Expanding Toolbox of Imageable Protein-Gold ... - ACS Publications

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Expanding Toolbox of Imageable Protein-Gold Hybrid Materials Han Ding,*,†,‡ Hongwei Li,† Xiaoliang Wang,† Yufeng Zhou,† Zhenhua Li,† J. Kalervo Hiltunen,†,§ Jiacong Shen,† and Zhijun Chen*,† †

State Key Laboratory of Supramolecular Structure and Materials, Institute of Theoretical Chemistry, and International Joint Research Laboratory of Nano-Micro Architecture Chemistry, Jilin University, 2699 Qianjin Street, 130012, Changchun, P. R. China ‡ Institute for Translational Medicine, College of Medicine, Qingdao University, Deng Zhou Road 38, Qingdao 266021, P. R. China § Faculty of Biochemistry and Molecular Medicine and Biocenter Oulu, University of Oulu, Oulu FI-90014, Finland S Supporting Information *

ABSTRACT: As a new class of bio-abiotic hybrid materials, a series of highly fluorescent self-assembled protein-gold hybrid materials (PGHMs) with different emission wavelengths (blue (B), green (G), yellow (Y), and red (R) colored under UV irradiation) are synthesized by using various combinations of proteins and free amino acids as stabilizer and reducing agents. The synthesis process was controllable by modulating the pH of the reaction solutions. The synthesized PGHMs (PGHMBlue (PGHM-B), PGHM-Green (PGHM-G), PGHM-Yellow (PGHM-Y), and PGHM-Red (PGHM-R)) exhibited distinct fluorescent properties and showed low cytotoxicity as justified by a bacteria-based test system. The sizes of PGHMs are approximately 100 nm with gold core diameters 0.8−1.8 nm as shown by SEM and TEM images. The assembly of PGHMs was dynamic and occurred through a free radical associated cross-linking process. This general strategy expanded the toolbox of protein-gold hybrid materials. The photostable bio-abiotic hybrid fluorescent nanomaterials can be an alternative to conventional fluorescent probes such as organic dyes and quantum dots for bioimaging studies.

1. INTRODUCTION Self-assembly is a ubiquitous phenomenon in living systems where building blocks are assembled to form supramolecular structures through ordered arrangement.1 In cells, proteins interact with their partners to form supramolecular structures in spatial and time-dependent fashion as exemplified by proteinchaperone interaction,2 heterometric enzyme assembly,3 and signaling complex formation.4 Inspired by this principle, a variety of self-assembling materials have been built by combining a limited number of building modules. Among them, protein-based materials have emerged recently as attractive building blocks, and the area has drawn substantial research interest. For instance, using tools of synthetic biology, a number of protein supramolecular structures have been designed and constructed using proteins as assembly modules and applying various supramolecular recognitions such as histidine-nickel, protein-zinc, and Phe-Gly-Gly (FGG)-cucurbituril interactions (for references, see below). These approaches have yielded products such as protein-quantum dots,5,6 proteingold nanoparticle,7 protein-cucurbit[7]uril (CB7)-dye,8 protein-polymer conjugates,9 nanowires,10,11 nanorings,12,13 nanotubes,14 and nanosheets.15 Also, various protein-based hydrogels have been constructed.16−18 These studies demonstrate that proteins are excellent building blocks to make various synthetic bio-abiotic hybrid materials in addition to their functions under physiological conditions. © 2017 American Chemical Society

As a charming technology, bioimaging has advanced life sciences in analyses of various processes in biological systems and characterization of diseases.19 Most of the currently used fluorescent probes are organic dyes,20−22 fluorescent proteins,23 inorganic quantum dots,24,25 rare earth materials,26−28 and polymers.29,30 However, these fluorescent materials have frequently intrinsic weaknesses such as photobleaching (organic dyes and fluorescent proteins) or cytotoxicity (quantum dots).31−35 As a new generation of fluorescent materials, noble metal nanoparticles as well as nanoclusters (NCs) have drawn much attention due to their unique optical and physicochemical properties.36−38 Nowadays, ultrasmall gold and silver NCs have been widely used in various sensing, catalysis, and bioimaging applications.39−42 Traditional gold NC (Au NC) synthesis methods include either a reduction step using thiol containing compounds or polymers as protection agents or etching of large gold nanoparticles.36,37 These conventional synthesis approaches frequently required harsh treatment by adding environmentally unfriendly reagents such as sodium borohydride or tetrabutylammonium borohydride to the reaction solution. Recently, new routes have been developed to Received: July 18, 2017 Revised: September 3, 2017 Published: September 7, 2017 8440

