Coupling Microbial Growth with Nanoparticles: A Universal Strategy

May 5, 2016 - Macroscale assembly of nanoscale building blocks is an intriguing way to translate the unique characteristics of individual nanoparticle...
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Coupling Microbial Growth with Nanoparticles: A Universal Strategy To Produce Functional Fungal Hyphae Macrospheres Wen-Kun Zhu,†,§ Huai-Ping Cong,†,∥ Qing-Fang Guan,† Wei-Tang Yao,§ Hai-Wei Liang,† Wei Wang,† and Shu-Hong Yu*,† †

Division of Nanomaterials and Chemistry, Hefei National Laboratory for Physical Sciences at Microscale, Collaborative Innovation Center of Suzhou Nano Science and Technology, CAS Center for Excellence in Nanoscience, Department of Chemistry, University of Science and Technology of China, Hefei, Anhui 230026, People’s Republic of China ∥ School of Chemistry and Chemical Engineering, Hefei University of Technology, Hefei, Anhui 230009, People’s Republic of China § Joint Laboratory for Extreme Conditions Matter Properties, Southwest University of Science and Technology, Mianyang, Sichuan 621000, People’s Republic of China S Supporting Information *

ABSTRACT: Macroscale assembly of nanoscale building blocks is an intriguing way to translate the unique characteristics of individual nanoparticles into macroscopic materials. However, the lack of the efficient universal assembly strategy seriously hinders the possibility of macroscale architectures in practical applications. Herein, we develop a general, environment-friendly, and scalable microbial growth method for the construction of macroscopic composite assemblies with excellent mechanical strength by in situ integrating various types of nanoparticles into fungal hyphae (FH) macrospheres. Notably, the size of the FH-based composite spheres and the loading amount of the nanoparticles with different dimensions can be well tuned by controlling the cultivation time and the dosage of nanoparticles, respectively. Interestingly, bifunctional FH-based core-shell macrospheres can also be achieved by programmed assembling two different kinds of nanoparticles in the cultivation process. The produced multifunctional FH-based composite spheres exhibit wide potential applications in magnetic actuation, photothermal therapy, and contaminant adsorption, etc. KEYWORDS: fungal hyphae, nanocomposite, macroscale, self-assembly, functionality

1. INTRODUCTION With the development of nanotechnology, high-quality nanomaterials with tunable structures and unique properties are expected to have tremendous impacts on numerous scientific and industrial fields.1−4 In view of growing demands for practical applications, it is of great importance to integrate these nanoscale building blocks into macroscopic architectures, allowing for an efficient route to translate unique properties and functionalities of individual nanoparticles into advanced hierarchical ensembles.5,6 In particular, this research field is enabled by the emergence of various assembly techniques, including layer-by-layer (LBL) deposition,7,8 vacuum filtrationassisted assembly,9,10 freeze-drying,11,12 interface-assisted assembly,13,14 and Langmuir−Blodgett (LB) technique.15,16 Despite of the significant achievements, it has been challenging to achieve control over the organization of nanoparticles on multiple length scales and produce new functional materials with ordered hierarchical structures. Recently, researchers have learnt from nature to integrate micro/nanoscale objects with biological systems for the creation of sophisticated functional macrostructures in a predicable fashion.17,18 Microbes such as viruses, bacteria, and fungus are attractive biotemplates for the self-organization of nanoscale materials © 2016 American Chemical Society

due to their unique, living structural forms and quick, low-cost reproduction.19−21 Furthermore, the inherent uniform structure of these microorganisms allows highly ordered assembly conformation for optimizing the properties and functionalities of the integrated nanoparticles at macroscopic scales. For example, Belcher et al. developed an interesting cathode material for Li-ion battery, exhibiting much enhanced specific capacity and rate capability using a hybrid CoO3/M13 virus inorganic/biological system.22 In another work, Yeh et al. produced the biocompatible bacteria@Au composites for photothermal therapy by using bacteria as the template.23 Recently, fungal hyphae (FH) with special hollow tube structures and dynamically controlled diameters and lengths has shown practical feasibility as living templates in aligning presynthesized nanoparticles into ordered microscale assemblies through a fast and facile cultivation process.24−28 To the best of our knowledge, previous studies have been only focused on the construction of nanostructures by using FH as a template, whereas coupling growth of FH in the presence of Received: March 20, 2016 Accepted: May 5, 2016 Published: May 5, 2016 12693

