Directed Biofabrication of Nanoparticles through Regulating

Aug 21, 2017 - Biofabrication of nanomaterials is currently constrained by a low production efficiency and poor controllability on product quality com...
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Directed Biofabrication of Nanoparticles through Regulating Extracellular Electron Transfer Li-Jiao Tian,† Wen-Wei Li,*,† Ting-Ting Zhu,† Jie-Jie Chen,† Wei-Kang Wang,† Peng-Fei An,§ Long Zhang,§ Jun-Cai Dong,§ Yong Guan,‡ Dong-Feng Liu,† Nan-Qing Zhou,† Gang Liu,‡ Yang-Chao Tian,‡ and Han-Qing Yu*,† †

CAS Key Laboratory of Urban Pollutant Conversion, Department of Chemistry, University of Science and Technology of China, Hefei 230026, China ‡ National Synchrotron Radiation Laboratory, University of Science and Technology of China, Hefei 230026, China § Beijing Synchrotron Radiation Laboratory, Institute of High Energy Physics, Chinese Academy of Science, Beijing 100049, China S Supporting Information *

oneidensis MR-1, a model DMRB. In particular, high-quality CdSe nanoparticles were fabricated in the cytoplasm at rates far exceeding the existing biosynthesis systems.4 Our findings imply a high potential of using DMRB for fine-controlled, efficient biosynthesis processes. In this proof-of-concept study, Cd and Se ions were selected as the precursors for nanoparticles biosynthesis. These ions are environmentally ubiquitous, abundantly available on the earth, and the resulting fluorescent CdSe nanoparticles are economically valuable and easily traceable to allow an in vivo study.5 Selenite reduction by S. oneidensis MR-1 with concomitant Se0 nanoparticles production has been demonstrated in previous studies.6 However, it is unclear whether CdSe nanoparticles could also be synthesized by this strain. Here, we found that, in addition to the abundantly formed Se0 within the wild-type (WT) bacterial cells after exposure to Na2SeO3 and CdCl2, the signal of CdSe was also detected by the Se K-edge X-ray absorption near-edge structure (XANES) and extended X-ray absorption fine structure (EXAFS) spectroscopy (Figure 1a,b). The presence of small-amount CdSe nanoparticles was also supported by the featured yellow fluorescence4b,5b emitted by the WT compared to the control (Figure 1c, Supporting Information (SI), Figures S1, S2). To clarify where and how CdSe was synthesized within the cells, we then constructed mutants with the CymA-encoding gene deleted (ΔcymA) or overexpressed (PYYDT-cymA) (SI, Figure S3a). CymA is a key membrane-anchor protein channeling the electron transfer from cytoplasm to periplasm in S. oneidensis.6 Thus, the ΔcymA severely impaired EET ability compared to the WT, whereas the PYYDT-cymA performed oppositely. This was validated by the different reduction performances of methyl orange, as an extracellular electron acceptor, by the strains (SI, Figure S3c). The EET ability changes remarkably affected the Se reduction products. The Se K-edge white line peak position, which is indicative of the Se redox states,7 shows the highest production of CdSe (12,662 eV) and lowest production of Se0 (12,664 eV) by ΔcymA among all the strains (Figure 1a). The production of hexagonal CdSe by ΔcymA was further validated by the three

ABSTRACT: Biofabrication of nanomaterials is currently constrained by a low production efficiency and poor controllability on product quality compared to chemical synthetic routes. In this work, we show an attractive new biosynthesis system to break these limitations. A directed production of selenium-containing nanoparticles in Shewanella oneidensis MR-1 cells, with fine-tuned composition and subcellular synthetic location, was achieved by modifying the extracellular electron transfer chain. By taking advantage of its untapped intracellular detoxification and synthetic power, we obtained high-purity, uniformsized cadmium selenide nanoparticles in the cytoplasm, with the production rates and fluorescent intensities far exceeding the state-of-the-art biosystems. These findings may fundamentally change our perception of nanomaterial biosynthesis process and lead to the development of finecontrollable nanoparticles biosynthesis technologies.

