A one-pot and modular self-assembly strategy for high-performance organized enzyme cascade bioplatforms based on dual-functionalized protein–PtNP@mesoporous iron oxide hybrid

Abstract

Inspired by the delicate structure and prominent efficiency of natural multiple-enzyme systems, combining nanotechnologies such as nanomaterials, self-assemblies, and enzyme mimics is fascinating for the development of next-generation high-performance organized enzyme cascade bioplatforms. In our facile and convenient design, a dual-functionalized β-casein-Pt nanoparticles@mesoporous-Fe₃O₄ (CM-PtNP@m-Fe₃O₄) hybrid acts as both a nanozyme with outstanding peroxidase-like activity and a scaffold to immobilize and stabilize a natural oxidase, resulting in a high-performance organized enzyme cascade bioplatform for a one-pot assembly procedure. Owing to special physicochemical surface properties, the multipoint attachment of various interactions between natural enzymes and protein/inorganic hybrids leads to efficient immobilization of the enzyme with retained activity. The proposed cascade bioplatform provides superior cholesterol sensing, including simplicity (one-step detection), reusable enzymes (peroxidase mimic and oxidase), and excellent sensitivity (detection limit, 0.05 μM). To our knowledge, the bioplatform presented in this work shows the highest sensitivity for cholesterol detection among all reported colorimetric methods based on nanozymes. Therefore, the highly rationally designed protein/inorganic hybrid and dual-functional strategy used in this study will provide a facile one-pot and effective high-performance organized enzyme cascade bioplatform with potential applications in biosensing, biotransformation, decontamination, and biofuel.

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Introduction

In a natural system, enzyme cascade reactions are efficient methods for improving the catalytic performance of enzymes.^1–3 Owing to their extraordinarily high efficiency, enzyme cascade reactions with promising applications in biocatalysis and biosensing are of great interest both academically and industrially.^4–6 Cooperating enzymes are often confined in the same compartment to perform a cascade reaction efficiently by mainly providing a high local concentration of enzymes and substrates, and efficient mass transfer. Currently, the construction and application of organized enzyme cascade systems with synergistic and complementary functions remains at the preliminary stage, because some drawbacks, including high cost, low stability, and inactivation, can be amplified when natural enzymes are used to fabricate an enzyme cascade system.^7–9

Recently, nanomaterial-based artificial enzymes (nanozymes) have attracted increasing attention and shown potential in biological, environmental, food, and medical applications owing to advantages such as low cost, stability, and tunable catalytic activities.^10–15 Integrating nanozymes and natural enzymes into appropriate scaffolds has attracted much attention as an effective approach to developing enzyme cascade platforms.^16–20 However, many methods require scaffold preparation or complicated surface linkages between the scaffold and natural enzyme, which inevitably affects the enzyme activity and leads to the cascade system having low efficiency. Furthermore, hindered substrate access and diffusion within the scaffold can also lead to low efficiency in the cascade system.

The direct combination of nanozymes and natural enzymes to fabricate enzyme cascade systems without a scaffold via self-assembly is convenient and facile. However, the adsorption of desired enzymes directly onto naked nanomaterial surfaces might result in denaturation or a loss in bioactivity.^21–24 Furthermore, the nonspecific attachment of biomolecules might lead to a loss in nanozyme activity.^25,26 Therefore, a key challenge in fabricating enzyme cascade systems through the direct binding of natural enzymes to nanozymes is retaining the activity of both natural enzymes and nanozymes. To this end, tailoring the physicochemical properties of nanozymes, including size, shape, component, and surface chemistry, allows interactions between the nanozyme and natural enzyme to be adjusted, providing the possibility of retaining both their activities.

Among various nanozymes, Pt nanozyme has attracted sustained attention and has been shown to be effective and useful in biosensing applications owing to its superior activity and excellent biocompatibility.^27–29 However, cascade systems concerning Pt nanozymes have usually involved multiple separate processes.^30–34 Evidently, multistep analysis and unrecoverable enzymes lead to complicated detection processes and enzyme waste (both of natural oxidase and Pt nanozyme). Although organized cascade systems based on Pt/Fe₃O₄ hybrid peroxidase nanozyme and glucose oxidase have been developed,³⁵ the peroxidase activity of the nanozyme and efficiency of the integrated enzyme cascade system has not been studied. In previous work, the binding process of the natural enzyme to the hybrid has been complex and slow, which might lead to the bound nature oxidase having reduced activity. Therefore, the meticulous design of Pt nanozymes with extremely high enzyme-like activity and the ability to immobilize a natural enzyme to fabricate high-performance organized enzyme cascade systems is challenging.