DOI: 10.1021/acsami.6b03399 ACS Appl. Mater. Interfaces 2016, 8, 12693−12701

Research Article

ACS Applied Materials & Interfaces

2.4. In Vitro Adsorption/Release of DOX on FH/GO Composite Spheres. The in vitro adsorption/release tests of DOX were conducted in a 10 mL DOX aqueous solution that contained 1 mg mL−1 of DOX and 10 mg of FH or FH/GO composite spheres. The mixture was shook for 72 h at 37 °C. The absorbance of the solution at 480 nm was monitored using a UV−vis spectrometer, and the loading capacity of DOX was calculated based on the concentration−absorbance calibration curve. Then, the FH/GO composite spheres were washed with distilled water for 3 times and dried for the release test. To perform the DOX release test, 5 mg of DOX-adsorbed FH/GO was dispersed in 10 mL of phosphate buffer with different pH values (pH 5.4 and 7.4, corresponding to acidic nature of tumor and normal tissue, respectively),29,30 and then transferred into a dialysis tube. Afterward, the tube was placed in a 500 mL beaker containing 290 mL of phosphate buffer for DOX release at 37 °C, with a proper shaking speed. The absorbance was monitored by taking out an aliquot sample (3 mL each time) at set intervals, and buffer solution with the same volume was added for compensation. The release rate was then calculated based on the concentration− absorbance calibration curve. 2.5. Adsorption/Desorption Tests of MG and Cu2+ on FH/ CNT Composite Spheres. Glassware used in adsorption and desorption tests was cleaned by soaking in 50% HNO3 solution for 24 h, then washed with DI water and dried. MG and Cu2+ solutions were prepared with a concentration of 100 mg L−1. The pH was adjusted with 0.1 mol L−1 HCl and 0.1 mol L−1 NaOH. The prepared dye solution was transferred to conical flasks containing 50 mL of the dye solution and 25 mg of FH or FH/CNT composite spheres. The flasks were shaken in a shaker with a rotation speed of 150 rpm at 25 °C for 48 h. Then the solution was filtrated using 0.45 μm microporous membrane and tested by ICP-AES and spectrometer to determine the concentrations of Cu2+ and MG, respectively. After the adsorption experiment, the FH/CNT composite spheres were soaked in 50 mL of desorption solution, placed in a shaker with a rotation speed of 150 rpm at 25 °C for 24 h to regenerate the adsorbent. The adsorption−desorption test was repeated for 6 times. Different desorption solutions were used, including salt solution, ethanol solution, hydrochloric acid solution, and their mixture. 2.6. Catalytic Reduction of 4-Nitrophenol (4-NP). First, 0.0139 g of 4-NP and 3.783 g of NaBH4 were dissolved in 100 mL of deionized water, respectively. Then, 3.7 mL of 4-NP and 3.65 mL of NaBH4 were mixed together in a 100 mL triangular flask and diluted with water to 40 mL, added 30 mg of hypha-based nanocomposite catalysts, and cultivated on a shaker. Since then, 3 mL of samples was collected every 2 min. Next, the samples were treated by microporous membrane filter, and then determined by UV−vis spectrophotometer at the wavelength from 250 to 500 nm. For testing recycling performance, the above processes were repeated 10 times. 2.7. Characterization. TEM images were performed on an H7650 (Hitachi, Japan) operated at an acceleration voltage of 100 kV. SEM images were performed with a field emission scanning electron microanalyzer (Zeiss Supra 40) at an acceleration voltage of 5 kV. XRD analyses were carried out on a Philips X’Pert PRO SUPER X-ray. Freeze-drying was carried out using freeze-drier (Labconco-195). The laser device (MDL-808 nm 2W) used in the photothermal tests was made from Changchun New Industries Optoelectronics Tech. Co., Ltd. Water temperature was measured by a digital thermometer (TES1310, TES Electrical Corp.). ICP-AES measurements were conducted using an Optima7300 DV spectrometer, PerkinElmer Inc., USA. The UV−vis spectra were recorded on a UV-2550 (Shimadzu Corporation, Japan) at room temperature by using BaSO4 as the calibration reagent. Sample sectioning was conducted on a Leica CM1950 after the sample was embedded and frozen.

