Introduction  导言

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.
1-3过氧化物酶是一种天然酶，它能催化电子从电子供体向过氧化物（电子受体）的转移。^4-6 方便地检测过氧化氢具有重要的实际意义，因为它在食品、制药和塑料工业、环境化学、生物分析和临床诊断等多个领域发挥着重要作用。^7-10 过氧化物酶在诊断学中的应用最为突出，是大多数基于 ELISA 检测方法的基础，过氧化氢在发色底物氧化成有色化合物的过程中充当电子受体。^1,11-13 然而，天然酶固有的缺点，如成本高、pH 值敏感性高、热稳定性差等，对其实际应用提出了挑战。^14-18 因此，人工过氧化物酶模拟材料作为天然酶的替代品近年来引起了广泛关注。^14,19-22 为此，各种纳米粒子作为天然酶模拟物出现了，它们能够在没有天然酶的情况下催化反应。这些被称为纳米酶的纳米粒子具有更好的稳定性、易于合成和修饰、催化活性相当、成本较低等特点，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
人们研究了各种纳米材料（如金属纳米颗粒、金属氧化物和盐、导电聚合物纳米颗粒和量子点）在免疫测定和谷胱甘肽、抗坏血酸、葡萄糖和胆固醇等生化物质检测等应用中的过氧化物酶模拟活性。^1,19,23-25 氧化铁纳米粒子作为可模仿过氧化物酶催化特性的人工非蛋白性纳米酶引起了广泛关注。^26-28 这些纳米粒子能在过氧化氢存在的情况下氧化底物 3,3′,5,5′-四甲基联苯胺（TMB），从而产生从无色到蓝色的比色变化，这种变化可以用肉眼观察到，也可以用紫外-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被蛋白分解，也不受其他环境因素（如温度、pH 值、离子强度和重金属）的影响。

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.
尽管氧化铁纳米粒子通常作为纳米酶进行研究，但它们在水环境中单独使用时会发生聚集，从而降低其总表面积，并因此大大降低其催化活性。³¹ Geleto et al.32此外，还制作了含有氧化铁纳米颗粒的电纺复合材料，显示出很高的催化活性。³³ Satvekar et al.的另一篇报告展示了二氧化硅/壳聚糖有机-无机混合材料的制备，该材料与氧化铁磁性纳米粒子同化，用于过氧化氢生物传感，表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粒子，以克服纳米粒子聚集引起的活性损失。然而，这些基于基底的方法无法提供任何可调性或按需催化反应。

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薄膜的开发情况。我们采用原位小型乳液聚合法制备了胶体稳定的聚（苯乙烯-丙烯酸-n丁酯）/氧化铁-嵌入二氧化硅纳米粒子（P(St-Stat-nstat-nBA)/FeSiNP) 纳米复合材料。多孔 FeSiNPs 被用作过氧化物模拟纳米酶传感填料。值得注意的是，据我们所知，具有可调节和可激活的纳米酶活性的纳米复合薄膜以前从未被探索过。

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 纳米复合薄膜具有几个优点：(i) 二氧化硅纳米颗粒中的共轭作用限制了氧化铁纳米颗粒的团聚程度；(ii) 二氧化硅外壳的亲水性使其有可能作为稳定聚合物颗粒的表面活性剂用于微型乳液聚合；(iii) 纳米复合膜可拉伸至不同长度，露出新鲜的 FeSiNPs，从而按需定制和增强催化反应。目前的纳米复合薄膜策略使这种方法具有高度的通用性，从而挑战了传感和工业应用功能复合材料的现状，为开发具有可调响应的长效传感器提供了渠道。

