Bacteria-Mediated Ultrathin Bi2Se3 Nanosheets Fabrication and Their

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Bacteria mediated ultrathin Bi2Se3 nanosheets fabrication and its application in photothermal cancer therapy Hao Zhou, Lin Che, Zhaoming Guo, Minghuo Wu, Wenqing Li, Weiping Xu, and Lifen Liu ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b04321 • Publication Date (Web): 14 Mar 2018 Downloaded from http://pubs.acs.org on March 15, 2018

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ACS Sustainable Chemistry & Engineering

Bacteria mediated ultrathin Bi2Se3 nanosheets fabrication and their application in photothermal cancer therapy Hao Zhou*a, Lin Chea, Zhaoming Guob, Minghuo Wua, Wenqing Lib, Weiping Xua and Lifen Liua a

Key Laboratory of Industrial Ecology and Environmental Engineering (Ministry of Education),

School of Food and Environment, Dalian University of Technology Panjin, China. E-mail: [email protected] b

School of Life Science and Medicine, Dalian University of Technology, 124221, Panjin, China.

ACS Paragon Plus Environment

ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT

Bismuth selenide (Bi2Se3) attracts a lot attention nowadays due to its unique electronic and thermoelectric properties. In this study, fabrication of Bi2Se3 nanosheets by selenite reducing bacterium (SeRB) was firstly reported. Morphology, size and location of the biogenic Bi2Se3 are bacteria-dependent. It is difficult to separate Bi2Se3 generated by Bacillus cereus CC-1 (Bi2Se3-C) from the biomass due to strong interaction with the cell membrane. However, Bi2Se3 produced by Lysinibacillus sp. ZYM-1 (Bi2Se3-Z), are highly dispersed in extracellular space with high stability. Further characterization by X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM) and atomic force microscopy (AFM) on Bi2Se3-Z indicates that the product is rhombohedral phase, ultrathin nanosheet-like structure with an average size of ~100 nm. Subsequently, the photothermal performance of Bi2Se3-Z with the irradiation of 808 nm near infrared (NIR) laser was determined. When the Bi2Se3-Z concentration was 26 mg L-1, and irradiation power power was 2W, the photothermal converion efficiency was calculated as 30.7%. At the same condition, 100% of the MCF7 and A549 cancer cells were killed within 10 min irradiation in vitro. Moreover, using 1% (v/v) PVP as surfactant, a novel nanodumbbells structure of Bi2Se3 was obtained. Overall, this bacteria driven Bi2Se3 fabrication pave a new way for biocompatible photothermal nanomaterials.

KEYWORDS: Bi2Se3; bacteria; photothermal material; biogenic; cancer cells.


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INTRODUCTION

Photothermal therapy (PTT) is an attractive non-invasive technology for cancer treatment.1 For PTT, the specific nanomaterials absorb the light energy and convert it to heat, which is directly responsible for the ablation of cancer cells. For an ideal photothermal nanomaterial, it needs excellent near infrared (NIR) optical performance, proper size, and good biocompatibility.2 Bi2Se3, i.e. bismuth selenide, is one of the most versatile metal chalcogenides. When its size decreases to nanoscale, it can be used in photodetection, photoelectrochemical cells, thermoelectric devices and topological insulators.3 Advantages of Bi2Se3 as PTT agents are recently noticed. Bismuth itself is a heavy element (Z=83) and has a photoelectric adsorption coefficient of 5.3 cm2 g-1 at 100 keV, which made it suitable for cancer radio sensitizer and X-ray contrast agent. On the other hand, selenium has been long-recognized as an anticancer agent in a low dose.4 Moreover, like other 2D nanomaterials, Bi2Se3 has a broad absorption band at the NIR region, which made it became an excellent candidate for PTT.5