DOI: 10.1021/acs.chemmater.7b03011 Chem. Mater. 2017, 29, 8440−8448

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Chemistry of Materials synthesize fluorescent Au NCs using biomolecules as stabilizer and reducing agents. Among them, proteins have showed great promise to fulfill this task.43−46 Moreover, other biomolecules like amino acids,47,48 lipoic acid,49 and glutathionate50 are also capable of protecting Au NCs. Unfortunately, most of the reported Au NCs were red emitting upon UV irradiation. The monotonic emission properties of Au NCs severely limit their application in bioimaging or other experiments. The latest advances in this field concern that Kawasaki et al. synthesized green and blue emitting fluorescent Au NCs in a pH-dependent manner by using pepsin as a template.51 In addition, Yang et al.52 and Mu et al.53 synthesized blue fluorescent emitting Au NCs by using histidine and proline as protective compounds. However, these methods suffer from several drawbacks in terms of photostability of Au NCs, and being laborious, unstable, and costly. Moreover, compared to other types of Au NCs, it remains challenging to control the size and other properties of biomolecule capped Au NCs. Therefore, it is highly favored to develop a generic approach that is amenable for synthesis of a series of protein conjugating Au NCs with distinct photophysical properties. We describe here a new approach to design and construct protein-gold hybrid materials (PGHMs) with different gold core sizes and optical properties by using a combination of proteins and certain amino acids (AAs) as capping and reducing agents and varying the pH of the reaction solutions. Unexpectedly, during the Au NC synthesis, proteins were assembled into higher structures. Notably, a ring-like structure was formed in the early phase, and then shifted into a capsulelike construction. Free radicals appeared during the synthesis process and were associated with the protein-gold hybrid assembly mimicking a free radical polymerization. The formation of Au NCs and the assembly of protein supramolecular structures were simultaneous and interrelated with each other. The synthesized PGHMs can be readily internalized by living mammalian cells and they can also be used for microbial cell fluorescent labeling in a pH-dependent recyclable manner. In this context, four highly fluorescent PGHMs exhibiting distinct fluorescent emissions were prepared by using a protein-AA combination approach. This type of materials is a new member among bio-abiotic hybrid materials that are promising in imaging, drug delivery, and other biomedical applications.