Owing to their unique magnetic properties and relatively good biocompatibility, iron oxide nanozymes have received widespread attention since they were found to possess intrinsic peroxidase mimic activity by the Yan group.¹⁰ Compared with Fe₃O₄ nanoparticles (NPs), mesoporous Fe₃O₄ nanospheres (m-Fe₃O₄) have received considerable attention owing to their large surface areas, high saturation magnetization, and abundant active surface sites.^36,37 Furthermore, magnetic nanomaterials are superior for enzyme immobilization owing to easy separation by magnetic fields. As an effective approach, combining the individual properties of each component will contribute to the multifunctionality and appealing properties of Pt NP/m-Fe₃O₄ hybrid nanozymes.^38,39 However, PtNP/m-Fe₃O₄ hybrid-based integrated cascade platforms have remained unexplored.

Significantly, the hydrophilic–hydrophobic properties of nanozyme surfaces are heavily related to the mass transfer and enzyme activity of the enzyme cascade system.^40,41 Therefore, the careful design of nanozymes with balanced hydrophilic/hydrophobic properties is required. In addition to bearing many functional groups, such as thiol, disulfide, amino, carboxylic, and imidazole groups, proteins are complex amphiphilic biopolymers with hydrophobic and hydrophilic patches on their surfaces. Desired features and functions can be achieved by introducing amphiphilic proteins into inorganic nanozymes, providing an opportunity for molecular level regulation.^42–44 This will contribute to the development of nanozyme-based integrated cascade platforms with extremely high activity and selectivity.

Cholesterol detection is an ongoing concern in biomedical fields and has played a crucial role in improving public health. Among various technologies, colorimetric detection has attracted considerable attention because it provides a simple and convenient platform, and does not require sophisticated instruments, highly trained operators, or complex processes. However, to our knowledge, no investigations into the colorimetric detection of cholesterol using an integrated Pt nanozyme/nature oxidase cascade system have been performed.

Herein, for the first time, we adopted a structural-design approach to create an organized cascade platform based on a β-casein-Pt nanoparticles@mesoporous-Fe₃O₄ (CM-PtNP@m-Fe₃O₄) hybrid nanozyme (Scheme 1). The model protein used, β-casein, has a high content of acidic amino acid residues (such as glutamic acid and aspartic acid residues), is negatively charged at neutral pH, and contains a negatively charged hydrophilic N-terminal region and strongly hydrophobic C-terminal region.^45,46 Specifically, compared with pure mesoporous Fe₃O₄ nanospheres and Pt nanoparticles, the synergistic effect of PtNP and m-Fe₃O₄ results in the hybrid having significantly enhanced peroxidase-mimicking activity. Furthermore, the meticulously designed CM-PtNP@m-Fe₃O₄ nanohybrid has a versatile hierarchical nanoporous structure and unique surface physicochemistry, which allows multipoint attachment through various interactions for immobilizing and stabilizing the natural oxidase and optimizing the enzymatic cascade reaction. This high performance enzyme cascade platform led to one-step cholesterol detection with excellent sensitivity and selectivity. These findings provide a new strategy and direction for the customization of protein/inorganic hybrids with ingenious hierarchical nanostructures and unique surface physicochemical properties, and a one-pot self-assembly method for fabricating high-performance enzyme cascade bioplatforms.

Scheme 1 Schematic illustration of fabrication of the ChOx/CM-PtNP@m-Fe₃O₄ cascade platform, and the corresponding enzyme cascade reaction.

Experimental section

Reagents and chemicals

β-Casein was purchased from Sigma (>99%). Chloroplatinic acid hexahydrate (H₂PtCl₆·6H₂O, 99%), 3,3′,5,5′-tetramethylbenzidine (TMB), and casein enzyme hydrolysate were purchased from Sigma. Cholesterol oxidase from Microbe (5 U mg⁻¹) was purchased from Worthington Biochemical Company. Ferric chloride (FeCl₃), ethylene glycol, trisodium citrate (C₆H₅N₃O₇·2H₂O), sodium acetate (CH₃COONa), and 30% H₂O₂ were purchased from Shanghai Chemical Reagent Company (Shanghai, China). All other reagents and chemicals were of reagent grade and used directly in experiments. Deionized water (18.2 MΩ) was used throughout this study.

Instrumentation and characterization

X-ray diffraction was conducted on a Bruker AXS D8 ADVANCE X-ray diffractometer. The composite morphology was examined by field-emission scanning electron microscopy (Zeiss Supra 55 VP FEG). Fourier transform infrared (FT-IR) spectra of samples were recorded in the range 400–4000 cm⁻¹ using an FT-IR spectrometer (Nicolet-740), with samples prepared in pellet form using spectroscopic-grade KBr. X-ray photoelectron spectroscopy (XPS) was performed using an ESCALAB 250Xi spectrometer (Thermo Fisher Co., USA) with an Al X-ray source (1350 eV). An ultraviolet-visible (UV-vis) spectrophotometer (UV-2501, Shimadzu Corp., Japan) was used to record absorption spectra and measure absorbance.