Previous reports have proved that the PTT performance of Bi2Se3 can be further promoted by using capping agent (PVP), changing sizes, or forming complex core-shell structure (CdSe/Bi2Se3, MnSe@Bi2Se3).6,7 The pristine Bi2Se3 is usually prepared through photochemical synthesis, hydrothermal treatment and sonochemical method. For instance, Bi2Se3 nanosheets with thicknesses of 50-100 nm can be simply synthesized by microwave heating in an ionic liquid, while about 6 nm thick Bi2Se 3 nanoplates were fabricated with the assist of PVP after being heated from room temperature to 190 oC.8,9 In another work, ultrathin (4-6 nm) Bi2Se3 nanodiscs and nanosheets were synthesized by hydrothermal



method using PVP as surfactant, and the growth mechanism is attributed to the 2D self-attachment of small Bi2Se 3 nanoparticles followed by epitaxial recrystallization into single crystals.10 Nevertheless, these methods are high-energy consuming, needing O2-free condition or using toxic precursors. Moreover, extra agents are usually needed to develop the biocompatibility.11 Therefore, a facile and environmentally benign method will be attractive for practical application of Bi2Se3.

Utilizing the detoxification process or anaerobic respiration of selenite reducing bacteria (SeRB) to obtain elemental selenium nanoparticles (SeNPs) have been widely reported.12 These bacteria employ various enzymes (nitrate reductase, sulphite reductase and glutathione reductase) or reduced thiols like glutathione (GSH) to reduce selenite (SeO32-) or selenate (SeO42-) into Se0.13 Morphology and size of biogenic SeNPs can be modulated by various environmental conditions.14 In addition, some metal chalcogenides have been reported to be synthesized by SeRB. Li et al demonstrated the importance of glutathione metabolic pathway (γ-glutamylcysteine ligase, glutathione synthetase and glutathione reductase) in intracellular CdSe quantum dots synthesized by yeast.15 By this yeast, selenite was immediately reacted with GSH to form selenodiglutathione (GS-Se-SG), and GS-Se-SG is reduced by glutathione reductase (GR) or thioredoxin reductase (ThR) to produce GS-Se-, which is an active intermediate to react with metal ions. On the other hand, Jiang et al reported that Shewanella putrefaciens 200 can reduce Hg2+ and SeO32- into Hg0 and Se0, and the biogenic Hg0 was captured by the α-Se nanospheres to form stable HgSe nanoparticles.16 Therefore, the biosynthesis mechanism of metal chalcogenides may be diverse. However, limited types of biogenic Se-based metal


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chalcogenides were reported up to now (only CdSe, ZnSe, PbSe and HgSe).17,18 Due to the unexplored diversity of SeRB in metabolism and metal resistance, it is interesting to investigate: (1) whether SeRB can produce Bi2Se3, (2) the characterization of biogenic Bi2Se3, and (3) the possible synthesis mechanism.

In this study, two SeRB were used to attempt Bi2Se3 synthesis using Bi(NO3)3 and NaSeO3 as precursors. Both of the SeRB can produce rhombohedral phase Bi2Se3 in mild condition, but the location of the products was different. The characterization and photothermal performance of extracellular Bi2Se3 produced by Lysinibacillus sp. ZYM-1 were investigated in detail. Under the NIR laser irradiation, this biogenic Bi2Se3 has little cytotoxicity and can kill cancer cells effectively in a relatively low concentration, which made it an ideal photothermal nanomaterial.

EXPERIMENTAL SECTION Reagents and Bacteria

Sodium selenite (NaSeO3, 98%), bismuth nitrate (99.5%) and polyvinylpyrrolidone (PVP, MW is about 10000) were purchased from Aladdin (Shanghai, China). Glutathione (GSH), NADPH and NADH were from Beyotime (Shanghai, China). Tryptic Soytone Broth (TSB) medium was from Aobox (Beijing, China). Phosphate buffer saline (PBS) was purchased from Biosharp (Hefei, China). Dulbecco’s modified Eagle medium (DMEM) and RPMI-1640 cell culture media were obtained from HyClone. Calcein-AM and propidium iodide (PI) double stain kit was from Yesen (Shanghai, China). All of the reagents were used without further purification. The bacteria used in this study, i.e. strain



Bacillus cereus CC-1 (CICC 24251) and Lysinibacillus sp. ZYM-1 (CGMCC 1.15346), were isolated from marine sediments and stored at -80 oC freezer in our lab.