12.5. The mixture was incubated at an incubator shaker (IS-RDS3, Crystal, Suzhou, P. R. China) at 37 °C for 12 h. The resulting solutions were centrifuged at 11 000g for 2 min. The supernatants were dialyzed against suitable buffers. C. Cytotoxicity of PGHMs. The cytotoxicity of these PGHMs materials was evaluated using an Escherichia coli system.45,54−56 An E. coli DH5α colony was inoculated into LB medium and incubated for overnight (12 h) at 37 °C (with rotating shaking at 150 rpm) until OD600 of the culture reached approximately 3.5. Bacteria were harvested by centrifugation and washed 3 times using PBS buffer and resuspended with fresh LB medium to OD600 = 0.3. Cells were then mixed either with PGHMs (0.2 mM) or with an equal volume of PBS buffer which serves as a control. These cells samples were further incubated at 37 °C (shaking at 150 rpm). OD600 was measured at intervals until the cells reached a steady growth. D. Cell Culture and Imaging. HELA cells were maintained in DMEM medium supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin. Approximately 5 × 104 cells were seeded into each well in a 24-well plate and incubated overnight at 37 °C with 5% CO2 in a humidified atmosphere. The next day, the culture medium was replaced by 200 μL of medium containing PGHMs (0.2 mM). Cellular uptake was detected by a high-content imaging fluorescent microscope (LSM710, Carl Zeiss Jena, German) after 6 h of incubation at 37 °C and then incubated with LysoTracker Deep Red or LysoSensor Green for another 10 min and washed with PBS buffer (pH 7.4) twice. A fixed excitation wavelength of 405 nm was used for all the confocal fluorescent microscopy (CFM) experiments. E. Adhering of PGHMs to Bacteria and Yeast Cells. Bacteria cells (E. coli DH5α) were inoculated into Luria−Bertani broth (LB) media and incubated at 37 °C with shaking at 180 rpm for 12 h. Yeast cells (Saccharomyces cerevisiae BY4741) were inoculated into yeast extract peptone dextrose (YPD) media and incubated at 30 °C with shaking for 24 h. Bacteria and yeast cells were harvested by centrifugation for 10 min at 4000g at 4 °C. The pelleted cells were washed three times with sodium acetate buffer (pH 4.7) including the collection of the cells by repeating the centrifugation step. The pH of the PGHM solutions was also adjusted to pH 4.7. Au NC and cell solutions were mixed to final concentrations of 1 mg/mL (Au NCs) and 1.1 × 109 cells/mL (E. coli) or 1.1 × 108 cells/mL (S. cerevisiae). The mixtures were incubated at 22 °C for 2.5 min and washed 3 times to remove unspecifically bound materials using sodium acetate buffer (pH 4.7). F. Materials Characterization. Fluorescence spectra were measured using a Shimadzu 5301PC Fluorescence Spectrophotometer. UV−visible absorption spectra were measured using a UV-3600 spectrophotometer. Fluorescence lifetime of the samples was recorded on an Edinburgh instruments fls920 spectrofluorometer equipped with a continuous (450 W) xenon lamp. For size and morphology analysis, field emission transmission electron microscopy (TEM) images were acquired on a JEM-2100F Transmission Electron Microscope. Scanning electron microscopy (SEM) images were acquired on a SU8020 scanning electron microscope. X-ray photoelectron spectroscopy (XPS, Thermo Fisher Scientific ESCALAB 250Xi XPS System) was applied to analyze. A SHIMADZU−UFLC LC-6AD was used for the analysis of amino acids. The Malvern ZEN 3600 Zetasizer was used to investigate the particle sizes and Zeta potential. A laser confocal inverted microscope (FV1000) was used for fluorescent imaging related data collections. A Bio-Rad Mini-PROTEAN Tetra was used for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

2. EXPERIMENTAL METHODS A. Materials and Methods. Chemical compounds such as sodium hydroxide (NaOH), hydrochloric acid (HCl), phosphate buffer, sodium chloride (NaCl), Tris-Base, glucose, sucrose, and HEPES were purchased from Beijing Chemical Reagent Company (Beijing, P. R. China). Double-distilled water (dH2O) used throughout the experiments was produced by a Milli-Q system (Millipore, Bedford, MA, USA). Bovine serum albumin (BSA), human serum albumin (HSA), chymotrypsin, lysozyme, glycine, histidine, tryptophan, and other AAs and Sephadex gel 200 were ordered from DingGuoChangsheng Biotechnology (Beijing, P. R. China). Tetrachloroauric acid (HAuCl4) was from Sinopharm Chemical Reagent (Shanghai, P. R. China). The small peptide (Gly-Gly-Gly) was ordered from SigmaAldrich (Beijing, P. R. China). LysoTracker Deep Red and LysoSensor Green DND-189 were ordered from Thermo Fisher Scientific. B. Synthesis of PGHMs. All glassware used in the reaction process was soaked in aqua regia and then washed using dH2O and dried before use. AAs, BSA, and chlorine acid were added into dH2O solution to a final concentration of 100 mM, 5 and 1.6 mg/mL, respectively, and then vigorously stirred for 5 min. The pH of the reaction solutions was adjusted to a desired pH ranging from pH 1.5 to

3. RESULTS AND DISCUSSION Although protein conjugated Au NCs have been extensively investigated and widely used in laboratories in the past few years, surprisingly, so far a standardized approach to make protein conjugated Au NCs with distinct fluorescent emission properties is still missing. Consequently, it is hard to extend their use in biological research in a versatile manner. This 8441


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Chemistry of Materials Scheme 1. Preparation of PGHMs for Cell Imaging Studies