Preparation of m-Fe₃O₄ nanospheres, CM-PtNP@m-Fe₃O₄, and ChOx/CM-PtNP@m-Fe₃O₄ nanohybrid

m-Fe₃O₄ nanospheres were synthesized according to the solvothermal method with some modifications.⁴⁷ FeCl₃ (0.2 M) and C₆H₅N₃O₇·2H₂O (32 mM) were dissolved in ethylene glycol (25 mL) and then CH₃COONa (0.73 M) was added with stirring. The mixture was continually stirred at 1000 rpm for 120 min at 90 °C and transferred into a Teflon-lined stainless-steel autoclave. The autoclave was then heated at 200 °C for 12 h. The resulting Fe₃O₄ nanospheres were washed with ethanol and deionized water several times and then stored in deionized water at room temperature.

The CM-PtNP@m-Fe₃O₄ nanohybrid was prepared by mixing chloroplatinic acid solution (400 μL, 3 mM) with β-casein solution (40 μL, 1 mg mL⁻¹) in PBS buffer (2.76 mL, 10 mM) containing m-Fe₃O₄ (pH 7). After stirring for 3 h at 35 °C, ice-cold NaBH₄ solution (400 μL, 10 mM) was added under stirring. After reaction completion (10 h), the hybrid was separated using a permanent magnet and washed with water three times.

ChOx (1 mL, 5 mg mL⁻¹) was added to the as-prepared CM-PtNP@m-Fe₃O₄ nanohybrid solution (1 mL). The resulting mixture was incubated at ambient temperature under ultrasonication for 2 h. The ChOx/CM-PtNP@m-Fe₃O₄ nanohybrid was separated using a permanent magnet. To estimate the amount of ChOx adsorbed on the CM-PtNP@m-Fe₃O₄ nanohybrid, the supernatant (obtained from a solution containing CM-PtNP@m-Fe₃O₄ nanohybrid and ChOx) was measured by UV adsorption.

Peroxidase-like activity of CM-PtNP@m-Fe₃O₄ nanohybrid

The peroxidase-like activity of the as-prepared CM-PtNP@m-Fe₃O₄ nanohybrid was investigated in the catalytic oxidation of peroxidase substrate TMB in the presence of H₂O₂. In a typical experiment, TMB (30 μL, 8.0 mM), CM-PtNP@m-Fe₃O₄ stock solution (10 μL), and H₂O₂ (60 μL, 4 M) were added into acetate buffer (2.9 mL, 0.2 M, pH 4.0) at 25 °C. The solution was transferred for UV-vis spectrophotometry after incubating for 15 min.

Determination of H₂O₂

The CM-PtNP@m-Fe₃O₄ nanohybrid (20 μL) was introduced into acetate buffer (2880 μL, pH 4.0), followed by the addition of TMB solution (50 μL, 40 mM) and H₂O₂ (50 μL, various concentrations). The mixture was incubated at 25 °C for 20 min with continuous oscillation. The final reaction solution was used to perform absorption spectroscopy measurements.

Cholesterol detection using ChOx/CM-PtNP@m-Fe₃O₄ nanohybrid

Cholesterol (30 μL, various concentrations) was added to acetate buffer (2.9 mL, 0.1 M, pH 4.0) containing the ChOx/CPtNP@m-Fe₃O₄ nanohybrid (20 μL) and TMB (50 μL, 48.0 mM). The resulting solution was incubated for 50 min at 40 °C. The ChOx/CPtNP@m-Fe₃O₄ nanohybrid was then isolated from the reaction solution using an external magnetic field. The final reaction solution was used to perform adsorption spectroscopy measurements.

Determination of cholesterol in human serum samples

Human serum samples from a local hospital were used to measure the amount of cholesterol. TMB (50 μL, 48 mM), ChOx/CPtNP@m-Fe₃O₄ nanohybrid solution (20 μL), and serum sample (30 μL) were added into acetate buffer (2.9 mL, 0.2 M, pH 4.0) at 40 °C. The solution was transferred for UV-vis spectrophotometry after incubating for 50 min.