Materials and methods  材料和方法

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.
六水氯化铁（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%）用于合成纳米氧化铁颗粒，均购自 Sigma-Aldrich（澳大利亚）。十六烷基三甲基溴化铵（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）、正硅酸四乙酯（TEOS）、镁粉（99%）和盐酸（HCl，37%）购自 Sigma-Aldrich（澳大利亚），用于合成氧化铁包埋二氧化硅纳米颗粒。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 抗坏血酸购自 Sigma-Aldrich（澳大利亚）。苯乙烯（St，Sigma-Aldrich，≥99%）和n 丙烯酸丁酯（nBA, Sigma-Aldrich，≥99%）通过过滤柱纯化，过滤柱中填充活性碱式氧化铝粉末（Sigma-Aldrich，Brockmann I）以去除抑制剂。偶氮二异丁腈（AIBN，Sigma-Aldrich）在水中从丙酮中重结晶。十六烷（HD，Sigma-Aldrich，99%）和十二烷基硫酸钠（SDS，Sigma-Aldrich，≥99%）按原样使用。二甲基亚砜（DMSO）和甘氨酸购自 Chem-Supply（澳大利亚）。醋酸钠缓冲液（0.2M）的制备方法是将醋酸钠溶解在 Milli-Q 水中，然后用醋酸和 NaOH 调节 pH 值。所有实验中使用的水都是 Milli-Q 水（25 °C 时为 18.2 MΩ.cm）。

Synthesis of FeSiNPs  合成 FeSiNPs

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 ）纳米粒子是根据先前报告的方法通过共沉淀合成的。³⁵ 氯化铁（iii ）六水合物（2.703 克）和氯化铁（ii ）四水合物（0.994 克）在一个 150 毫升的烧杯中混合，保持 2 : 1 的亚铁比。在 400 转/分钟的搅拌下，将溶于 40 毫升去离子水的 NaOH（1.6 克）加入铁混合物中 1 分钟。将溶液加热至 50 °C，并搅拌 30 分钟。观察到 pH 值不足时，在 10 mL 水中再加入 0.4 g NaOH，然后在相同的温度下以相同的速度再搅拌 30 分钟。用磁力分离形成的纳米颗粒，并用 Milli-Q 水洗涤三次，然后离心（8000 rpm）。将含有纳米颗粒的糊状物在 100 °C 下烘干 12 小时，得到 1.211 克干产品。

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 的合成是根据之前描述的方法进行的（图 S1，ESI† ）。然后将分散的纳米颗粒转移到一个 150 毫升的锥形烧瓶中，该烧瓶中含有 8 毫升 CTAC、360 毫克三乙醇胺和 72 毫升去离子水。混合物以 400 rpm 的转速搅拌 1 小时，温度保持在 60 °C。随后加入 32 毫升环己烷和 8 毫升 TEOS，在相同条件下陈化 14 小时。然后用丙酮洗涤样品，并以 10 000 rpm 的转速离心，直至上清液变得清澈。颗粒在通风橱中风干 72 小时后，使用强磁棒将磁响应颗粒分离出来，以便进行进一步分析。

Preparation of P(St-stat-nBA)/FeSiNP nanocomposite latexes using miniemulsion polymerisation
利用微型乳液聚合法制备 P(St-stat-nBA)/FeSiNP 纳米复合胶乳

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).
将 FeSiNPs（相对于单体的浓度分别为 5、10 和 20 wt%）与 15 mL 水混合 15 分钟，然后在冰上以 20% 的振幅超声 5 分钟（Branson 数字超声仪），制备 FeSiNPs 的水分散液。有机相包括 St 和 nBA 作为单体，重量比为 1 ：1（相对于水的总重量为 7 wt%），HD 作为共稳定剂（相对于单体的重量比为 5 wt%），AIBN 作为引发剂（相对于 HD 和单体的重量比为 0.25 M），SDS 作为表面活性剂（相对于有机相--单体、HD 和 AIBN 的重量比为 1 wt%）。随后，在 20 mL 玻璃瓶中将 FeSiNP 分散液与有机相在冰上混合 15 分钟，然后在冰上以 20% 的振幅超声 10 分钟，以获得装饰有 FeSiNP 的单体液滴。之后，获得的微型乳液在冰上脱气 20 分钟。最后，在 70 °C 下聚合 24 小时（方案 1）。

Nanocomposite film preparation
纳米复合薄膜制备

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).
为了制备 P(St-stat-nBA)/FeSiNP 纳米复合薄膜，首先将合成的纳米复合胶乳在真空室中脱气 30 分钟以去除微气泡。随后，将 10 mL 胶乳滴入 25 × 25 mm 的硅胶模具中，然后在环境条件（20 °C∼和 1 atm）下静置约 2 周，以形成坚固的独立薄膜（方案 1）。