nanoscale building blocks during the cultivation process and thus integration of FH with nanoscale building blocks into macroscale functional spheres has been rarely reported. Herein, we propose a universal, green, and low-cost fungal hyphae growth strategy for coupling the microbial growth with functional nanoparticles, which allows it possible for the scalable integration of a variety of nanoscale building blocks into multifunctional 3D macroscale composite spheres with high mechanical strength. Notably, the diameter of macrospheres and loading density of nanoparticles can be well controlled by changing the cultivation time and the amount of nanoparticles. Bifunctional FH-based core−shell macrospheres can be prepared by programmed assembly of two different kinds of nanoparticles in cultivation process, making these FHbased assemblies particularly attractive for applications in magnetic actuation, photothermal therapy, drug delivery, contaminant adsorption, and many others.

2. EXPREIMENTAL SECTION 2.1. Assembly of FH-Based Composite Spheres. FH-based composite spheres with different building blocks, namely FH/Fe3O4 NPs, FH/Au NPs, FH/CNTs, FH/Au NRs, FH/GO, and FH/MTM, were obtained by growing FH in the corresponding nanomaterial suspensions. Briefly, the as-synthesized Fe3O4 NPs (or other nanoobjects) were dispersed in sterile water at first. And then, 10 mL of the above nanomaterial suspension (0.5 mg mL−1) was added into 110 mL of FH medium, before which 5 mL of cultivated FH was inoculated. The medium was allowed to grow for 72 h in a shaker with a rotation speed of 145 rpm at 28 °C. The obtained FH-based composite spheres were collected and washed with deionized water for 3 times, then soaked in 0.5% NaOH solution for 1 day and washed again until the pH value was neutral. Afterward, the FH-based composite spheres were frozen by liquid nitrogen and dried using a Labconco-195 freeze-drier. All the materials used in this section were autoclaved at 121 °C, 1.5 MPa for 20 min with a Sanyo MLS-3750 autoclave. 2.2. Photothermal Effect of FH/Au NPs and FH/Au NRs in Aqueous Solution. FH integrated with different amounts of Au NPs and Au NRs was immersed in a 1.5 mL tube containing 1.0 mL of distilled water, respectively. Then near-infrared (NIR) light with a wavelength of 808 nm and an output power of 2.0 W was irradiated to the FH/Au NPs and FH/Au NRs. The temperatures were measured every 1 min over a period of 10 min by using a digital thermometer (TES-1310, TES Electrical Corp.). An equal size of Au-free FH sample was used as the control, and 1 mL of distilled water was used as the blank control. 2.3. Cell Cytotoxicity Assays of FH/GO Composite Spheres. RAW264.7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum (Life Technologies, Inc., Grand Island, NY) and 1% antibiotics (100 U mL−1 penicillin and 100 μg mL−1 streptomycin; Life Technologies), and incubated at 37 °C with 5% CO2. The FH and FH/GO composite spheres were cut into small pieces (∼3 × 3 mm) and bedded in 12-well plates. Wells without any film were set as the control group. Then, they were dried at 37 °C with 5% CO2 incubation for 12 h. 12 000 RAW264.7 cells with 1000 μL of medium were seeded in each well. The cells were then stained with Calcein, showing green fluorescence on live cells by binding with calcium and propidium iodide, showing red fluorescence on dead cells by intercalating between the strands of DNA and RNA for the confirmatory Live−Dead test. The stained cells were washed, fixed, and observed under inverted microscopy (Nikon ECLIPSE Ti-S). In cytotoxicity assays, 12 000 RAW264.7 cells in 1000 μL medium were seeded onto an FH or FH/GO composite spheres coated well of a 12well plate. After 24, 48, and 72 h of incubation, samples were treated with trypsin to harvest cells. Cell numbers of each well were counted by a Countstar automated cell counter.