Results and discussion

Characterization of CM-PtNP@m-Fe₃O₄ nanohybrid

The as-prepared CM-PtNP@m-Fe₃O₄ nanohybrid was first identified from the X-ray diffraction (XRD) pattern. For comparison, the standard position and relative intensities of Pt (JCPDS 04-0802) and Fe₃O₄ (JCPDS 19-0629) crystals have been provided in Fig. S1A and B (ESI†).^48,49 In addition to the identified Fe₃O₄ peaks, the CM-PtNP@m-Fe₃O₄ nanohybrid showed all major peaks contributed by Pt, indicating that it was composed of Fe₃O₄ and Pt. Fig. 1A shows TEM images of the CM-PtNP@m-Fe₃O₄ nanohybrid, which displayed mesoporous spherical nanostructures formed by the assembly of small-sized nanospheres. The SEM image confirmed the mesoporous spherical assembled nanostructure of the hybrid (Fig. S2, ESI†). A similar structure was observed for the m-Fe₃O₄ nanosphere sample (Fig. S3, ESI†). This indicated that Pt nanoparticles of similar sizes were grown on the m-Fe₃O₄ nanosphere, which could not be distinguished from Fe₃O₄ nanoparticles in the TEM images. HRTEM images showed that the lattice fringes had lattice spacing distances of 0.24 and 0.33 nm (Fig. 1B), attributed to the (111) plane of Pt and (311) plane of Fe₃O₄ nanoparticles, respectively. The as-prepared CM-PtNP@m-Fe₃O₄ nanohybrids were also characterized by HAADF-STEM. The elemental mapping images showed Fe, Pt, O, N, and C elements within the nanostructure (Fig. 1D–H). Energy-dispersive X-ray spectroscopy (EDX) confirmed that the CM-PtNP@m-Fe₃O₄ nanohybrid was composed of C, O, N, Pt, and Fe without other impurities (Fig. S1C, ESI†). The ratio of Fe and Pt atoms in the hybrid was about 100 : 2.6, as estimated from the EDX analysis. Notably, the very low Pt content in the hybrid reduced the use of Pt, which will enhance the application of Pt-based nanozymes in practice. The inset in Fig. 1A shows a digital image of the CM-PtNP@m-Fe₃O₄ nanohybrid in the absence and presence of a magnetic field in aqueous solution. The excellent magnetic response of the CM-PtNP@m-Fe₃O₄ nanohybrid allowed instantaneous separation using a magnet, because m-Fe₃O₄ in the CM-PtNP@m-Fe₃O₄ nanohybrid was composed of many primary Fe₃O₄ particles.⁵⁰ The nanohybrid exhibited an excellent magnetic response, which contributed to the recycling of CM-PtNP@m-Fe₃O₄.

Fig. 1 (A) TEM image and (B) HRTEM image of CM-PtNP@m-Fe₃O₄ nanohybrid. (C) HAADF-STEM image, and corresponding TEM elemental mappings of (D) C, (E) N, (F) O, (G) Fe, and (H) Pt signals.

X-ray photoelectron spectroscopy (XPS) was used to further clarify the nanohybrid composition and surface information. The XPS spectrum showed the presence of C, O, N, Pt, and Fe (Fig. S4A, ESI†). In Fig. S4B (ESI†), two broad peaks at binding energies of around 710.9 and 724.5 eV are assigned to Fe 2p_3/2 and Fe 2p_1/2, respectively, which are characteristic of the Fe₃O₄ phase. As shown in Fig. S4C (ESI†), the Pt 4f_7/2 electron spectrum of the CM-PtNP@m-Fe₃O₄ nanohybrid could be deconstructed into Pt⁰ and Pt²⁺ components with binding energies of 71.4 eV and 72.4 eV, respectively. Furthermore, the PtNPs peak (Pt²⁺ 4f_7/2, 72.4 eV) shifted toward a lower binding energy (approx. 0.8 eV) compared with that of Pt²⁺ (73.2 eV), suggesting electron transfer from the carboxylic group of the proteins to the Pt surface. The existence of Pt²⁺ species might be due to coordination of Pt nanoparticles to –COO⁻ groups in casein, which contains a high content of acid amino acid residues, such as aspartate and glutamate residues.

Fig. S5A (ESI†) shows the FTIR spectra of native β-casein and the CM-PtNP@m-Fe₃O₄ nanohybrid. The bands at 1447 and 1398 cm⁻¹ due to the COO⁻ groups of Asp and Glu residues 3334 had significantly changed in the CM-PtNP@m-Fe₃O₄ nanohybrid, demonstrating that Asp and Glu residues bind with metal via carboxyl groups. Furthermore, the band shift from 1645 to 1632 cm⁻¹ indicated that an unordered structure transformed into the extended β-casein chain in the CM-PtNP@m-Fe₃O₄ nanohybrid. Furthermore, the appearance of a strong peak at 1052 cm⁻¹, contributed by alkoxy stretching vibrations, demonstrated the interaction between metal and OH groups. For comparison, the FTIR spectra of CM/m-Fe₃O₄ and CM-PtNP are shown in Fig. S5B (ESI†). The spectrum of the CM-PtNP@m-Fe₃O₄ nanohybrid was different from those of both CM/m-Fe₃O₄ and CM-PtNP, which indicated that the protein might bind to m-Fe₃O₄ and CM-PtNP together. Functional groups in proteins, including –NH₂, –COOH, and –OH, exhibit high affinities for metal ions. β-Casein contains about 200 amino acid residues, including a high content of acidic amino acid residues (such as glutamic acid and aspartic acid residues). All these functional groups, including –NH₂, –COOH, and –OH (especially the high content of carboxyl groups), can bind to Fe₃O₄ and Pt nanoparticles through complexation.

The specific surface area and pore volume of the CM-PtNP/m-Fe₃O₄ nanohybrid were characterized using the nitrogen sorption technique, with a typical isotherm shown in Fig. S6 (ESI†). The Brunauer–Emmett–Teller (BET) specific surface area of the hybrid was measured as 79.3 m² g⁻¹, which was similar to that of many reported mesoporous Fe₃O₄ structures.^51–53 The Barrett–Joyner–Halenda (BJH) average pore diameter calculated from the adsorption branch of the isotherms was 3.8 nm in the hybrid.