Characterisation  特征

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，通过重量分析计算单体转化率。将 1.5 克 P(St-stat-nBA)/FeSiNP 胶乳放入一个预先称重的铝盘中称重，然后盖上带孔铝箔，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分。初始胶乳和真空干燥胶乳的重量差即为单体转化率。

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.
使用动态光散射（DLS，Malvern Zetasizer Ultra）测量单体液滴和聚合物颗粒的水动力直径。DLS 样品的制备方法是将 1-2 滴微型乳液或纳米复合胶乳稀释在水中，然后进行三次运行，每次运行进行 5 次测量。三次运行的平均强度数据与多分散性一并列出。

Gel permeation chromatography (GPC)
凝胶渗透色谱法（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 LC-20AT 泵（岛津）和 SIL-20A HT 自动进样器（岛津）。洗脱液为四氢呋喃（THF，HPLC 级，RCI Labscan Ltd.），温度为 40 °C，流速为 1.0 mL/min^-1 ，进样量为 50 μL。系统根据线性聚苯乙烯标准进行校准。GPC 样品的制备方法是将重量分析得出的干燥纳米复合材料以 1 mg : 2 mL 的比例溶解在 THF 中，然后使用注射器过滤器（13 mm-Ø，PTFE 膜，0.45 μm，Adelab Scientific）过滤以去除 FeSiNP。

Tensile testing  拉伸试验

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.
使用 Mark-10 ESM303 仪器对 P(St-stat-nBA)/FeSiNP 薄膜进行了拉伸测试。硅胶模具中的成品薄膜被修剪成约 5 毫米宽、20 毫米长。每层薄膜两端夹紧，以 20 mm min^-1 的速度拉伸，直至断裂。使用数字卡尺测量薄膜的宽度和厚度，以及两个夹具之间的初始距离或测量长度。这些参数用于将原始数据（以载荷（牛顿）与行程（毫米）表示）转换为应力应变图。每种薄膜至少测量三个样本，数据以平均值 ± 标准偏差表示。

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.
在未拉伸和拉伸条件下，对制作的薄膜进行了扫描电镜观察。薄膜表面涂有用于 SEM 成像的铂（10 nm）和用于 EDS 制图的 20 nm 碳。使用 FEI Nova NanoSEM 230 FE-SEM 系统在 5 kV 的加速电压下拍摄图像。为了进行拉伸成像，薄膜在镀膜前拉伸了 ∼ 100%。

Scanning transmission electron microscopy (STEM) and electron energy loss spectroscopy (EELS)
扫描透射电子显微镜（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 STEM（ADF-STEM）成像和 EELS 光谱分析是使用配备有 K3 检测器的 Gatan Continuum 能量滤波器的 JEOL GrandArm2 在 300 kV 下进行的。将超声处理过的 FeSiNP 悬浮液直接滴铸在涂有花边碳膜的 TEM 铜栅格上，然后在真空中干燥 20 分钟，就制备出了 FeSiNP 的 TEM 样品。

Optimisation of conditions for peroxidase-mimicking activity of bare FeSiNPs
优化裸 FeSiNPs 过氧化物酶模拟活性的条件

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.
为了优化 FeSiNPs 过氧化物酶样活性的条件，我们进行了一系列实验。通过将醋酸钠缓冲液的 pH 值从 2.5 调整到 7.5，确定了 pH 值对反应的影响。此外，还研究了温度在 25 °C 至 45 °C 范围内的影响。实验过程包括：在醋酸钠缓冲液（pH 3.5）中的 TMB（1000 μM，来自溶于 DMSO 的 10 mM 储存液）和 H₂O₂ （700 mM）存在下，将裸露的 FeSiNPs 暴露在不同温度（25 至 45 °C）下，在黑暗中持续 20 分钟。随后使用平板阅读器（Multiskan Sky，Thermo Fisher Scientific）在 652 纳米波长处测量吸光度。同样，在室温、黑暗条件下，在 2.5 至 7.5 的 pH 范围内评估了 pH 对 FeSiNPs 纳米酶活性的影响。