Figure 1. UV−visible, fluorescent spectra, and TEM images of Au NCs of PGHMs. Upper panels: UV−vis, excitation and emission spectra of PGHM-B (a), PGHM-G (b), PGHM-Y (c), and PGHM-R (d). Insets: Au NCs of PGHMs in aqueous solution under UV irradiation (365 nm). Lower panels: TEM morphology and size characterization of Au NCs of PGHM-B (e), PGHM-G (f), PGHM-Y (g), and PGHM-R (h). Insets: size distribution (left ordinate) of the relevant Au NCs of PGHMs with indicated standard deviation (right ordinate).

combination route produced three different types of PGHMs with distinct photophysical properties: PGHM-Green (PGHMG) (Figure 1b), PGHM-Yellow (PGHM-Y) (Figure 1c), and PGHM-Red (PGHM-R) (Figure 1d), which were synthesized at pH 3.5, 1.5, and 12.5, respectively. It was known that AAs can interact with gold atom through forming N−Au, O−Au, S−Au, or H···Au and O−H···Au bonds.57−60 In particular, His and gold ion can form a complex in acidic solution via interaction of gold atom with the α-amino group and the imidazole ring.61 Concerning Gly, it also contains an α-amino group and is the simplest and the only achiral amino acid in nature. In this project, small peptides such as Gly-Gly-Gly and glutathione were also used to tackle the possible mechanism of BSA-AA Au NC synthesis. However, these small peptides did not lead to formation of Au NCs as compared to free Gly (Figures S23, S24), suggesting the size of AAs might be an important factor for the protein-AA combination synthetic process. Indeed, small-sized AAs (e.g., Gly) can be free from steric hindrance of side chains, thus easily enter the cavities of proteins and function as a stabilizer. The role and mechanism of Gly in the synthesis of Au NCs await further studies, raising a possibility for a potential use of Gly in synthesizing other materials.

report describes a novel method for making a kind of bioabiotic hybrid materials, namely, PGHMs, and the method utilizes a protein-amino acid (AA) combination approach (Scheme 1). Briefly, tetrachloroauric acid (1.6 mg/mL) was mixed with different ratios of BSA (5 mg/mL) and AAs (100 mM) and then adjusted to various pHs (1.5−12.5). These mixtures were incubated by shaking at 37 °C for 12 h and then dialyzed against dH2O. UV−vis absorption spectra showed that BSA and tetrachloroauric acid had peaks centered at 280 and 320 nm, respectively. These characteristic peaks were diminished after the synthesis process (Figure S1), indicating the formation of Au NCs containing PGHMs. Using this approach, a set of protein conjugated Au NCs with distinct emission wavelengths can be obtained via changing the combination of protein/AA and pH conditions. To synthesize PGHMs, the 20 natural common AAs were evaluated by using this approach at different pH conditions (Figures S3−S24). Notably, PGHMs with distinct emission wavelengths were synthesized when glycine (Gly) or L-histidine (His) was used as stabilizer/reducing agent in the presence of BSA (Figures S3, S4). Specifically, PGHM-Blue (PGHM-B) was formed at pH 5.5 via the BSA-His combination route (Figure 1a). Strikingly, by changing pH, the BSA-Gly 8442


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Chemistry of Materials

Figure 2. SEM images of PGHMs:PGHM-B (a), PGHM-G (b), PGHM-Y (c), and PGHM-R (d).