Peroxidase-like activity of CM-PtNP@m-Fe₃O₄ nanohybrid

Among the most common chromogenic substrates of peroxidase, TMB was chosen to demonstrate the peroxidase-like activity of the CM-PtNP@m-Fe₃O₄ nanohybrid. As shown in Fig. 2A, the CM-PtNP@m-Fe₃O₄ nanohybrid could catalyze the oxidation of TMB by H₂O₂ to produce the typical blue color with maximum absorbance at 652 nm. Control experiments showed that the TMB–H₂O₂ system and the CM-PtNP@m-Fe₃O₄ nanohybrid–TMB system had no obvious color change, confirming the peroxidase-like activity of the CM-PtNP@m-Fe₃O₄ nanohybrid. For comparison, we studied the peroxidase activity of m-Fe₃O₄ nanospheres and CM-PtNPs (Fig. 2B). Significantly, CM-PtNP@m-Fe₃O₄ showed a much higher activity toward TMB than individual m-Fe₃O₄ nanospheres and CM-PtNPs, which indicated that the synergistic effect of PtNP and m-Fe₃O₄ efficiently improved the peroxidase-like activity of the hybrid.

Fig. 2 (A) UV-vis spectra of different reaction systems: (a) TMB + H₂O₂ + CM-PtNP@m-Fe₃O₄ nanohybrid, (b) H₂O₂ + TMB, and (c) CM-PtNP@m-Fe₃O₄ nanohybrid + TMB in pH 4.0 acetate buffer at 25 °C. Inset: Corresponding photographs. (B) Enzyme activities of CM-PtNP@m-Fe₃O₄, CM-PtNP, and m-Fe₃O₄. (C) Typical UV-vis absorbance spectra of TMB and H₂O₂ in the presence of CM-PtNP@m-Fe₃O₄ nanohybrid with different H₂O₂ concentrations. (D) Linear calibration curve of corresponding absorbance at 652 nm vs. H₂O₂ concentration.

Similar to peroxidase and other nanomaterial-based peroxidase mimics, the catalytic activity of CM-PtNP@m-Fe₃O₄ was also dependent on pH, temperature, and substrate concentration. The optimal pH and temperature were pH 4.0 and 25 °C, respectively (Fig. S7A and B, ESI†). As shown in Fig. S7A (ESI†), the enzyme activity of CM-PtNP@m-Fe₃O₄ changed less than 20% between 15 to 35 °C, which indicated that the hybrid can be used more freely among a range of ambient temperatures. The apparent steady-state kinetic parameters were measured to assess the peroxidase activity of CM-PtNP@m-Fe₃O₄. In a certain substrate concentration range, typical Michaelis–Menten curves were obtained for both TMB and H₂O₂ (Fig. S8, ESI†). The Michaelis–Menten constant (K_m) and maximum initial velocity (V_max) were obtained using a Lineweaver–Burk plot, with the results shown in Table S1 (ESI†). The small apparent K_m value of CM-PtNP@m-Fe₃O₄ with both TMB (0.257 mM) and H₂O₂ (0.036 mM) as substrates indicated that CM-PtNPs had a high affinity for both TMB and H₂O₂. Meanwhile, the V_max values of the CM-PtNP@m-Fe₃O₄ nanohybrid with TMB and H₂O₂ as substrates were higher than those of the other catalysts, suggesting a higher peroxidase-like activity toward the catalytic reaction due to the synergistic effect of protein, PtNPs, and m-Fe₃O₄. Notably, caseins can be thought of as amphiphilic block copolymers consisting of blocks with high levels of hydrophobic or hydrophilic amino acid residues.^45,46 Therefore, both hydrophobic interactions and electrostatic attraction between casein and TMB led to the high affinity of TMB for the hybrid. The high affinity of H₂O₂ for the hybrid was not due to its facile adsorption onto Fe₃O₄, but mainly the synergistic effects of every component in the hybrid (the protein, PtNPs, and Fe₃O₄). Furthermore, the microporous structure of the m-Fe₃O₄ NPs caused the reactant and product molecules to diffuse freely in and out the hybrid.^27,50 These factors contributed to the high peroxidase-like activity of the CM-PtNP@m-Fe₃O₄ nanohybrids synergistically.

H₂O₂ is an important enzymatic intermediate produced by many enzyme–substrate reactions and substances used in various areas. As the CM-PtNP@m-Fe₃O₄ hybrid possesses outstanding peroxidase-like activity and a high affinity for H₂O₂, it can be used to quantitatively detect H₂O₂ concentrations using TMB as substrate. Fig. 2C shows the gradual increase in the absorbance at 652 nm of the TMB system with increasing H₂O₂ concentration. As shown in Fig. 2D, the linear range for H₂O₂ detection was 0.01–1000 μM, with a detection limit of 1 nM (signal-to-noise ratio = 3). Compared with other nanozymes in earlier studies (Table S2, ESI†), the H₂O₂ sensor using the CM-PtNP@m-Fe₃O₄ nanohybrid was much more sensitive and had a much wider linear range.