Synthesis of biogenic Bi2Se3

For Bi2Se3 fabrication, 1 mL pre-incubated bacteria liquid (OD600 ~ 2.0) was injected into 100 mL sterilized TSB medium, and incubated in an incubator with 10 mM Na2SeO3 and 1 mM Bi(NO3)3 at 30 oC, 150 rpm. After 48h, the Bi2Se3 associated with bacteria cells were collected by centrifugation (8000 rpm, 10 min). For strain ZYM-1, the extracellular Bi2Se3 was further collected by centrifuging the supernatant at 20,000 rpm for 30 min, and washed the collected pellets twice with ultrapure water (Milli Q reverse osmosis water purificator, 18 MΩ·cm at 25 oC). The collected products were dried in vacuum at 80 oC for 8h, and then grinded for further characterization and PTT application. For morphology modulation, 1% PVP (v/v) was added into the sterilized TSB medium, and the other conditions were the same with the previous mentioned.

Characterization of as-synthesized Bi2Se3

The crystal structure and phase purity of the biogenic Bi2Se3 NPs were determined by X-ray diffractometer (Shimadzu, XRD-7000S) using Cu Kα as a radiation source at a scan rate of 6o min-1. Bismuth and selenium quantitative analysis were performed using ICP-OES (Thermo ICAP-QC). A FEI NanoSEM450 scanning electron microscope (SEM) was used to examine the sample morphology. Transmission electron microscope (TEM) and spot scanning were carried out by FEI Tecnai G2 F30 equipped with EDX (Bruker super-X). Chemical compositions and elemental chemical states were determined through


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X-ray photoelectron spectroscopy (XPS), which was conducted on the Thermofisher ESCALAB 250Xi. UV-vis-NIR spectra were obtained by a PerkinElmer LAMBDA 950 spectrophotometer. The absorption coefficient of Bi2Se3 (α) at 808 nm was calculated from the absorbance (A) of different concentration Bi2Se3 (0, 6.5, 13 and 26 mg L-1) using Lambert-Beer law (A/L = αC), where L is the cell length (1 cm). The thickness of Bi2Se3 was measured with atomic force microscopy (Bruker Dimention ICON) using the tapping mode. The size distribution of nanoparticles was obtained by Zetasizer Nano ZS (Malvern).

Details for photothermal conversion efficiency calculation

After collected the Bi2Se3 nanoparticles, the pellets were resuspended using PBS buffer (10 mM), and then centrifuged (5000 rpm, 60 min) and washed with PBS buffer in a 50 mL ultrafiltration centrifuge tube ( Millipore, 10 kDa) for 6 times. A quartz cuvette filled with 1 mL Bi2Se3 dispersions (0, 6.5, 13 and 26 mg L-1) was irradiated with a fiber-coupled continuous semiconductor diode laser (808 nm, Changchun New Industries) with specific irradiation power (2 W) for 600 s, then naturally cooled to the ambient temperature. The solution temperature change was determined by both thermocouple and infrared thermal imaging camera (FLIR One). For evaluating the photothermal conversion efficiency η of 26 mg L-1 Bi2Se3-Z, we calculated through the following equations:

η= hS (Tmax-Tmax,water) / I (1 - 10 −A808)

(1)

hS =∑ mCp/τs

(2)

τs= −t / ln θ

(3)



θ = (Tabm -T)/(Tabm -Tmax)

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(4)

where h is the heat transfer coefficient, S is the surface area of the container. Tmax and Tmax, water

are the maximum temperatures of Bi2Se3 and water (61.9 and 28.1 oC, respectively). I

is the incident laser power (2 W). A808 is the absorbance of Bi2Se3 at 808 nm (A808 = 0.425). m is the mass (1.0 g) and Cp is the heat capacity of water [4.2 × 103 kJ (kg oC)−1]. τs is the sample system time constant, which was calculated as 357 s according to Figure S1. θ is the dimensionless driving force temperature, Tabm is the ambient surrounding temperature.19

Cytotoxicity and in vitro photothermal cancer therapy

For cytotoxicity studies of Bi2Se 3, human breast cancer cells MCF-7, human breast epithelial cells MCF10A, and human lung cancer cells A549 were respectively seeded into 96-well plates at a density of 5,000 cells per well and cultured for 24 h. The medium was then removed and the cells were cultured with fresh medium containing serial concentrations of Bi2Se3. After further incubation for 8 h at 37 °C, 10 µL of CCK8 solution was added into each well. The cells were incubated for 1 h at 37 °C and then the absorbance was determined using a 96-well plate reader (BioTek, Synergy H1, USA). All data were shown as the percentages of viable cells relative to the survival of the control group (cells treated with medium).