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iofabrication presents a promising sustainable way to produce metallic nanoparticles,1 with many environmental, economic and operational merits over conventional chemical synthetic routes. However, the utilization of such biogenic nanoparticles is generally limited by a low yield and quality, arising from the biotoxicity of precursors and a poor process controllability. One intensively studied bioproducer is the dissimilatory metal-reducing bacteria (DMRB), which are widely present in natural environment and various engineered biosystems. DMRB can respire on multiple metal ions to produce nanoparticles,2 relying mainly on their ability of extracellular electron transfer (EET). However, the usually sluggish EET process and the complexity of metabolic pathway and environment matrix severely constrain the yield and quality of resulting nanoparticles.3 Here, we discovered in DMRB an amazing intracellular synthetic power that could be exploited for high-rate production of nanoparticles. This finding makes it possible to tune the biosynthesized products through simply regulating the EET process. A directed production of cadmium selenide (CdSe) or elemental selenium (Se0) nanoparticles by enforcing such an EET regulation strategy was demonstrated in Shewanella © 2017 American Chemical Society

Received: July 17, 2017 Published: August 21, 2017 12149

DOI: 10.1021/jacs.7b07460 J. Am. Chem. Soc. 2017, 139, 12149−12152

Communication

Journal of the American Chemical Society

Figure 2. Morphology, size distribution and element composition of purified nanomaterials from (a−c) ΔcymA and (d-f) PYYDT-cymA. (a, d) TEM image, (b, e) size distribution histogram, (c, f) EDS spectra.

Figure 1. Redox states, coordinate bonds of Se content and fluorescence properties of biogenic materials by different strains. (a) Se K-edge normalized XANES spectra, (b) Fourier transformed of Se K-edge EXAFS k2χ(k) functions spectra of references Se (Se, SeCys2: selenocystine, SeMet: selenomethionine, CdSe, Na2SeO3) and samples. (c) Strains under UV light irradiation. (d) Fluorescence emission spectra were recorded at 303 nm excitation wavelength.

controlled biosynthesis of nanoparticles with distinctly different compositions and morphologies was enabled by modifying the CymA-centered EET pathway. The critical regulatory role of CymA and its unique position in the EET chain suggest that CdSe was most likely formed mainly in the cytoplasm whereas Se0 in the periplasm. The cytoplasmic fabrication of CdSe was verified by the SEM, TEM and soft X-ray tomography (SXT) characterizations at the subcellular level. The ΔcymA cells showed smooth surface without visible nanoparticles (Figure 3b), but intensive Se and Cd signals were detected by EDS (Figure 3c). Interestingly, large aggregates of spherical Se-containing nanoparticles were abundantly found at the vicinity of the PYYDT-cymA cells (Figure 3n,o), and small aggregates were also observed in the WT samples (Figure 3h,i), indicating that Se0 nanoparticles were obtained at outside the cells. An aggregate of the nanoparticles with a high electron density and rich Se and Cd contents was observed in the cytoplasm of all the strains whereas Se-rich particles were found mainly at the surface of WT and PYYDTcymA cells. Similar subcellular locations of formed nanoparticles were also revealed by the SXT analysis (SI, Figure S7, Video S1). Together, these results confirm the formation of CdSe nanoparticles aggregates inside the cells and the presence of Se0 aggregates at the cell surface. Here, the aggregation of biogenic NPs should be mainly attributed to the presence of capping proteins, as can be seen from the TEM image of the extracted yet insufficiently purified samples (SI, Figure S8). The largest-sized aggregates of cytoplasmic CdSe and extracellular Se0 were observed in ΔcymA and PYYDT-cymA (Figure 3), respectively, which were in a good agreement with the above results of the product composition analysis. Notably, the abundant extracellular Se0 aggregates obtained by the PYYDTcymA seemed to contradict the fact that many SeO32− reductase were located in the periplasm.6 It is likely that the extracellular Se0 nanoparticles were mainly synthesized in the periplasm and then exported out of the cells as a detoxification mechanism. Such a secretion of intracellularly formed Se0 nanoparticles has been found in many other microorganisms.10 Though the periplasmic synthesis of Se0 was firmly supported by our study, the possibility of its cytoplasmic formation and further conversion into CdSe cannot be fully excluded yet according to the literature.11 To clarify the route of cytoplasmic CdSe formation, we calculated the standard Gibbs free energy changes of this reaction route by density functional theory (DFT) calculations. The results clearly indicate that, when Cd is present, the direct formation of CdSe is thermodynamically