3. RESULTS AND DISCUSSION 3.1. Microbial-Growth Mediated Assembly of FHBased Composite Spheres. In a typical process, the fungal was added into a nutrient culture medium containing different types of nanoparticles (NPs) such as zero-dimensional (0D), 12694

DOI: 10.1021/acsami.6b03399 ACS Appl. Mater. Interfaces 2016, 8, 12693−12701

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particles in the cultivation process (Figure 1). Typical SEM images of the FH-based composite spheres are depicted in

one-dimensional (1D), and two-dimensional (2D) nanoscale building blocks. During the growth of filamentous fungi, these nanoparticles were postulated to adsorb onto the lipids and glycoproteins of the hyphae surface. Under shear force, the filaments covered with the nanoparticles were intertwisted together and subsequently assembled to the porous 3D FHbased composite spheres as illustrated in Scheme 1. Without Scheme 1. Schematic Illustration of the Fungal Growth Method for Assembly of FH-Based Composite Spheresa

Figure 1. XRD patterns of the FH spheres and different FH-based composite spheres.

Figure 2. It was found that 0D Au NPs, Fe3O4 NPs, 1D CNTs, and Au NRs were uniformly coated onto the hyphae surface. In comparison, 2D GO and MTM nanosheets were assembled in the porous structure of the hyphae, forming an interconnected network. Notably, the loading amount of the nanomaterials in the spheres and the size of the spheres could be well tuned by varying the dosage and the cultivation time, respectively. For example, as the dosage of GO increased, the color of the resulting FH/GO composites became darker, indicating the increased amount of GO loaded in the spheres (Supporting Information, Figure S5). Meanwhile, prolongation of the cultivation time led to the larger FH/GO composite spheres (Supporting Information, Figure S6). Importantly, compared with the traditional physical-adsorption method by soaking FH spheres in the nanomaterial dispersion, the assemblies with a higher and more uniform loading of the nanoscale building blocks can be achieved through the facile fungal hyphae growth method proposed here. For example, Au NPs were sparsely located on the outer surface of the hyphae by traditional adsorption methods, thereby exhibiting a nonuniform red color (Supporting Information, Figure S7). On the contrary, the FH/Au NP composite spheres gave an intense color owing to the uniform coverage of Au NPs on each hypha with a much higher loading amount during the process of fungal hyphae growth. The assembly mechanism of FH-based composite spheres through such hyphae growth strategy could be explained in terms of physical and chemical interactions. In detail, for physical interactions, on the one hand, the porous-structured FH spheres offer large surface area for the direct adsorption of the nanomaterials.35,36 On the other hand, the surface potential of the hyphae also plays an important role in the formation of the macroscopic assembled structure. In the absence of nanoparticles, the ζ-potential of the hyphae was slowly shifted from negative charge to positive charge during the growth of FH spheres (Supporting Information, Figure S8). In this way, the negatively charged nanoparticles tended to be adsorbed and assembled on the positively charged hyphae surface in the cultivation process due to the strong electrostatic interaction (Table 1). In the case of FH/Au NR composite spheres, when the Au NRs were initially modified with the positively charged hexadecyl trimethylammonium bromide (CTAB), the assembly

a

The fungal spore and various kinds of nanoparticles (0D, 1D, and 2D) were cocultured in medium, during which the nanoparticles adsorbed onto the surface of hyphae to form 3D FH-based composite spheres.