that of Rh 6G and even higher than that of reported BSA-Au NCs43 (Figure S26). The synthesized PGHMs were carefully evaluated by using transmission electron microscopy (TEM). It was shown that the gold core of PGHM-B, PGHM-G, PGHM-Y, and PGHM-R was 0.9, 1.7, 1.3, and 1.8 nm, respectively (Figure 1e−h). This observation was consistent with UV−vis spectra, confirming the formation of small-sized Au NCs and the absence of large gold nanoparticles within PGHMs. The valence states of these Au NCs of PGHMs were investigated by using XPS. It was shown that the gold cores of PGHMs were mainly composed of Au0 and Au+ (Au1) (Figure S27). Interestingly, the Au0 content of PGHM-B, PGHM-G, PGHM-Y, and PGHM-R was 50.53, 48.88, 48.28, and 41.62%, respectively, whereas the Au+ content of this set of PGHMs was 49.47, 51.12, 51.72, and 58.88%, respectively. These results hinted that the difference of emission wavelengths of these materials might at least in part be attributed to the difference of the ratio of Au+/Au0. This observation is consistent with several other reports where Au +/Au0 playing a role in determining emission properties of fluorescent materials was suggested65,66. A relationship was observed putatively between the ratio of Au+/Au0 and emission wavelengths of PGHMs (Figure S27). Therefore, we proposed a model to interpret the distribution of the luminescence emission wavelengths of PGHM-B, PGHMG, PGHM-Y, and PGHM-R (Figure S28). In this model, the emission wavelengths of these materials can be tuned through modulating the Au+/Au0 ratio via changing protein, AA, pH, or other conditions. We thus expect that Au NCs containing UV or near-infrared emission properties might be synthesized by

UV−vis spectra of PGHM-B, PGHM-G, PGHM-Y, and PGHM-R did not show a localized surface plasmon resonance peak at approximately 520 nm (Figure 1a−d), suggesting that the synthesized products were small-sized Au NCs instead of large gold nanoparticles.51,62 PGHMs synthesized with a combination of BSA and His/Gly at different pHs emitted distinct fluorescent signals as exemplified by the four different colors (blue, green, yellow, and red) under UV irradiation. Specifically, PGHM-B had excitation and emission peaks at 384 and 468 nm, respectively (Figure 1a). PGHM-G emitted green fluorescence with excitation and emission peaks at 370 and 515 nm, respectively (Figure 1b). The emission peak of PGHM-Y was approximately at 560 nm under irradiation at 383 nm (Figure 1c). PGHM-R had red fluorescence with excitation and emission localized at 372 and 607 nm, respectively (Figure 1d). The quantum yield (QY) of PGHM-B, PGHM-G, PGHM-Y, and PGHM-R was 10.04, 7.67, 6.30, and 6.41%, respectively, when rhodamine 6G (Rh 6G) was used as a reference (Table S1). The QYs of this set of Au NCs were reasonably high compared to those of other biomolecule-capped fluorescent Au NCs.43,63,64 Moreover, the lifetime of PGHM-B, PGHM-G, PGHM-Y, and PGHM-R was 5.57, 8.65, 31.79, and 101.82 ns, respectively (Table S1, Figure S25). Photostability is an important parameter for fluorescent materials. The photostability of recently reported Au NCs was higher than that of traditional fluorescent probes such as Rh 6G and other organic dyes,43 indicating their potential use in imaging or relevant fields. Fluorescence photobleaching experiments showed that the photostability of PGHM-B, PGHM-G, PGHM-Y, and PGHM-R was clearly better than 8443


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Figure 3. SEM morphology diagram showing the PGHM-B forming process along with time. a−i refer to 0, 0.5, 1, 2, 4, 6, 8, 10, and 12 h, respectively. The proteins were gathered and assembled into a ringlike structure in the beginning and then quickly shifted to capsule-like constructions. The dynamic process was not terminated until a smaller globular structure was formed.