We further studied the stability and reusability of the CM-PtNP@m-Fe₃O₄ nanohybrid. The response sensitivity was more than 97% retained over one month and the catalytic activity was 90% maintained after five cycles, indicating the high stability of the as-prepared CM-PtNP@m-Fe₃O₄ nanohybrid (Fig. S9, ESI†). Repeated use of the hybrid nanostructure did not significantly alter its morphology (Fig. S10, ESI†), indicating the excellent structural stability of the hybrid nanostructure.

Fabrication of organized ChOx/CM-PtNP@m-Fe₃O₄ cascade bioplatform

Cholesterol monitoring is among the most studied topics in biosensing, as determining the cholesterol concentration in blood is a considerably important factor in human health. Encouraged by the outstanding performance of the CM-PtNP@m-Fe₃O₄ nanohybrid, incorporating cholesterol oxidase (ChOx) into the hybrid was a facile and novel strategy for constructing an enzyme cascade reaction system for cholesterol detection with excellent sensitivity.

Enzyme immobilization is a key factor affecting biosensor performance. The nanostructure and surface physicochemical features of the CM-PtNP@m-Fe₃O₄ nanohybrid could affect the immobilization of ChOx and its catalytic properties. When ChOx was incubated with the CM-PtNP@m-Fe₃O₄ nanohybrid in phosphate buffer solution, ChOx molecules were spontaneously entrapped in the CM-PtNP@m-Fe₃O₄ nanohybrid, as confirmed by UV-vis and FTIR spectra results (Fig. 3A and B). The enzyme-coated hybrids were purified from the excess enzyme via three-fold magnetic separation/redispersion. The complete removal of unbound (free) enzymes from the enzyme-coated nanohybrid was confirmed by the supernatant of the third purification step not showing a protein absorbance peak (280 nm for ChOx) in the UV-vis spectrum. The immobilization yield was about 60%, as determined by UV-vis spectroscopy, indicating that the hybrid exhibited significantly higher immobilization efficiency for ChOx. FTIR spectroscopy has been well established as the method of choice for analyzing protein secondary structure.^54,55 As shown in Fig. 3B, compared with the FTIR spectra of individual ChOx and CM-PtNP@m-Fe₃O₄ nanohybrids, the spectrum of the ChOx/CM-PtNP@m-Fe₃O₄ nanohybrid indicated that ChOx had binded to the CM-PtNP@m-Fe₃O₄ nanohybrid. Furthermore, the peak intensity increased in the range 554–640 cm⁻¹, attributed to Fe–O vibration, demonstrating the interaction between the enzyme and m-Fe₃O₄ in the CM-PtNP@m-Fe₃O₄ nanohybrid. Simultaneously, bands at 1453 and 1399 cm⁻¹ attributed to COO⁻ of Asp and Glu residues 3334 had changed significantly after the binding of ChOx to the CM-PtNP@m-Fe₃O₄ nanohybrid (Fig. 3B), which indicated that Asp and Glu residues of ChOx contributed to the natural enzyme binding to the CM-PtNP@m-Fe₃O₄ nanohybrid. Significantly, compared with the FTIR spectrum of ChOx, the band at 1656 cm⁻¹ indicated that the α-helix structure of ChOx was retained in the hybrid. Therefore, interaction with the CM-PtNP@m-Fe₃O₄ nanohybrid did not lead to the compact α-helix structure being lost, which is important for preserving ChOx activity. Fig. S11 (ESI†) shows the XPS survey scan spectrum of the ChOx/CM-PtNP/m-Fe₃O₄ nanohybrid. Compared with the CM-PtNP/m-Fe₃O₄ nanohybrid XPS spectrum, the intensity of peaks assigned to C, O, and N (Fig. S11A, ESI†) had increased, and the Pt⁰ 4f_7/2 and Fe 2p peaks had shifted (Fig. S11B and C, ESI†), confirming binding of the oxidase to the hybrid. Compared with the SEM image of CM-PtNP/m-Fe₃O₄, the SEM image of the ChOx/CM-PtNP/m-Fe₃O₄ nanohybrid indicated the binding of oxidase to the hybrid (Fig. S12, ESI†).

Fig. 3 (A) UV-vis spectra of ChOx before and after blending with CM-PtNP@m-Fe₃O₄ (enzyme solution diluted 10-fold). (B) FTIR spectra of CM-PtNP@m-Fe₃O₄ nanohybrid, ChOx, and ChOx/CM-PtNP@m-Fe₃O₄ nanohybrid. (C) UV-vis absorbance spectra of TMB + H₂O₂ system catalyzed by CM-PtNP@m-Fe₃O₄ and ChOx/CM-PtNP@m-Fe₃O₄ nanohybrids. (D) Oxidase activity of ChOx/CM-PtNP@m-Fe₃O₄ nanohybrid and natural ChOx.