well-defined feature peaks (a′, b′ and c′) that matched the CdSe standard. Meanwhile, the suppressed Se0 production in the ΔcymA implies that the Se0 formation might mainly occur at places outside the cytoplasm, which is consistent with a previous report.6 The different fates of Se in these strains were further evidenced by the Fourier transforms of the Se K-edge EXAFS and the least-squares EXAFS curve fitting analysis, which reveals detailed bond information (SI, Figure S4). The peak intensities of Cd−Se bond followed the order of ΔcymA > WT > PYYDTcymA, whereas those of Se−Se bond were in the opposite order (Figure 1b). This result is consistent with the lowest Se−Se coordination (0.8) at a distance of 2.32 Å and the highest Se− Cd coordination (2.5) at a distance of 2.60 Å observed in ΔcymA compared to the other strains (SI, Table S1). In situ micro-Raman spectrum further confirmed the presence of CdSe (203 and 406 cm−1)8 and Se0 (253 cm−1)9 in the ΔcymA cells (SI, Figure S5f). In addition, among all the strains, the ΔcymA showed the weakest reddish color (typical of Se0) under bright field (SI, Figure S6) and highest fluorescence intensity (typical of CdSe nanoparticles) (Figure 1c). The peak intensity at 570 nm band (indicative of CdSe nanoparticles) was 170.1% higher than that of the WT, and 466.0% higher than that of the PYYDTcymA (Figure 1d). All this evidence confirms that CdSe nanoparticles were the main products in ΔcymA whereas Se0 nanoparticles dominated in PYYDT-cymA. The nanoparticles obtained from different strains exhibited significantly different morphological properties. The transmission electron microscopic (TEM) and high angle annular dark field (HAADF) microscopic images show that the purified products from ΔcymA were mainly ultrafine and uniform-sized particles (average diameter = 3.3 ± 0.6 nm), with lattice fringe (distance of 0.21 nm) consistent with the (110) interplanar spacing of wurtzite structure CdSe (Figure 2a,b, SI, Figure S5d). In addition, the purified nanoparticles contained abundant Cd and Se elements according to the X-ray energy-dispersive spectrum (EDS) (Figure 2c) and showed slow fluorescence decay (99.80 ± 1.770 ns in emission lifetime) (SI, Figure S5e). Compared with the ΔcymA-derived products, aggregates of larger-sized (104.6 ± 8.4 nm), spherical Se0-rich nanoparticles were derived from PYYDT-cymA (Figure 2d−f). Therefore, a 12150

DOI: 10.1021/jacs.7b07460 J. Am. Chem. Soc. 2017, 139, 12149−12152

Communication

Journal of the American Chemical Society

Figure 3. Fluorescence, morphology and elemental composition of the biosynthesized nanomaterials in vivo by (a−f) ΔcymA, (g−l) WT, and (m−-r) PYYDT-cymA. (a, g, m) Fluorescence microscopic images show the yellow fluorescence emitted by the cells; (b, h, n) SEM images of the cells with synthesized nanoparticles; (c, i, o) EDS spectra of samples at the arrow marked point in the SEM images; (d, e, j, k, p, q) TEM images of single cell show the location of formed nanoparticles aggregates; (f, l, r) EDS spectra of the samples at the arrow-marked point in the TEM images.

more favorable than the Se0 generation under the physiological conditions (SI, Figure S9). The inability of cytoplasmic Se0 formation and further conversion into CdSe in the presence of Cd is also supported by the experimental results with different time intervals between Se and Cd addition (SI, Figure S10). Therefore, it can be concluded that CdSe was the sole NPs product formed in the cytoplasm, and the small amount of Se0 should be mainly formed in the periplasm. The CdSe and Se0 formation processes both involve a series of Se reduction steps and might proceed in parallel (Figure 4), leading to a mixed product of nanoparticles .