(HPLC) showed that tracer amounts of free amino acids such as Gly were associated with PGHMs (Figure S39), while the rest of supplemented AAs might be covalently associated with PGHMs. The overall morphology of the synthesized PGHMs was characterized by using SEM. The sizes of PGHM-B, PGHM-G, PGHM-Y, and PGHM-R were around 140, 120, 60, and 100 nm, respectively (Figure 2a−d), which may correlate to the cross-linking between BSA−BSA and BSA−AA that coat the outside of Au NCs. As control, we also investigated the BSA structure under the same conditions, which showed an amorphous morphology (Figure S42). To explore the mechanism of this cross-linking facilitating assembly, a photoelectron spectrograph was used. Interestingly, free radicals were detected from the samples collected during the intermediate process (Figure S43), suggesting the generation of radicals in the synthetic course. The radicals might play a role in the cross-linking and polymerization of the protein-gold conjugates. As a representative of PGHMs, the assembly process of PGHM-B was carefully monitored using SEM. Significantly, the assembly of PGHM-B was a multistep and dynamic process, where ringlike structures appeared approximately within half an hour, and thereafter quickly shifted to capsule-like constructions and finally spherical nanoballs were formed and stabilized (Figure 3). During this process, the fluorescent signal was gradually enhanced until the stable nanoballs were developed (Figure S44). Additionally, TEM-energy dispersive spectrometry (EDS) (Figure S45) and SEM-EDS (Figure S46) analyses showed that all the tested nanoballs and nanocubes contained gold elements (Table S2), suggesting these particles were PGHMs. The size, morphology, optical, and other properties of PGHMs suggested a possibility that these bio-abiotic hybrid materials might be considered for biological applications. As a precondition, the cytotoxicity of PGHMs was evaluated by using bacterial cells (E. coli) as a model system. Interestingly, PGHMs showed rather low toxicity toward bacterial cells at least up to 0.2 mM (Figure S47).

using this protein-AA combination method. It is worthy to note that further research regarding the fluorescent mechanism of the set of Au NCs is needed before drawing a further conclusion. To our best knowledge, the pepsin-Au NCs were the only reported green emitting protein-conjugated Au NCs synthesized at approximately pH 1 before this study.51 However, the synthesis condition described for pepsin-Au NCs did not work for BSA or several other proteins tested in our laboratory (Figure S29). Protein sequence in silico analysis showed that Gly content within BSA (bovine) and pepsin (porcine) was 2.8% and 9.4%, respectively (sequences 1 and 2 and Figure S30). Therefore, the difference in Gly content of these proteins might somehow explain their different performance in Au NC synthesis. Indeed, as an example, the content of Gly was associated with the fluorescence of PGHM-G (Figure S31). Collectively, our approach can serve as a new platform to synthesize Au NCs within a given protein via supplementing AAs (e.g., Gly or His) and modulation of pH conditions (Figure S32). The metal ion response properties of these Au NCs containing PGHMs were carefully analyzed (Figures S33− S36). The data demonstrate that PGHM-G specifically recognized Fe3+, while PGHM-R selectively responded to Hg2+ and Fe2+. The concentration-dependent quenching effect of PGHM-G and PGHM-R toward these metal ions was also investigated (Figures S37−S39), and the findings exhibited their distinct metal ion response properties as compared to other protein conjugated Au NCs. The protein coronas of the synthesized Au NCs and their overall structures were analyzed by using SDS-PAGE, Superdex 200 chromatography, and SEM. Of note, after completion of the synthesis, the BSA monomers of PGHMs were covalently linked (Figure S40a), suggesting cross-linking events have occurred during the synthesis process. On the basis of the difference in size, these PGHMs can be separated from the excessive amount of free BSA by using size exclusion chromatography on a Superdex 200 column (Figure S40b− d). Moreover, high performance liquid chromatography 8444


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Figure 4. PGHMs can be readily internalized by mammalian cells likely through endocytosis. CFM micrographs show that PGHMs entered HELA cells (a, e, i, m), which were partially co-localized with LysoTracker Deep Red (b, f, j) or LysoSensor Green DND-189 (n) and can be also observed in the merged image (d, h, l, p).

The fluorescence and internalization properties of PGHMs were investigated using mammalian cells (Helen Lane (HELA) cells). The HELA cells were incubated with PGHMs (0.2 mM) at 37 °C for 6 h and then incubated with lysosomal markers (either LysoTracker Deep Red or LysoSensor Green) for another 10 min. Then cells were washed for multiple times using phosphate buffered solution (1 × PBS, pH 7.4) and CFM. Interestingly, PGHMs can be easily taken up and internalized by HELA cells, as shown by the strong emitting signals within the HELA cells that partially co-localized with lysosomes (Figure 4). The other possibility of implementing PGHMs for biosystems was tested by using both bacteria (E. coli DH5α) and yeast cells (S. cerevisiae BY4741) as model systems. Surprisingly, PGHMs were not able to get through the plasma membrane of the bacteria and yeast cells (data not shown). Notably, the zeta potentials of these four distinct PGHMs were positive at low pH and negative at high pH (Table S3), whereas E. coli and S. cerevisiae cells were mainly negatively charged under all the test conditions (Table S4). Therefore, via modulation of the pH of the solution, PGHMs can interact with cells by forming cell-PGHM hybrid supramolecular complexes in a controllable fashion (Figure S48). The following fluorescent cell labeling experiments were carried out at pH 4.7 if not otherwise stated. E. coli cells were incubated with PGHMs for 3 min, followed by centrifugation/ washing steps to separate cells and nonintegrated fluorescent materials. As shown on CFM images, E. coli cells were efficiently labeled by PGHM-B (Figure S49a,b), PGHM-G