The binding of polymers to nanoparticles is known to lead to reduced nanoparticle activity. Simultaneously, direct interactions between nanoparticles and enzyme can induce conformational changes and reduced activity in natural enzymes. Therefore, both the peroxidase and oxidase activities of the ChOx/CM-PtNP@m-Fe₃O₄ nanohybrid were measured. As shown in Fig. 3C, the peroxidase activity of the CM-PtNP@m-Fe₃O₄ nanohybrid was decreased to about 80% of the original value due to ChOx binding. As the CM-PtNP@m-Fe₃O₄ nanohybrid exhibited extremely high activity, the ChOx/CM-PtNP@m-Fe₃O₄ nanohybrid still possessed sufficiently high activity for fabrication of the following cascade system. The activity of ChOx in the ChOx/CM-PtNP@m-Fe₃O₄ nanohybrid was measured using a two-step method, namely, HRP to detect H₂O₂ generated from the oxidation of cholesterol by O₂ in the presence of the ChOx/CM-PtNP@m-Fe₃O₄ nanohybrid. Compared with native ChOx at the same concentration, the activity of CM-PtNP@m-Fe₃O₄-bound ChOx was slightly higher (Fig. 3D), indicating that the hybrid provided a favored microenvironment to immobilize oxidase and preserve the oxidase activity.

To confirm the role of amphiphilic protein in fabricating the cascade platform, control experiments were conducted. The catalytic efficiencies of bare PtNP@m-Fe₃O₄ nanohybrid and a casein enzymatic hydrolysate (CEH)-modified PtNP@m-Fe₃O₄ nanohybrid were assessed under the same experimental conditions (Fig. 5B). Casein hydrolysates are mixtures of small peptides that lack the amphiphilicity of a protein. As shown in Fig. 5B, the response of cholesterol to treatment with the bare PtNP@m-Fe₃O₄ and CEH/PtNP@m-Fe₃O₄ nanohybrid systems with ChOx was very low, which indicated that these two hybrids did not provide an environment suitable for immobilizing ChOx or preserving the ChOx activity. The UV-vis absorption spectra indicated that both PtNP@m-Fe₃O₄ and the CEH/PtNP@m-Fe₃O₄ nanohybrid could not immobilize ChOx (Fig. S13, ESI†). These results were in agreement with previous reports that natural enzymes are preferentially immobilized and retain their activity in microdomains with balanced hydrophilic/hydrophobic properties.^56,57 Therefore, the presence of amphiphilic protein provides an optimal environment for immobilizing the natural enzyme (Scheme 1), which could potentially reduce unwanted direct interactions between inorganic particles and oxidase. This will also contribute to preserving the activity of the natural enzyme and the peroxidase-like activity of the hybrid.

Cholesterol detection using CM-PtNP@m-Fe₃O₄ nanohybrid-triggered cascade platform

When the cholesterol solution was added, cascade catalysis of cholesterol oxidation and subsequent TMB oxidation occurred (Fig. 4A). Control experiments were performed in the absence of the hybrid, with no notable color change observed. This oxidation was promoted by increased cholesterol concentrations, which supplied more H₂O₂ for TMB oxidation catalyzed by the cascade system. To obtain optimal assay performance, the reaction temperature and pH were optimized (Fig. S14, ESI†). The optimal conditions were set as pH 4.0 and 40 °C. The formed cascade platform was used to detect cholesterol. The absorbance of the reaction products at 652 nm was proportional to the cholesterol concentration in the range 0.1–400 μM (Fig. 4B), with a linear correlation of 0.999. The estimated detection limit for cholesterol (S/N = 3) was 50 nM. The analytical results obtained using our method, including the linear range and cholesterol detection limit, show outstanding progress compared with previously reported detection limits of 5 μM¹⁷ (Table S3, ESI†). Compared with a previous Fe₃O₄ nanoparticle-based cascade platform,¹⁷ the CM-PtNP@m-Fe₃O₄ nanozyme-based cascade platform exhibited much higher sensitivity. This sensitivity was mainly due to: (i) the CM-PtNP@m-Fe₃O₄ nanozyme having outstanding peroxidase-like activity; (ii) the hybrid nanozyme serving as a scaffold to immobilize and stabilize natural oxidase with retained activity; (iii) the amphiphilic protein in the hybrid contributing to binding of the hydrophobic cholesterol substrate to the hybrid; and (iv) the diffusion-limited kinetics in the silicon dioxide scaffolds being eliminated and the hybrid contributing to efficient mass transfer.

Fig. 4 (A) UV-vis absorbance spectra of TMB and cholesterol in the presence of CM-PtNP@m-Fe₃O₄ nanohybrid with different cholesterol concentrations. (B) Linear calibration curve of corresponding absorbance at 652 nm vs. cholesterol concentration. [TMB] = 0.8 mM, pH = 4.0, T = 40 °C.