Determination of catalytic parameters
催化参数的测定

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:
通过改变 H₂O₂ (100-1000 mM)，同时将 TMB 的浓度固定在 1000 μM，以及改变 TMB 的浓度 (100-1000 μM)，同时将 H₂O₂ 的浓度固定在 700 mM。在黑暗中培养 20 分钟后，在 652 纳米波长处测量反应混合物的吸光度，通过将吸光度数据拟合到 Michaelis-Menten 公式，确定催化参数如下： (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 表示最大转化率（当底物使酶的活性位点饱和时）、[S] 是底物的浓度，K_m 表示迈克尔斯-门顿常数。

Mechanism of bare FeSiNPs activity
裸 FeSiNPs 的活性机制

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.
利用羟基自由基清除剂抗坏血酸来研究裸FeSiNPs的酶模拟活性机制。在含有 1000 μM TMB 和 700 mM H₂O₂ 的 FeSiNP 溶液（pH 值为 3.5）中加入一定量的抗坏血酸（50 μg）。在黑暗中培养 20 分钟后，在 652 纳米波长处测量吸光度。

Calibration curve with bare FeSiNPs
裸 FeSiNPs 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，得到了校准曲线。将 FeSiNPs 分散在醋酸钠缓冲液（pH 3.5）中，然后用醋酸钠缓冲液对分散液进行连续稀释。在 48 孔组织培养板中，每孔加入 556 μL 0.2 M 醋酸缓冲液，然后加入 100 μL FeSiNP 分散液。最后，加入 64 μL H₂O₂（700 mM，来自 30% w/w 的储备液）和 80 μL TMB（1000 μM，在 DMSO 中的 10 mM 储备液）。每孔中纳米颗粒的最终浓度范围为 0-1.25 mg/800 μL。每孔取 150 μL 反应混合物，立即用平板阅读器（Multiskan Sky，赛默飞世尔科技公司）在 652 纳米波长处测量吸光度。

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.
在拉伸和未拉伸条件下评估了 P(St-stat-n-BA)/FeSiNP 薄膜的催化活性。纳米复合薄膜被切割成 5 × 5 毫米的小块，每组分析三块。在拉伸实验中，用手将薄膜拉伸到所需的长度（50% 和 100%），并将其固定在 PDMS 块中预定长度的两根针棒上。在 48 孔组织培养板中，将所有样品浸入 656 μL 0.2 M 醋酸缓冲液中，然后加入 64 μL H₂O₂ （30% w/w 储量）和 80 μL TMB（DMSO 中 10 mM 储量）。样品在黑暗中孵育 20 分钟。然后，将每个孔中的 150 μL 反应混合物转移到 96 孔组织培养板中，用平板阅读器在 652 纳米波长处记录吸光度。

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.
对 P(St-stat-nBA)/10 wt% FeSiNP 薄膜进行了基于时间的研究。如上所述，薄膜片（5 × 5 毫米）100% 地拉伸在 PDMS 块上，然后浸入含有 656 μL 0.2 M 醋酸缓冲液（pH 3.5）、64 μL H₂O₂ （30% w/w 的储备液）和 80 μL TMB（10 mM 的 DMSO 储备液）的 48 孔组织培养板中。反应进行 2 小时，使用 Ocean Optics HR 4000 光谱仪在正常入射条件下实时记录吸光度，聚焦光斑尺寸为 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.
将纳米复合薄膜（4 片，5 × 5 mm）浸入 48 孔组织培养板中的 656 μL 0.2 M 醋酸缓冲液中，然后加入 64 μL H₂O₂ （取自 30% w/w 储存液）和 80 μL TMB（取自 DMSO 中的 10 mM 储存液）。室温下暗处培养 18 小时后，从每个孔中抽取 150 μL 体积，在 652 纳米波长下读数，一式两份。用两把镊子将第一片薄膜在试剂下用手拉伸 20 分钟，其余三片不拉伸。20 分钟后，再次读取所有薄膜的吸光度。然后将第二片薄膜同样拉伸 20 分钟并读取吸光度。再进行两次测量（间隔 20 分钟）后，同样拉伸第三块薄膜。总共记录读数至 200 分钟。第四片薄膜从实验开始到结束都没有拉伸。