To test the feasibility of photothermal ablation of cancer cells, MCF7 cells and A549 cells (1 × 104 cells per well) were respectively seeded into 96-well plates and incubated in a humidified 5% CO2 atmosphere overnight at 37 °C. Then cells were cultured with fresh


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medium containing different concentrations of Bi2Se3-Z (0, 6.5, 13, 26 mg/L) for 5 h at 37 °C and then illuminated by an 808 nm laser with a power of 0.5 W or 2 W for 10 min. The laser spot was adjusted to fully cover the area of each well. After irradiation, the samples were incubated at 37 °C in 5% CO2 and 95% air humidified atmosphere for 20 h. The treated cells were rinsed with PBS, co-stained with Calcein AM and PI for 30 min, and then imaged with inverted fluorescence microscope (Leica DMI4000B). Quantitative results of live/dead cells were measured via counting green and red cells using imageJ software (https://imagej.nih.gov/ij/).

Preliminary formation mechanism of biogenic Bi2Se3

To find possible reaction location of precursors and bacteria, the bacteria cells were first incubated in TSB medium for 24 h, and then the cells were collected through centrifuging at 10,000 rpm for 20 min, the supernatant was designated as “extracellular”. After then, the collected cells were washed using ultrapure water and centrifuged repeatedly, and the cells were lysed by 20 min ultrasonication, then centrifuged at 22,000 rpm for 40 min. Pellets were mainly the membrane fraction, and the supernatant contained intracellular proteins. After that, 200 µL of each sample was incubated in 96-well plates, and different concentrations of NADH and NADPH were added into samples.Then, 10 mM Na 2SeO3 and 1 mM Bi(NO3)3 were added into each well and further incubated for 12h. Meanwhile, Different concentrations of GSH (1 mM and 5 mM) were added into 20 mL pre-incubated ZYM-1 or CC-1 cells (OD600 is about 2.0) to investigate the role of GSH in Bi2Se3 formation. For time-course TEM imaging, 10 mM Na2SeO3 and 1 mM Bi(NO3)3



were added into 100 mL cultures of ZYM-1, and incubated at 30 oC, 150 rpm. Samples were taken at a specific time interval (2h, 4h, 12h and 48h) and dropped into copper grids.

RESULTS AND DISCUSSION

Two SeRB isolated from marine sediments, i.e. Lysinibacillus sp. ZYM-1 and Bacillus cereus CC-1, were used to investigate the possibility of synthesis Bi2 Se3. Both of the culture solutions turned to black within 12h. Interestingly, after centrifuging at 8,000 rpm for 10 min, the supernatant of strain ZYM-1 was still black, indicating small extracellular nanoparticles formed (Figure S2). The as-synthesized nanoparticles were centrifuged, washed, and dried in vacuum at 80 oC for 8 h, then collected for further characterization. As shown in Figure 1, the products generated by strain CC-1 (hereafter named Bi2Se3-C) have the main peaks around 18.5o, 25.0 o, 29.3o, 40.3o, 43.7o, which can be assigned to the (006), (101), (015), (1010) and (110) crystal planes of rhombohedral phase of Bi2Se3 (JCPDS 89-2008).20 Meanwhile, the XRD peaks of both extracellular and intracellular products of strain ZYM-1 exhibit two weaker peaks corresponded to the (015) and (110) crystal planes of rhombohedral Bi2Se3 phase. Therefore, both SeRB used in this study can synthesis Bi2Se3 nanoparticles. Production of extracellular nanoparticles indicates that strain ZYM-1 may have a secretion system to pump out Bi2Se3. It has been reported that yeast Saccharomyces cerevisiae can expulse biogenic SeNPs to extracellular space by vesicle-like structures.21 However, it is still unclear that which proteins involve the intracellular nanoparticles secretion in SeRB.22 In order to compare the possible effects of crystallinity and biomass on further application, the extracellular Bi2Se3 from strain ZYM-1(hereafter named Bi2Se3-Z) and Bi2Se3-C were used for the following investigation.