organelles might be responsible for the nanoparticle bioassembly and storage.5b,12 The above results demonstrated an EET-dependent, selective production of nanoparticles in S. oneidensis MR-1 at different subcellular locations and unveiled the underlying mechanisms. In particular, the EET-impaired ΔcymA exhibited an amazing cytoplasmic CdSe synthesis ability, with the CdSe nanoparticles yield (dictated by the intensity of yellow fluorescence) far exceeding the state-of-the-art Escherichia coli (E. coli)-based biosynthesis system.12a,13 This is likely benefited from the superior metal resistance and detoxification ability of S. oneidensis species (SI, Figure S11). In addition, the synthesis process of this mutant took only 4 h, much faster than other CdSe biosynthesis systems reported so far (SI, Table S2). The cytoplasmic synthesis of CdSe nanoparticles might be an important antagonistic mechanism of S. oneidensis for detoxifying Cd,14 because otherwise the imported Cd ions at high concentrations would cause severe oxidative damage to cellular structures.15 The cell viability tests with fluorescence microscopic analysis confirm that the WT cells all became dead after 4 h of exposure to Cd alone (SI, Figure S12), but a markedly high cell viability of 77.09% was sustained under coexposure to Se and Cd (Figure 5b,e). In line with such a CdSe associated mechanism, the ΔcymA cells showed the highest viability (85.48%) among all the strains (Figure 5), coinciding with its highest CdSe nanoparticles production. Therefore, the synthesis of metallic nanoparticles, which typically have a drastically decreased toxicity than their ionic counterparts, offers an important route for biodetoxification of heavy metals, especially for the nonreducible ones such as Cd2+ and lead ions.16 In summary, we propose an EET regulation strategy to tune the bioproduction of nanoparticles (Se0 or CdSe) in S. oneidensis MR-1 based on an insight into the untapped intracellular detoxification and synthetic power in this bacterium. Impairing the EET by deleting cymA gene significantly improved the cytoplasmic CdSe production. The ΔcymA exhibited the fastest biosynthesis of high-purity CdSe nanoparticles ever, implying a

Figure 4. Schematic diagram of the EET-dependent synthesis of nanoparticles by S. oneidensis MR-1.

Thus, the possibility to regulate these two competitive routes through EET pathway modification opens up an unprecedented opportunity for facilely tuning the composition of biosynthesized nanoparticles. In particular, the selective fabrication of high-purity, valuable CdSe nanoparticles in the cytoplasm is highly desirable. Here, the CdSe aggregates were found to locate at one or two defined zones in the cytoplasm of each cell (Figure 3, SI, Figure S5a), implying that some specific cytoplasmic 12151

DOI: 10.1021/jacs.7b07460 J. Am. Chem. Soc. 2017, 139, 12149−12152

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Horiba Co., Japan for the technical support on Raman characterization.

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Figure 5. Fluorescence images (a−c) and flow cytometry analysis (d− f) showing the cell viabilities of different strains after 4 h of exposure to Se and Cd. The blue signals represent the alive cells and the purple red signals represent the dead cells.

great potential to utilize such engineered DMRB for finecontrolled biosynthesis applications. It is expected that such an EET regulation strategy may also be applied to other DMRB and, given their respiration diversity and environmental ubiquity, hopefully lead to more diverse and valuable nanomaterial biofactories. Notably, the molecule-level mechanisms of the biosynthesis processes remain unclear, and the production efficiency and process controllability still need substantial improvement to be competitive with chemical processes. In addition, low-cost, facile methods for extraction and purification of intracellular nanoparticles will be required for large-scale applications, which warrant further investigations.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.7b07460. Experimental details, EXAFS fitted results, bright and UV light images, 3D fluorescence spectra, SXT (PDF) Video showing 3D SXT of ΔcymA synthesized CdSe nanoparticles (MPG)

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*[email protected] *[email protected] ORCID

Wen-Wei Li: 0000-0001-9280-0045 Dong-Feng Liu: 0000-0003-4782-0033 Han-Qing Yu: 0000-0001-5247-6244 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the Natural Science Foundation of China (21477120 and 51538011), the Collaborative Innovation Center of Suzhou Nano Science and Technology of the Ministry of Education of China for the support of this study. The authors thank Prof. Dai-Wen Pang at the Key Laboratory of Analytical Chemistry for Biology and Medicine (MOE), Wuhan University, China for helping nanoparticles purification and 12152

DOI: 10.1021/jacs.7b07460 J. Am. Chem. Soc. 2017, 139, 12149−12152