Results and discussion  结果和讨论

Characterisation of FeSiNP
FeSiNP 的特性

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）观察到的 FeSiNPs 形态。我们观察到树枝状多孔二氧化硅纳米粒子结构中嵌入了氧化铁纳米粒子。从 DLS 和 STEM 数据（表 S1 和图 S2，ESI†）可以看出，FeSiNPs 制剂中使用的氧化铁纳米粒子是通过沉淀法制备的，其尺寸约为 100 nm。FeSiNPs 的平均直径约为 100 nm。利用 EELS 分析（图 1b）和 STEM-EELS 制图（图 1c）进一步证实了铁和硅的氧化态以及 FeSiNP 纳米粒子中氧化铁颗粒的分布。FeSiNP 的 Si L_2,3 和 Fe L_2,3 边缘的 EELS 光谱显示了与二氧化硅和氧化铁相对应的特征光谱。STEM-EELS 图谱进一步证实了嵌入 FeSiNPs 二氧化硅中的氧化铁颗粒的存在和形态。

Characterisation of polymer latex
聚合物胶乳的特性

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
利用微型乳液聚合法合成了胶体稳定的聚（苯乙烯-stat-n 丙烯酸丁酯）（P(St-stat-nBA) ）/FeSiNP 纳米复合胶乳。小型乳液聚合法是一种制备聚合物/填料纳米复合胶体的单锅合成策略。在微型乳液聚合过程中，聚合是通过在亚微米级单体液滴中的液滴成核进行的，这种液滴是通过高能量混合产生的，理想的结果是单体液滴一对一地转化为聚合物颗粒。³⁷ 当使用氧化石墨烯等填料时，微型乳液聚合通常会在纳米复合材料的界面上形成填料装饰的聚合物颗粒。

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 丙烯酸丁酯（nBA）的统计共聚物，这是因为我们和其他人以前曾报告过所产生的聚合物能够在环境温度下形成薄膜。^44-49 St 和 nBA 的重量比保持在 1 ：⁵⁰ 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）在 70 °C 下聚合 24 小时。在所有填料负载条件下，我们都获得了胶体高度稳定的胶乳，单体转化率大于 85%（Fig.2a 和表 S2，ESI†）。聚合物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，M_n 在 100 至 110 kg mol^-1 之间呈单峰分布（Fig.2b、c和表 S3，ESI† ）。使用 DLS 测定的聚合物粒度也在用微型乳液聚合法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†).
接着，将获得的包含不同浓度 FeSiNPs 的 P(St-stat-nBA)/FeSiNP 纳米复合胶乳滴铸在硅胶模具中，在环境温度下制备纳米复合薄膜（方案 1）。对未拉伸和拉伸条件下获得的薄膜的表面轮廓进行了扫描电子显微镜成像，以观察拉伸对表面轮廓造成的任何变化。在未拉伸薄膜的情况下，SEM 图像显示所有薄膜的表面都很粗糙，与 FeSiNP 的负载无关（图 3）。在所有薄膜中，我们都观察到薄膜表面有类似裂缝的特征，暴露出嵌入的 FeSiNPs。更高倍率的成像显示，在这些缝隙中存在更多聚集的球形纳米粒子，这表明在薄膜形成过程中可能存在干扰，导致其覆盖不完全，这也是出现缝隙的原因。这些裂缝并不是薄膜开裂造成的，相反，我们假设在薄膜形成过程中，装饰聚合物粒子开始凝聚，增强了 FeSiNP 之间的相互作用，导致 FeSiNP 聚集。这种 FeSiNP 聚集反过来又会在一定程度上干扰聚合物颗粒的凝聚，导致薄膜表面覆盖不完全。正如预期的那样，随着 FeSiNP 负载的增加，薄膜中的这些裂缝特征也会增加（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 FeSiNPs 都暴露出来。如图 3 所示，所有薄膜上的 FeSiNPs 都变得清晰可见，随着填料负载的增加，暴露的 FeSiNPs 数量也在增加。 对未拉伸和拉伸的 P(St-stat-nBA)/FeSiNP 薄膜进行的 SEM-EDS 分析证实，薄膜表面存在来自 FeSiNPs 的硅和铁（图 S4 和 S5，ESI†）。EDS 图谱显示，这些缝隙内的硅浓度可能更高（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.
使用单轴拉伸测试研究了 FeSiNP 对独立纳米复合薄膜机械性能的影响。拉伸强度随着 FeSiNP 浓度从 5 wt% (0.78 ± 0.08 MPa) 到 10 wt% (1.71 ± 0.21 MPa) 的增加而增加（表 S4，ESI†）。然而，进一步增加到 20 wt%后，拉伸强度（1.15 ± 0.09 兆帕）没有进一步提高。相反，随着 FeSiNP 浓度从 5 wt% (∼1448%) 增加到 10 wt% (847 ± 27%)，断裂伸长率下降。然而，当 FeSiNP 浓度增加到 20 wt% (860 ± 46%) 时，拉伸强度没有进一步变化（表 S4，ESI†）。我们推测，将填料浓度从 10 wt% 提高到 20 wt%，可能会在薄膜形成过程中诱发更大范围的 FeSiNPs 聚结。与 10 wt% 相比，20 wt% 添加量下的 FeSiNP 聚结程度更高，这一点得到了扫描电镜数据的支持，在拉伸薄膜条件下，纳米粒子聚结在缝隙中更加清晰可见。这种团聚会影响纳米复合薄膜随着应变的增加而消散应力的能力，从而影响拉伸强度和断裂伸长率。