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Figure 1. XRD patterns of the synthesized nanoparticles. (a) Products associated with cell debris of Lysinibacillus sp. ZYM-1, (b) extracellular products of strain ZYM-1, and (c) products associated with cell debris of Bacillus sp. CC-1. The black vertical lines at the bottom correspond to the peaks of rhombohedral phase of Bi2Se3 (JCPDS 89-2008).

Then, XPS was employed to evaluate the composition and chemical valence of the products (Figure 2). The survey scan spectrum revealed that element Bi, Se, C, N, O and S existed in both products, indicating the coexistence of biomolecules and nanoparticles.23 The high resolution Se 3d scan of Bi2Se 3-Z showed the binding energies of Se 3d3/2 and Se 3d5/2 are 55.28 eV and 53.68 eV, which suggested the existence of Se2- and the absence of Se0. It could be also observed that the significant S 2p1/2 and S 2p3/2 peaks were located nearly at the Bi 4f5/2 and Bi 4f7/2 in both products. The S source may be the thiol group of GSH or cysteine in proteins, which have been reported are important for biogenic CdSe formation.15 In comparison, the elemental Se peak was observed in the products of strain CC-1.24 A tentative explanation was that excess selenite are alternatively reduced to Se0 by



strain CC-1, and it is also suggest that different formation mechanism of Bi2Se3 exist in SeRB.

Figure 2. High resolution XPS spectra of as-synthesized products by strain ZYM-1 (a, b) and CC-1 (c, d). (a) and (c) correspond Se 3d signal, (b) and (d) correspond Bi 4f signal.

As the size and morphology of Bi2Se3 affect its photothermal performance, SEM, TEM and AFM were employed to observe the morphology and size of products. For Bi2Se3-Z, it can be seen that the sheet-like structure is associated with the microbe cells or dispersed at the extracellular space (Figure 3a and 3b). The extracellular dispersed Bi2Se3 exhibits a crapy state, and the result of EDX shows the atomic proportion of Bi and Se is nearly 2:3 (Figure S3a and S3b). The FTIR spectrum in Figure S3c shows that peaks at 1396 and 1648 cm-1 are attributed to the symmetric and stretching vibrations of carboxylic group, and the peak at 1532 cm-1 belongs to the amide-II band.25 This result confirms that


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proteins are coated on the Bi2Se3-Z. Moreover, Figure S3d shows that the as-synthesized Bi2Se3-Z are highly stable on various medium and buffer and its hydraulic sizes are all around 60 nm. According to the AFM image, the average thickness of nanosheets was about 5-6 nm, which corresponded to 5 to 6 quintuple layer (five atomic basic units in sequence of -Se-Bi-Se-Bi-Se-).26 This thickness is hardly obtained by traditional chemical methods, nevertheless in mild condition. Interestingly, some holes were found in Bi2Se3-Z, suggesting the nanosheets were assembled from smaller nanoparticles (Figure 3c). The SEM and TEM images show that Bi2Se 3-C displays a similar sheet-like strcuture with Bi2Se3-Z, but the as-synthesized nanomaterials were implanted into the cell wall and difficult to purify from biomass, which limits its further application (Figure S4).

Figure 3. Morphology of the as-synthesized Bi2Se3 by strain ZYM-1 (Bi2Se3-Z). (a) Bi2Se3 nanosheets associated with bacterium cells, (b) extracellular Bi2Se3, and inset



was the SAED image of Bi2Se3, and (c) AFM image of extracellular Bi2Se3. The A, B, C, D were four randomly selected nanosheets for thickness measurement.