According to all aforementioned results and discussions, a schematic illustration of the enzyme mimic cascade system on the ChOx/CM-Pt NP@m-Fe₃O₄ nanohybrid is proposed in Scheme 1, which aids understanding of the high-efficiency generation of active radicals by mimicking an enzyme cascade pathway. The combination of the porous structure and the physicochemical surfaces properties of the CM-Pt NP@m-Fe₃O₄ nanohybrid provides an optimal environment for immobilizing and stabilizing ChOx, resulting in an oxidase/peroxidase bienzyme cascade system, as shown in Scheme 1. Benefiting from activation of the immobilized ChOx, cholesterol will be oxidized to form H₂O₂ as the oxygen-containing product. This H₂O₂ then acts as the substrate for the ChOx/CM-PtNP@m-Fe₃O₄ nanohybrid, which oxidizes TMB to a colored product, TMB_ox (λ = 650 nm). Here, the absorbance change at 650 nm (owing to the quantity of generated TMB_ox) is indirectly related to, and could be used to quantify, the cholesterol concentration. During the reaction process, the hydrophobic microdomain provided by casein molecules contributes to the binding of cholesterol to the CM-PtNP@m-Fe₃O₄ nanohybrid via hydrophobic interactions because cholesterol is hydrophobic, resulting in a high affinity of the substrate towards the cascade system. Furthermore, the nanoporous structure of the CM-PtNP@m-Fe₃O₄ nanohybrid promotes an increase in the reaction diffusion rate of reactants from the solution to the active site. The ChOx/CM-PtNP@m-Fe₃O₄ hybrid containing CM-PtNP@m-Fe₃O₄ as the artificial peroxidase and ChOx as the oxidase provides a new approach to constructing multiple enzyme systems mainly using a self-assembly method instead of chemical processes.

To assess the selectivity of the developed method for cholesterol determination, we investigated the influence of ascorbic acid, cysteine, glucose, histidine, and uric acid on double the concentration of cholesterol. The results of selectivity testing are shown in Fig. 5A. Notably, none of these compounds caused any obvious interference, as also confirmed by the solution colors shown in the inset of Fig. 5A. These results clearly demonstrated the excellent selectivity of the colorimetric bioassay toward cholesterol.

Fig. 5 (A) Determination of cholesterol detection selectivity with 1 mM ascorbic acid, cysteine, glucose, histidine, uric acid, and 0.5 mM cholesterol. Inset: Color change of different solutions. (B) Enzyme activities of ChOx/CM-PtNP@m-Fe₃O₄, ChOx/CEH-Pt@m-Fe₃O₄, and ChOx/Pt@m-Fe₃O₄ nanohybrids. (C and D) Relative activities of ChOx/CM-PtNP@m-Fe₃O₄ nanohybrid after (C) storage in refrigerator at 4 °C for one month and (D) recycling five times.

We further studied the stability and reusability of the ChOx/CM-PtNP@m-Fe₃O₄ nanohybrid (Fig. 5C and D). The as-prepared biosensor was stored in the refrigerator for 30 days at 4 °C and the response sensitivity was more than 96% retained after one month, illustrating that the ChOx/CM-PtNP@m-Fe₃O₄ cascade platform possessed excellent stability. Furthermore, the catalytic activity maintained 92% of the original value after five cycles, demonstrating the excellent reproducibility of the as-prepared ChOx/CM-PtNP@m-Fe₃O₄ cascade platform.

The feasibility of the biosensor for cholesterol detection was tested by analyzing different real blood serum samples. The serum samples were injected into the cell instead of cholesterol without any pretreatment. The cholesterol contents of the biosensor were estimated from the calibration curve. As shown in Table S4 (ESI†), the results were in good agreement with the values obtained from a local hospital. Therefore, the biosensor is a reliable and accurate tool for cholesterol determination in real samples.

Conclusions

A meticulously designed protein–PtNP@m-Fe₃O₄ nanohybrid has a versatile hierarchical nanoporous structure and unique surface physicochemical properties for optimizing the enzymatic cascade reaction. The hybrid provides a favored microenvironment that can immobilize oxidase and preserve the oxidase activity, which resulted in significantly enhanced peroxidase-like activity. The nanoporous structure and the hydrophobic microdomain of the CM-PtNP@m-Fe₃O₄ hybrid contributes to the high substrate affinity and ensures the product of the first reaction can be consumed by the second reaction in a timely fashion, thus activating the cascade reaction. The CM-PtNP@m-Fe₃O₄ nanohybrid-triggered cascade platform exhibited excellent sensitivity with a wide linear range of 0.1–400 μM and a detection limit of 0.05 μM for cholesterol. This strategy provides a universal and easily operated route for using amphiphilic protein-functionalized nanozymes to immobilize natural enzymes for efficient enzyme cascade platform.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was supported by the National Nature Science Foundations of China (21573190), Nature Science Key Basic Research of Jiangsu Province for Higher Education (15KJA150009), and PAPD.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c8tb02162g

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