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.
随后以 TMB 为底物对纳米复合薄膜的过氧化物酶模拟催化纳米酶活性进行了研究。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 的存在下会产生羟基自由基 (˙OH)、与 TMB 反应生成蓝色氧化产物（TMB_ox），在 652 纳米波长处吸收光，可用于定性和定量分析（图 4a）。⁵⁴ 在测试催化活性之前，我们对合成的 FeSiNPs 的反应条件进行了优化。首先，研究了缓冲液 pH 值和反应温度的影响，将 pH 值从 2.5 改为 7.5，将反应温度从室温（25 °C）改为 45 °C。催化活性最初有所提高，在 pH 值为 3.5 时达到最大活性（图 4b）。然而，pH 值进一步升高会导致催化活性持续显著降低。催化活性随着反应温度的升高而系统性降低，室温（25 °C）下表现最佳，45 °C下表现最低（图 4c）。除非另有说明，所有后续实验均选择并维持这些条件。接下来，研究了合成的纯净 FeSiNPs 的过氧化物酶模拟活性。正如预期的那样，FeSiNPs 浓度与催化性能之间呈线性相关，回归系数（R² ）为 0。973（图 4d）。为了确定 FeSiNPs 催化活性的机理，使用抗坏血酸作为氧自由基（˙OH 和 O₂˙^- ）清除剂。⁵⁵ 在加入抗坏血酸的情况下，催化反应的进行与上述类似，并对生成物的吸光度进行量化，以确定催化活性的变化。随着抗坏血酸的加入，吸光度明显下降（图 4e）。这些结果表明，抗坏血酸与 ˙OH 发生了反应，抑制了 TMB 的氧化，从而减少了蓝色产物的形成，证实其作用机制确实是由˙OH 自由基介导的。根据上述结果，催化机理与之前发表的报告一致，如图 S7 和公式 (S1)-(S4) 所述（ESI†）。⁵⁶ 总之、FeSiNP 中的铁（Fe²⁺ ）在第一步催化 H₂O₂ 还原，生成羟基自由基（˙OH）和氧化的 Fe³⁺ 。该反应是限制速率的步骤。接下来，生成的 ˙OH 自由基与 TMB 反应，形成蓝色的氧化产物（TMB_ox ）。在第三步中，˙OH 自由基与 H₂O₂ 反应生成 HO₂˙ 自由基、然后还原 Fe³⁺ 生成催化剂 Fe²⁺ 和一个氧分子。