addition of NPs, the FH grew into white spheres with an averaged diameter of 0.5 cm after 72 h of cultivation (Supporting Information, Figure S1a,b). SEM image showed that the freeze-dried FH spheres with the porous structure were composed of branched smooth hyphae with a diameter of ∼500 nm (Supporting Information, Figure S1c). It was noted that the as-obtained FH spheres could still maintain their structure with ultrasonication treatments for 5 h (100 W, 40 kHz), revealing their excellent mechanical strength (Supporting Information, Figure S2). Additionally, the size of the FH spheres could be controlled by varying the cultivation duration (Supporting Information, Figure S3). In the initial lag cultivation stage within 60 h, FH spheres were grown into a diameter of 0.3− 0.45 cm. In the following 24 h, the diameter of FH spheres was increased to 0.45−0.75 cm. Over 84 h, the hyphae was autolyzed in the core area, leading to the hollow structure, due to the diffusion limitation of nutrilite in the big sphere.31,32 To demonstrate the universality of the above-mentioned microbial growth strategy for macroscale assembly of nanoparticles, various types of uniform nanoparticles with different dimensions, including Au NPs, Fe3O4 NPs, carbon nanotubes (CNTs), Au nanorods (NRs), montmorillonite (MTM), and graphene oxide (GO) nanosheets, were selected as nanoscale building blocks and added into the culture medium, respectively (Supporting Information, Figure S4). In this case, magnetite Fe3O4 NPs highly dispersed in water was synthesized through a high-temperature hydrolysis process (JCPDS card no. 65-3107).33 Au NPs colloid solutions were prepared by using Frens’ method (JCPDS card no. 04-0784).34 XRD patterns of the obtained FH-based composite spheres showed the characteristic diffraction peaks of the corresponding nanostructures, indicating the effective integration of nano12695

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Figure 2. Photographs and SEM images of various FH-based composite spheres after 72 h of cultivation. (a, b) FH/Au NP, (c, d) FH/Fe3O4 NP, (e, f) FH/CNT, (g, h) FH/Au NR, (i, j) FH/MTM, and (k, l) FH/GO spheres. Insets are the corresponding magnified SEM images of FH-based composite spheres.

Table 1. ζ-Potentials of Several Types of Nanomaterials Used for the Assembly into FH Sphere at pH 7 sample Au NPs Fe3O4 NPs PSS/Au NRs CNT GO MTM

of FH/Au NR composite spheres failed because of the charge repulsion. After surface modification by poly(sodium-pstyrenesulfonate) (PSS), a negatively charged surfactant, the resulting PSS−Au NRs could be assembled on the FH sphere effectively. In addition, the extracellular polymeric substance (EPS) secreted by the fungi contained numerous of polysaccharide and proteins is beneficial to the immobilization of the nanoscale building blocks onto the hyphae surfaces.32,35 Importantly, abundant functional groups from FH spheres were characterized by the FT-IR spectra, including hydroxyl at 3423 cm−1, carboxyl at 1735 cm−1, and acylamide at 1646 and 1537

ζ-potential (mV) −23.82 −15.80 −12.30 −20.30 −18.03 −31.82

± ± ± ± ± ±

4.32 6.92 3.16 2.24 3.79 4.67

Figure 3. Properties and functionalities of the FH-based composite spheres after 72 h of cultivation. (a) Photographs of the magnetic responses of the FH/Fe3O4 NPs. (b) Hysteresis loops of the FH/Fe3O4 NPs at room temperature. (c) Photothermal effects of the FH, FH/Au NPs, and FH/Au NRs. (d) Adsorption capacity of Cu2+ and MG on the FH and FH/CNT. (e) Adsorption recycling capability curves of Cu2+ and MG on the FH/ CNT spheres. (f) TGA curves for the FH and FH/MTM spheres. 12696

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Figure 4. Photographs of (a) FH, (b) FH/Au NPs-0.25 mg mL−1, (c) FH/Au NPs-0.5 mg mL−1 composite spheres, (d) photothermal effects of FH, FH/Au NPs-0.25 mg mL−1 and FH/Au NPs-0.5 mg mL−1 composite spheres. The concentrations of Au NPs were 0.25 and 0.5 mg mL−1, respectively.