Then, as the surface coated biomolecules on Bi2Se3 may promote the biocompatibility in PTT, the photothermal conversion efficiencies of Bi2Se3-Z and Bi2Se3-C were firstly evaluated. For Bi2Se3-Z, the UV-vis-NIR spectra with different Bi2Se3 concentrations were shown in Figure 4a. The absorbance at 808 nm were used to calculate the mass extinction coefficient α. This value was calculated as 17 L g-1 cm-1, which is higher than the chemogenic Bi2Se3 (11.5 L g-1 cm-1), and was about 4-folds by commercial Au nanorods (3.9 L g-1 cm-1).2 Figure 4b shows the concentration-dependent photothermal performance of Bi2Se3-Z. When the solution was irradiated by an 808 nm NIR laser (2W) for 600 s, the temperature raised rapidly. The temperature change was 37.6 oC, 22.9 oC and 13.7 oC for 26 mg L-1, 13 mg L-1 and 6.5 mg L-1 Bi2Se 3-Z, respectively. The corresponding photothermal conversion efficiency for 26 mg L-1 Bi2Se 3-Z was calculated as 30.7%, as showed in Figure 4c. This efficiency was comparable with most of the reported chemogenic photothermal nanomaterials.27 Then, four runs of laser on/off cycles were performed to evaluate the PTT stability of Bi2Se 3-Z (Figure 4d). A slightly decrease of the highest temperature from 61.9 oC to 58.9 oC can be observed during the four cycles. Morphology and size of Bi2Se3-Z before and after 4 laser on/off cycles were similar (Figure S5). This result showed the Bi2Se3-Z has relatively high PTT stability. For comparison, 26 mg L-1 Bi2Se3-C only showed weakly photothermal conversion efficiency (data not shown). The temperature change was less than 10 oC after 10 min irradiation,


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which may be due to the tightly-associated membrane fractions with Bi2Se3 interfere the contact of laser and pristine Bi2Se3.

Figure 4. Photothermal characterization of Bi2Se3-Z. (a) UV-vis-NIR adsorption spectra of different concentrations of Bi2Se3. (b) temperature variation of Bi2Se3-Z solutions with different concentrations. (c) PT heating and cooling curve in the presence of 26 mg L-1 Bi2Se3. Inset were infrared thermal images during heating process. (d) temperature variation over four laser ON/OFF cycles with 808 nm NIR laser irradiation (2W).

Inspired by photothermal performance of Bi2Se3-Z, We further study the in vitro biocompatibility and PTT effeciency for human breast cancer MCF-7 cells and lung carcinoma A549 cells. With the coating of biomolecules, the Bi2Se3-Z showed no significant toxicity to both cells after incubation for 24h with different concentration of Bi2Se3-Z. Even the concentration was up to 26 mg L-1, the cell viability of MCF-7 and A549 cells could reach 89.2±3.1 % and 84.1 ± 15.6% (Figure 5a and 5b). Moreover,



Bi2Se3-Z exhibits a high biocompatibility to the normal cells, i.e. human breast epithelial cells MCF10A (Figure 5c). Then, after incubated with Bi2Se3-Z for 5 h, both cells were irradiated with the NIR laser (808 nm, 2 W or 0.5 W) for 10 min. It can be observed a Bi2Se3 dose and power dependent PT effect on both cancer cells. When the Bi2Se3 concentration is 6.5 mg L-1, less than 30% of A549 cells are killed. MCF-7 cells are more sensitive to Bi2Se3-Z, 56% of the cells are be killed with a 2 W NIR laser irradiation. When the Bi2Se3 concentration further increases to 13 mg L-1, more cell death are observed for both A549 (81%) and MCF-7 (49%) cells with 2 W NIR irradiation. 26 mg L-1 Bi2Se 3 leads to 75% of A549 cells and 87% of MCF-7 cells death even the irradiation power is 0.5 W. When the power increases to 2 W, all of the cancer cells are dead. For comparison, in the absence of Bi2Se3-Z, the cell viability of MCF-7 and A549 is not affected by NIR laser irradiation (Figure 6).

Figure 5. Relative viability of (a) MCF-7, (b) A549 and (c) MCF10A cells after incubating with different concentrations of Bi2Se3-Z (0, 6.5, 13 and 26 mg L-1) for 8 h by CCK8 assay.