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.
随后进行了稳态动力学研究，通过改变 H₂O₂ 或 TMB 的浓度来研究纯 FeSiNPs 的过氧化物酶催化活性，同时保持其他成分不变。动力学参数（K_m 和 V_max ）通过摩尔吸收系数（ε）为 39 000 M^-1 cm^-1 （在 652 纳米波长）计算出 TMB_ox 的摩尔吸收系数。⁵⁷ 由于天然过氧化物酶的酶活性通常遵循 Michaelis-Menten eqn (1)，本研究获得的数据被拟合到典型的 Michaelis-Menten 曲线（Fig.4f 和 g），使用非线性最小二乘拟合方法确定催化参数（K_m 和 V_max ）。K_m 表示酶对底物的亲和力；K_m 值越低，表明两者之间的亲和力越高。V_max 是转化为产物的最大速率。随着 H₂O₂ 和 TMB 浓度的增加，652 纳米波长处的吸光度值也随之增加（分别以速度、 图 4f 和 g 表示）。 在较高浓度的 TMB 和 H₂O₂ 中，催化过程没有受到抑制。此外，还生成了 Lineweaver-Burk 双折线图（ 图 4h 和 i），以估计纳米酶（FeSiNPs）对底物（TMB）的亲和力。计算得出的 K_m 和 V_max 值分别为 0.060 mM 和 0.672 × 10^-8 M s^-1 H₂O₂ 和 7.对于 TMB，分别为 143 mM 和 1.075 × 10^-8 M s^-1 。根据所得结果可以推断，FeSiNPs 对 H₂O₂ 的亲和力高于 TMB（K_m 更低）。与之前报道的其他纳米酶相比，这项工作中获得的 FeSiNPs 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（此处为 H₂O₂ ）的亲和力较高。 因此，与表 S5（ESI†）中列出的其他纳米酶相比，本研究中使用的 FeSiNPs 对 H₂O₂ 底物表现出更高的亲和力。仅考虑作为催化物种的铁成分，计算得出 FeSiNPs 的催化效率为 6.35 × 10^-4 s^-1 对 H₂O₂ 和 1.02 × 10^-3 s^-1 对 TMB 的催化效率。

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 负载的 P(St-stat-nBA)/FeSiNP 纳米复合薄膜的过氧化物酶模拟活性。制备的薄膜厚度相似，以消除厚度对催化性能的潜在影响（图 5a）。首先，我们研究了未拉伸薄膜，以获得其 "基线 "性能，并确定不同 FeSiNP 负载的影响。如图 5b-d 所示，FeSiNP 负载与催化性能（吸光度值）之间存在直接的相关性。正如预期的那样，FeSiNPs 含量的增加会导致催化性能随剂量的增加而增加，这可能是因为 SEM 分析（图 3）中观察到薄膜表面的缝隙中存在 FeSiNPs。在相同的实验条件下，不含 FeSiNPs 的纯 P(St-stat-nBA) 对照组的吸光度值与缓冲对照组相当（基线值≡0），这表明复合薄膜中的 FeSiNPs 确实起到了纳米酶的作用（图 S8，ESI†）。因此，可以推断出 P（St-stat-nBA ）/FeSiNP 薄膜通过使 FeSiNPs 发挥催化作用，具有作为纳米酶生物传感器的潜力。考虑到这些 P(St-stat-nBA)/FeSiNP 薄膜的高拉伸性，我们随后探讨了通过简单地将它们拉伸到不同程度（50% 和 100%）来调整催化性能的可能性。 随着拉伸度从 0 增加到 100%，P(St-stat-nBA)/FeSiNP (5 wt%)的蓝色产物（TMB_ox；催化性能的定量指标）的吸光度逐渐增加、而 P（St-stat-nBA）/FeSiNP（10 wt%）的吸光度则出现了急剧的线性增长。