cm−1 (Supporting Information, Figure S9).37,38 It is conceivable that these groups facilitated the assembly of nanoparticles with FH through the interactions of hydrogen bonding, van der Waals force or coordination bonding, etc. Owing to such multimode interactions, the FH-based composite spheres exhibited excellent stability. For example, the uniformity of the FH/GO spheres remained essentially the same even after soaking in deionic water for 3 months (Supporting Information, Figure S10). Moreover, the FH/CNT composite spheres could endure an ultrasonication treatment for 5 h with a power of 100 W, without noticeable breakdown in their structural integrity (Supporting Information, Figure S11). Furthermore, the FH-based composite spheres showed high loading amounts of different types of nanomaterials after cultivation for 72 h (Supporting Information, Table S1). 3.2. Functionalities of the FH-Based Composite Spheres. The above experimental results demonstrated that various nanoscale building blocks can be effectively assembled into macroscopic hierarchical structures by such a fungal hyphae growth method. The properties and functionalities of the FH-based composite spheres have been tested systematically for possible applications. Fe3O4 NPs were selected and represented as one 0D model of nanoscale building blocks for the assembly of the magnetic FH-based composites, due to their unique electronic and magnetic properties for the applications of high-density information storage, electronic devices and magneto fluid technology. Because these magnetic NPs were assembled into the FH spheres, the black composites exhibited excellent magnetic response to an external magnetic field, as shown from their movement controlled by a magnet in Figure 3a. The hysteresis loops in Figure 3b revealed a superparamagnetic behavior for the FH/Fe3O4 NP composite,

with the magnetic moment reaching 1.63 emu g−1. This result was in good agreement with the size of the Fe3O4 NPs within the superparamagnetic range. Thus, these FH/Fe3O4 NP composite spheres have great potential applications in biomedicine, environment, and catalysis.39 In the case of FH/Au NP and FH/Au NR composite spheres, their photothermal effect was investigated.40,41 As shown in Figure 3c, under the irradiation of near-infrared light, the FH/Au NPs and FH/Au NRs heated up the water to 68 and 82 °C within 5 min, respectively. By comparison, the water temperature was nearly unchanged in the presence of pure FH spheres. The electromagnetic coupling caused by the assembly of the Au NPs or Au NRs on the FH sphere surface may further enhance the photothermal conversion efficiency.42−45 Furthermore, the photothermal conversion efficiency can also be influenced by Au NP concentrations. With increasing Au NP concentrations, the photothermal conversion efficiency was greatly enhanced (Figure 4). For the FH/CNT composite spheres, water treatment application in terms of heavy metal ions and dyes was tested. Figure 3d,e revealed that the adsorption capabilities of FH/CNT spheres toward Cu2+ and malachite green (MG) were 154 and 50.2 mg g−1, 35% and 42% higher than pure FH spheres, respectively. Additionally, the higher amount of CNTs loaded in FH composite spheres attributed to the larger adsorption capacity of Cu2+ and MG (Supporting Information, Figure S12). This enhancement could be attributed to the strong interactions of the pollutants with the oxygen-containing groups on the functionalized-CNT. Recycling experiments were also conducted using 80% ethanol solution containing 1 M of HCl as the desorption solution (Figure 3e). It was found that the adsorption capability of the FH/CNT composites was almost 12697

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Figure 5. Cytotoxicity and drug-release performance of FH/GO composite spheres. Fluorescence microscopy evaluation of living (green-labeled) and dead (red-labeled) RAW264.7 cells grown in DMEM: (a) control sample, (b) FH, and (c) FH/GO. (d) Quantification of RAW264.7 cells proliferation rate on FH and FH/GO surface after being cultured for 0, 24, 48, and 72 h, respectively. Error bars indicate the standard deviation (SD). (e) Loading amounts of DOX on FH and FH/GO spheres versus time at 25 °C (pH 7). (f) DOX releasing rates on FH and FH/GO spheres at different pH at 37 °C in PBS (pH 5.4 and 7.4).

