间的推移，位于膜内部更深处的 FeSiNP 逐渐暴露于试剂中，从而显示出催化活性。如果膜表面有足够的 FeSiNP 可用以持续驱动催化反应，人们会期望吸光度达到平台期，而不是随着时间的推移继续增加。这些数据突显出所开发的膜可以作为一个持久的连续监测平台。

On-demand catalytic response
按需催化反应

To further validate stretching-mediated tuneability in the catalytic performance, we stretched P(St-stat-nBA)/FeSiNP (10 wt%) films at different time points during the catalytic reaction. We incubated films in the reaction buffer containing H₂O₂ and TMB for 18 h and recorded the absorbance value of ∼0.2, which was considered as a starting point before stretching the films. The unstretched film exhibited a marginal reduction in the catalytic performance with time (Fig. 6). These values at individual time points were considered as a ‘baseline’ to compare the change in catalytic response as a result of film stretching. Next, we stretched films after 0, 20, and 80 min of incubation and recorded the change in absorbance values compared to the respective baseline values.
为了进一步验证拉伸介导的催化性能可调性，我们在催化反应期间对 P(St-stat-nBA)/FeSiNP (10 wt%) 薄膜进行了不同时间点的拉伸。我们在含有 H₂O₂和 TMB 的反应缓冲液中孵育薄膜 18 小时，并记录了约 0.2 的吸光度值，这被视为拉伸薄膜前的起始点。未拉伸的薄膜在时间上表现出催化性能的轻微下降（图 6）。这些在各个时间点的值被视为“基线”，以比较由于薄膜拉伸而导致的催化响应变化。接下来，我们在孵育 0、20 和 80 分钟后对薄膜进行了拉伸，并记录了与各自基线值相比的吸光度值变化。

All films exhibited a significant increase in the absorbance values when stretched, and the increased absorbance values were similar in all stretched films regardless of whether the film was stretched at 0, 20, and 80 min after incubation (a marginally lower response was observed for 80 min stretched films). These data highlight the tuneability of the developed nanozyme-loaded films, i.e. activity can be enhanced on-demand or can be refreshed during its performance life. Taken together, we propose the developed film to be a highly tuneable platform with prolonged functional activity where the catalytic functionality can be made active by simply stretching the nanocomposite film.
所有薄膜在拉伸时的吸光值显著增加，且所有拉伸薄膜的吸光值增加相似，无论薄膜是在孵育后 0、20 还是 80 分钟拉伸（80 分钟拉伸薄膜的反应略低）。这些数据突显了所开发的纳米酶负载薄膜的可调性，即活性可以按需增强或在其使用寿命期间进行刷新。综合来看，我们提出所开发的薄膜是一个高度可调的平台，具有延长的功能活性，其中催化功能可以通过简单地拉伸纳米复合薄膜来激活。

Conclusions  结论

The aim of the present work has been to fabricate nanozyme-loaded tunable polymer nanocomposite films for on-demand, responsive, and sustained peroxidase-mimicking activity. P(St-stat-nBA) films containing iron oxide-loaded porous silica nanozymes (FeSiNP) were prepared using miniemulsion polymerisation where FeSiNP functions as a peroxidase-mimic. The nanozyme-loaded films exhibited nanozyme (FeSiNP) concentration-dependent catalytic activity with higher performance observed with increasing nanoparticle loading. When stretched, the catalytic performance increased with the extent of stretching from 0 to 100%. Furthermore, the films exhibited a sustained and linear response with time, confirming their ability to facilitate continuous detection. On-demand switching at different time points revealed successful activation by a sharp increase in the catalytic performance simply by stretching the 10 wt% FeSiNPs-loaded films. Overall, the developed nanocomposite films demonstrated responsive and tuneable peroxidase-mimicking activity. This study has for the first time demonstrated tuneable on-demand activatable films for enzyme-mimicking activity, thus setting up a platform for the future development of smart sensors for various diagnostic applications.
本研究的目的是制造负载纳米酶的可调聚合物纳米复合膜，以实现按需、响应和持续的过氧化物酶模拟活性。采用微乳液聚合制备了含有铁氧化物负载的多孔二氧化硅纳米酶（FeSiNP）的 P(St-stat-nBA)膜，其中 FeSiNP 作为过氧化物酶模拟物。负载纳米酶的膜表现出依赖于纳米酶（FeSiNP）浓度的催化活性，随着纳米颗粒负载的增加，性能提高。当膜被拉伸时，催化性能随着拉伸程度从 0%到 100%而增加。此外，膜在时间上表现出持续和线性的响应，确认其能够促进连续检测。在不同时间点的按需切换显示，通过简单地拉伸 10 wt%的 FeSiNPs 负载膜，催化性能显著提高，成功激活。总体而言，开发的纳米复合膜展示了响应性和可调的过氧化物酶模拟活性。 本研究首次展示了可调节的按需激活薄膜用于酶模拟活性，从而为未来开发用于各种诊断应用的智能传感器建立了平台。

Data availability  数据可用性

Data for this article are available in the UNSW public repository at https://doi.org/10.26190/unsworks/30229.
本文的数据可在 UNSW 公共存储库中获取，链接为 https://doi.org/10.26190/unsworks/30229。

Conflicts of interest  利益冲突

No conflict to declare.  没有冲突需要声明。

Acknowledgements 致谢

V. A. acknowledges the National Health and Medical Research Council (NHMRC), Australia, for an Early Career Fellowship (GNT1139060) and UNSW Safety Net Fellowship. The authors acknowledge the facilities and the scientific and technical assistance of Microscopy Australia at the Electron Microscope Unit (EMU), and the Solid State & Elemental Analysis Unit within the Mark Wainwright Analytical Centre (MWAC) at UNSW Sydney.
V. A. 感谢澳大利亚国家健康与医学研究委员会（NHMRC）提供的早期职业奖学金（GNT1139060）和新南威尔士大学安全网奖学金。作者感谢澳大利亚显微镜中心在新南威尔士大学悉尼的电子显微镜单位（EMU）以及马克·温赖特分析中心（MWAC）内的固态与元素分析单位提供的设施和科学技术支持。

References 参考文献