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Figure 6. In vitro photothermal therapy of cancer cells by biogenic Bi2Se3-Z. A549 (a) and MCF-7 (b) cells were incubated with different concentrations of Bi2Se 3-Z (0, 6.5, 13 and 26 mg L-1) and irradiated by NIR light (808 nm) with different power (0.5 and 2W) for 10 min.

Considering the location of Bi2Se3 nanosheets were different when using strain ZYM-1 and CC-1, the forming mechanism could be supposed to either a direct extracellular synthesis or self-attachment of intracellular small nanoparticles pumped out by bacteria. We firstly added 10 mM Na2SeO3 and 1 mM Bi(NO3)3 into the extracellular extracts, membrane fractions and intracellular extracts of pre-incubated bacterial cells with or without cofactors (NADH and NADPH) into 96-well plates, respectively. It can be seen that the cell membrane fraction of both bacteria are responsible for Bi2Se 3 formation. Moreover, the concentration dependence of NADH and NADPH suggested this process is an enzymatic reaction (Figure S6). The selenite reducing capacity in membrane fraction is



usually contributed by membrane proteins, such as nitrite reductase, sulfite reductase and fumarate reductase, as we found in the genome of strain ZYM-1.12 As previously reported, Bi3+ and Se2- can be metabolized by bacteria to form corresponding organometallic species, especially with the intracellular thiol GSH.28,29 Interestingly, adding 5 mM GSH into the culture solution of strain ZYM-1 made the Bi2Se3 formation faster than without extra GSH (less than 1 min versus more than 5 h). While the same concentration of GSH inhibits the Bi2Se3 formation by strain CC-1 significantly. The red color suggested only selenite was reduced, but no Bi2Se3 was formed (Figure S7). These results clearly shows the existence of different bacteria mediated Bi2Se 3 formation mechanism.

On the other hand, the time-course TEM images of Bi2Se3-Z fabrication during different reaction stages exhibited a morphology change from small nanoparticles to large nanosheet structure, indicating a self-attachments process occurred (Figure 7a to 7d). Meanwhile, even at the initial stage (2h reaction), small extracellular Bi2Se3 nanoparticles can be observed, indicating the production and pumping out of Bi2Se 3 by strain ZYM-1 takes place simultaneously. According to the previous report, the growth mechanism of Bi2Se3-Z may be similar with the PVP assisted hydrothermal method, which involves the self-attachment of small nanoparticles and further epitaxial recrystallization.10 Based on the above results, the possible synthetic and self-attachment mechanism for biogenic Bi2Se3-Z by strain ZYM-1 was shown in Figure 7e.


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Figure 7. TEM images of Bi2Se3-Z at different time intervals. (a) small extracellular and intracellular nanoparticles at 2h, (b) large amount of intracellular nanoparticles at 4h, (c) nanosheets formed and bacterial cell lysis at 12h, (d) ultrathin nanosheets obtained at 48h, and (e) schematic for biological synthesis and self-assemble mechanism of Bi2Se3-Z.

Moreover, a main drawback for biogenic nanoparticles is the difficulty of controlling morphology and size. Therefore, we also tried to modulate the crystallinity and morphology of biogenic Bi2Se 3 by adding surfactant. Interestingly, if 1% (v/v) PVP is added into the culture medium of strain ZYM-1, special Bi2Se3 nanodumbbells are obtained. The TEM images, obtained at different time intervals, exhibits that size and morphology change from small nanospheres (2-3 nm) to irregular nanosheets, turns into near spherical nanoparticles (about 100 nm), and finally self-assembles into nanodumbbells (Figure 8). High-affinity of PVP to both Bi2Se3 and bacterial proteins plays an important role in obtaining the nanodumbbells. Existence of PVP prevents the aggregation of Bi2Se3 nanospheres, and the bacterial proteins induce the formation and growth of this nanospheres.

The excess proteins

can

combine serveral


nanospheres into

the


nanodumbbells. This specific morphology of Bi2Se 3 has not been reported at previous work, and may have different photothermal or thermoelectric characterization.