Figure 6. Programmed assembly of bifunctional FH/Fe3O4 NP/Au NP composite spheres with two kinds of nanoobjects. (a) Photograph of FH/ Fe3O4 NP/Au NP composites. (b) Optical microscopy image of composite sphere after the Cryo-section. (c) XRD pattern of composite sphere. (d) SEM image of composite sphere. Magnified SEM images of (e) shell and (f) core of spheres. (g) Time-dependent UV−vis absorption spectra during the catalytic reduction of 4-NP by FH/Fe3O4 NP/Au NP composites. (h) Plot of ln(A0/At) versus reaction time for the reduction of 4-NP by FH/ Fe3O4 NP/Au NP composites. (i) Catalytic activity of composite spheres for reduction of 4-NP with 10 cycles. The inset photographs show the recovery of composite spheres with a magnet and reuse as the catalyst for the reduction of 4-NP.

unchanged after 6 cycles, indicating their promising potentials as candidates for pollutant removal. Because 2D MTM 12698

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Figure 7. Photographs of (a) FH/GO/Au NP, (b) FH/Fe3O4 NP/GO, and (c) FH/GO/Fe3O4 NP composite spheres. Optical microscopy images of (d) FH/GO/Au NP, (e) FH/Fe3O4 NP/GO, and (f) FH/GO/Fe3O4 NP composite spheres after the Cryo-section.

3.3. Programmed Growth of Bifunctional FH-Based Composite Spheres. Interestingly, the microbial growth method proposed here is also an effectively programmable strategy to assemble different kinds of nanoparticles into FH spheres successively, forming multifunctional composites with unique hierarchical structures. A series of the bifunctional FHbased composite spheres were produced and presented welldefined core−shell structures such as FH/Fe3O4 NP/Au NP, FH/GO/Au NP, FH/Fe3O4 NP/GO, and FH/GO/Fe3O4 NP composite spheres (Figure 6 and Figure 7). Taking the assembly of FH/Fe3O4 NP/Au NP composite as an example, Fe3O4 NPs were first integrated into FH spheres through the one-step cultivation for 60 h. Followed by washing and transferring into a fresh growth medium containing Au NPs for another 36 h of cultivation, the bifunctional composite spheres were constructed (Figure 6a). XRD pattern in Figure 6c strongly confirmed the integrations of Au NPs and Fe3O4 NPs into the spheres. Optical microscopy imaging (Figure 6b) and SEM imaging (Figure 6d) showed the clear core−shell structure in FH/Fe3O4 NP/Au NP sphere. As investigated from the magnified SEM images of Figure 6e,f, the first-cultivated Fe3O4 NPs were coated on the hyphae surface in the core of sphere and the following Au NPs were covered on the external hyphae, similar to the structures of FH/Au and FH/Fe3O4 spheres in Figure 2b,d, respectively. The obtained FH/Au NP/ Fe3O4 NP spheres exhibited excellent magnetic response, good photothermal effect, and high catalytic activity. Figure 6g displays the time-dependent evolution of UV−vis spectra when reduction of 4-nitrophenol (4-NP) catalyzed by FH/Au NP/ Fe3O4 NP spheres. 10 min later, the strong absorbance at 400 nm disappeared, indicating a nearly 100% reduction efficiency into 4-aminophenol. The linear relationship of ln(A/A0) vs time revealed that the reduction of 4-NP in the presence of FHbased nanocomposite spheres was in accordance with pseudofirst-order kinetics (Figure 6h). More important, the catalytic activity of FH/Au NP/Fe3O4 NP spheres could maintain ∼99% conversion of 4-NP with 10 successive cycles of reduction