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，薄膜表面（和缝隙）上存在的几乎所有 FeSiNPs 在未拉伸条件下都参与了催化作用，因此，拉伸仅导致催化活性略有增加（图 5b）。据推测，在纳米复合薄膜中，5 wt% 的 FeSiNPs 含量相当低，因此尽管进行了拉伸，仍有极少量的新表面或嵌入的 FeSiNPs 可用于催化。如果是这种情况，人们就会期望吸光度值会随着 FeSiNPs 含量的增加而提高，这正是在所有条件下（0、50 和 100% 拉伸）观察到的情况。在 20 wt% 的情况下，FeSiNPs 的含量过高，与较低的 NP 含量相比，会产生更强的催化反应，但同时也会导致 NP 在整个薄膜中产生更大的团聚。在拉伸条件下，这种聚结可能会限制薄膜中嵌入的先前未暴露的 NP 的数量，导致催化性能仅有微弱的提高（图 5d）。对于 10 wt% 的 FeSiNP 负载薄膜，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 聚结，但在从 0、50 到 100% 的拉伸条件下，先前未暴露的 NP 仍可持续用于催化（图 5c）。总之，这些结果突出表明，所开发的纳米复合薄膜提供了一个平台，通过简单的拉伸（图 5e）就能对催化性能进行前所未有的调整。

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.
如 (图 5f)所示，随着时间的推移，观察到线性催化反应（R2 = 0.994）。对这一观察结果的一个合理解释是，存在于薄膜深处的 FeSiNPs 随着时间的推移越来越多地暴露在试剂中，从而显示出催化活性。如果薄膜表面有足够的 FeSiNPs 随着时间的推移不断产生催化作用，那么吸光度就会趋于平稳，而不会随着时间的推移继续增加。这些数据突出表明，所开发的薄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，我们在催化反应的不同时间点拉伸了 P(St-stat-nBA)/FeSiNP (10 wt%)薄膜。我们将薄膜在含有 H₂O₂ 和 TMB 的反应缓冲液中培养了 18 小时，并记录了 0.2 以下的吸光度值，将其作为拉伸薄膜前的起点。随着时间的推移，未拉伸薄膜的催化性能略有下降（图 6）。各个时间点的这些值被视为 "基线"，用于比较薄膜拉伸后催化反应的变化。接下来，我们在培养 0、20 和 80 分钟后拉伸薄膜，并记录吸光度值与各自基线值相比的变化。

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.
所有薄膜在拉伸后的吸光度值都有明显增加，而且无论在培养后 0 分钟、20 分钟还是 80 分钟拉伸薄膜，所有拉伸薄膜的吸光度值都有相似的增加（80 分钟拉伸薄膜的反应略低）。这些数据凸显了所开发的纳米酶载薄膜的可调性，即可以按需增强活性，也可以在其性能寿命期间进行更新。综上所述，我们认为所开发的薄膜是一种高度可调的平台，具有长期的功能活性，只需拉伸纳米复合薄膜即可使催化功能变得活跃。

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.
本研究旨在制备纳米酶载可调聚合物纳米复合薄膜，以实现按需、灵敏和持续的过氧化物酶模拟活性。采用微型乳液聚合法制备了含有氧化铁负载多孔二氧化硅纳米酶（FeSiNP）的 P(St-stat-nBA) 薄膜，其中 FeSiNP 起着过氧化物酶模拟物的作用。负载纳米酶的薄膜表现出与纳米酶（FeSiNP）浓度相关的催化活性，随着纳米粒子负载量的增加，催化活性也随之提高。当拉伸时，催化性能随着拉伸程度从 0 到 100% 的增加而增加。此外，薄膜随着时间的推移表现出持续的线性响应，证实了其促进连续检测的能力。不同时间点的按需切换显示，只需拉伸 10 wt% FeSiNPs 负载的薄膜，催化性能就会急剧增加，从而成功激活。总之，所开发的纳米复合薄膜具有反应灵敏、可调的过氧化物酶模拟活性。这项研究首次展示了可按需调节酶模拟活性的可活化薄膜，从而为未来开发用于各种诊断应用的智能传感器搭建了平台。

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.
本文数据可在新南威尔士大学公共资料库https://doi.org/10.26190/unsworks/30229中查阅。

Conflicts of interest  利益冲突

No conflict to declare.  无冲突申报。

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）提供的早期职业研究奖学金（GNT1139060）和新南威尔士大学安全网奖学金。作者感谢悉尼新南威尔士大学马克-温莱特分析中心 (Mark Wainwright Analytical Centre, MWAC) 的澳大利亚显微镜研究所 (Microscopy Australia) 电子显微镜组 (EMU) 和固体与元素分析组 (Solid State & Elemental Analysis Unit) 提供的设施和科技协助。

References 参考资料

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