Yan
Liu
*,
Yuling
Qin
,
Yuanlin
Zheng
,
Yong
Qin
,
Mengjun
Cheng
and
Rong
Guo
*
School of Chemistry and Chemical Engineering, Yangzhou University, Yangzhou, 225002, Jiangsu, P. R. China. E-mail: yanliu@yzu.edu.cn; guorong@yzu.edu.cn; Fax: +86-514-87971802; Tel: +86-514-87971802
First published on 22nd November 2018
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.
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. |
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.
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 2p3/2 and Fe 2p1/2, respectively, which are characteristic of the Fe3O4 phase. As shown in Fig. S4C (ESI†), the Pt 4f7/2 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+ 4f7/2, 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.
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 (Km) and maximum initial velocity (Vmax) were obtained using a Lineweaver–Burk plot, with the results shown in Table S1 (ESI†). The small apparent Km 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 Vmax 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.
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 4f7/2 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†).
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.
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, TMBox (λ = 650 nm). Here, the absorbance change at 650 nm (owing to the quantity of generated TMBox) 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.
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.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c8tb02162g |
This journal is © The Royal Society of Chemistry 2019 |