nanosheets can also be assembled into the FH spheres, the thermal stability of the composites was obviously improved, as revealed from the TGA curves (Figure 3f). For the FH/GO composite spheres, they showed great promising application in the drug-release field. First, the cytotoxicity of the FH/GO spheres and the controlled samples was examined by cocultivation of RAW264.7 cells. As shown in Figure 5a−c, the similar morphologies of the cells were observed in blank medium, pure FH spheres and FH/GO composites through monitoring their growing states every 24 h. Cell counting further quantified that no apparent cytotoxicity was found on FH/GO composite spheres (Figure 5d). On the basis of their good biocompatibility, the drug-releasing performance of the FH/GO composites was investigated by using DOX as the model compound. Compared with pure FH spheres, the oxygenated group-functionalized GO nanosheets with large surface area in the FH/GO composites facilitated high adsorption capacity.46,47 By prolonging the adsorption time, the maximum loading amount of DOX in composite spheres reached to 0.99 mg mg−1 (Figure 5e). To mimic the in vivo drug release process, the DOX release tests were conducted for pure FH spheres and FH/GO composites under simulated physiological conditions (PBS solution) at different pH values (5.4 and 7.4) at 37 °C. As shown in Figure 5f, the DOX-releasing rate of FH/GO composites was much lower than that of FH spheres, presumably caused by the strong hydrogen bond interaction between GO and DOX. Furthermore, the released amount of DOX at pH 7.4 was considerably lower than that obtained at pH 5.4. As both the extracellular environment and intracellular lysosome were in weak acidic conditions for the cancer cells, such pH-controlled drug release was of great importance in clinical application.48,49 The facilitation of the DOX release in acidic conditions demonstrated here showed the promises of the FH/GO composite spheres in biomedicine and targeted drug delivery for tumor therapy.50 12699

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reactions (Figure 6i). Owing to the magnetic Fe3O4 NPs incorporated in the spheres, the catalysts could be easily recovered by a magnet, which would greatly promote their industrial application.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b03399. Experimental methods and data for assembly process, photothermal effect, mechanical stability, adsorption capacity, and bifunctional composite sphere (PDF).

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REFERENCES

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4. CONCLUSIONS In summary, a facile, green, and universal microbial growth strategy has been developed for the assembly of various nanoscale building blocks into the multifunctional FH-based composite spheres with hierarchical structures. The size of the FH-based composite spheres and loading density of nanoparticles with different dimensions are tightly related with the choices of the cultivation duration and the amount of NPs. Moreover, bifunctional FH-based composite spheres with core−shell structures and tunable components can be conveniently achieved for the first time in such a system by cultivating with two different kinds of nanoscale building blocks in a programmed fashion. The versatility offered by this strategy is expected to extend to other microorganism systems and provide tantalizing opportunities for assembling hierarchical macroscale structures with tunable optical, electronic, and magnetic properties attractive for broad applications in catalysis, sensors, energy conversion and storage, the biomedical field, and so on.

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Research Article

AUTHOR INFORMATION

Corresponding Author

*S.-H. Yu. E-mail: [email protected]. Author Contributions

W.-K.Z. and H.-P.C. planned and performed the experiments, collected and analyzed the data and wrote the paper. Q.-F.G., W.-T.Y., H.-W.L., and W.W. performed the experiments including the synthesis of nanoscale building blocks and assembly of FH-based composite spheres. S.-H.Y. supervised the project, conceived the experiments, analyzed the results, and wrote the paper. All the authors discussed the data and commented on the paper. W.-K.Z. and H.-P.C. contributed equally to this work. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is supported by the National Natural Science Foundation of China (Grants 21571046, 21431006), the Foundation for Innovative Research Groups of the National Natural Science Foundation of China (Grant 21521001), the National Basic Research Program of China (Grants 2014CB931800, 2013CB933900), the Users with Excellence and Scientific Research Grant of Hefei Science Center of Chinese Academy of Sciences (Grants: 2015HSC-UE007, 2015SRG-HSC038), and the Fundamental Research Funds for the Central Universities. 12700

DOI: 10.1021/acsami.6b03399 ACS Appl. Mater. Interfaces 2016, 8, 12693−12701

Research Article

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