Figure 8. TEM images and scheme about 1% PVP (v/v) assisted Bi2Se3 nanodumbbells formation by strain ZYM-1. (a) small nanospheres formed at 10h, (b) irregular nanosheets formed at 12h, (c) near spherical nanospheres coated by proteins formed at 24h, (d) and (e) larger assembled Bi2Se 3 nanodumbbells existed with bacterial cells, and (f) scheme about the formation process of Bi2Se3 nanodumbbells.

Finally, to evaluate the expandability of this biogenic approach for metallic selenides, we also test the possibility to fabricate other metal selenides using SeRB. Besides Bi2Se 3, highly crystallized Ag2Se and PbSe nanoparticles can also be synthesized by strain CC-1, which are also excellent photothermal and thermoelectric materials (Figure S8). However, ZnSe and CdSe which had synthesized by other SeRB were not obtained in this case.15,17 These results indicated the potential and reaction selectivity for fabricating versatile


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metallic selenides nanomaterials by specific SeRB, which may relate with the metal transformation and resistance genes.

CONCLUSION

In this study, we reported a novel microbial based synthetic approach for Bi2 Se3, an important phothothermal and thermoelectric material. The reaction was carried out in mild condition and ultrathin nanosheet-like structure could be obtained using Lysinibacillus sp. ZYM-1.The biogenic Bi2Se3 shows a dose and irradiation power dependent photothermal performance to kill cancer cells. When the concentration of Bi2Se3-Z was 26 mg L-1 and irradiation power was 2 W, both of the cancer cells were killed with 10 min irradiation. Meanwhile, the synthetic mechanism of biogenic Bi2Se3 was also explored. This bacteria driven approach has some advantages compared with chemogenic methods, such as: easy obtained Se precursor, mild reaction conditions (room temperature, neutral pH, and aqueous phase), and low energy cost. Moreover, this method can also apply to the fabrication of other metallic selenides. Overall, the “smart” bacteria can be a nanofactory to produce biocompatiable photothermal nanomaterials.

SUPPORTING IMFORMATION

Plot of cooling time versus negative natural logarithm of the temperature driving force

(Figure S1), Photographs of Bi2Se3 NPs synthesized by strain CC-1 and ZYM-1 after centrifugation (8000 rpm, 10 min) (Figure S2), (a) TEM image of extracellular Bi2Se3-Z. (b) EDX of Bi2Se3-Z. (c) FTIR of Bi2Se3-Z. (d) size distribution of Bi2Se3-Z measured by DLS in different matrices (Figure S3), SEM (a) and TEM (b) images of Bi2Se3-C (Figure S4), (a) TEM images of



extracellular Bi2Se3-Z before NIR laser irradiation. (b) TEM images of extracellular Bi2Se3-Z after 4 ON/OFF cycles of NIR laser irradiation. (c) size distribution of Bi2Se3-Z measured by DLS

(Figure S5), Forming location of Bi2Se3 by (a) strain CC-1 and (b) ZYM-1 (Figure S6), Effect of GSH to the Bi2Se3 formation by strain CC-1 and ZYM-1 (Figure S7), (a) XRD spectra of as-synthesized Ag2Se by strain CC-1. (b) SEM image of as-synthesized Ag2Se by strain CC-1. (c) EDX of as-synthesized Ag2Se by strain CC-1. (d) XRD spectra of as-synthesized PbSe by strain CC-1. (e) SEM image of as-synthesized PbSe by strain CC-1. (f) EDX of as-synthesized PbSe by strain CC-1 (Figure S8).

AUTHOR INFORMATION

Corresponding Author * E-mail: [email protected].

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS

The authors gratefully acknowledge the financial supports from the National Natural Science Foundation of China (No. 31500080), and “the Fundamental Research Funds for the Central Universities” (DUT17JC46). We also thank Dr. Michihiko Ike for the communication of biogenic Bi2Se3 in an international conference.


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Table of Contents


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Fabricating Bi2Se3 nanosheet using "smart" bacteria and use it as photothermal material 279x143mm (150 x 150 DPI)


Bacteria-Mediated Ultrathin Bi2Se3 Nanosheets Fabrication and Their

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