(Figure S49d,e), PGHM-Y (Figure S49g,h), and PGHM-R (Figure S49j,k). In contrast, E. coli cells were nonfluorescent in the absence of these materials, which served as controls (Figure S49c,f,i,l). Notably, these fluorescent materials can be easily recycled through shifting the pH of the solutions (Figure S50). Similarly, S. cerevisiae cells were also investigated using a similar procedure. Again, the yeast cells were efficiently labeled by PGHM-B (Figure S51a,b), PGHM-G (Figure S51d,e), PGHMY (Figure S51g,h), and PGHM-R (Figure S51j,k). The fluorescent labeling experiments for both prokaryotic and eukaryotic cells suggest that PGHMs provided a useful toolbox which has potential in microbial biolabeling studies, in addition to mammalian cell intracellular imaging.

4. CONCLUSION Protein-based materials have attracted much attention due to their unique properties such as evolutionary diversity and multiple functional groups of proteins, biocompatibility, and biodegradability. In particular, bio-abiotic hybrid materials hold the promise for future synthetic biology and material development. In the past, concerning protein conjugated metal NCs, most of the researchers focused on the investigation of the properties of NCs, whereas their protein coronas had been largely neglected. We described here that the assembly of Au NC conjugating protein modules underwent a dynamic supramolecular assembly. The proteins shifted from monomeric form to a ringlike structure, and then capsule-like, before they stabilized in nanoball/nanocubes. During this process, free radicals appeared, and cross-linking between protein building 8445



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blocks occurred, which may mimick radical triggered polymerization. Further research is required to understand the supramolecular assembly mechanism of this type of bio-abiotic materials. Collaborating with other laboratories, we are currently also working on drug/gene/protein delivery and nanodiagnostic studies by using PGHMs or the intermediate products of PGHM synthesis (e.g., capsules). Two factors are critical in Au NC synthesis: capping and reducing agents. However, we developed an avenue that a series of protein conjugating Au NCs can be constructed through modulating supplementing AAs and pH. Is this method applicable to other proteins? Our preliminary test was encouraging as several other proteins also showed particular Au NC synthesis capability in the presence of AAs (Figures S52−S63). PGHMs synthesized by using the protein-AA combination approach had potential as new fluorescent biomaterials involved in research tasks that require highly photostable fluorescent labels. PGHMs may be suitable for in vivo or ex vivo studies. They can be readily internalized by mammalian cells as an intracellular bioimaging tool. More efforts are needed to this end to develop new probes for human diseases based on this type of materials. Microbial cells can be labeled by PGHMs in a pH-dependent recyclable manner, suggesting a new role of these materials in bacterial and fungi fluorescent labeling. Taken together, we developed a novel method for the synthesis of a series of fluorescent PGHMs with distinct emission wavelengths. The morphology of PGHMs and their conjugated Au NCs was characterized by using TEM and SEM. The mechanism of multicomponent assembly may be associated with free radicals facilitated cross-linking as shown on SDS-PAGE and photoelectron spectrographs. Rings and capsule-like structures can be observed at the intermediate stage of PGHM-B assembly, suggesting a dynamic supramolecular assembly process. Moreover, XPS analysis suggested that the emission wavelengths of Au NCs of PGHMs were associated with the Au+/Au0 ratio which potentially can be tuned by modulating protein and AA composition, pH, or other conditions. Moreover, it is highly favored to have synthesized hybrid materials with different gold core sizes and optical properties for various uses.67−69 In particular, this type of bioabiotic hybrid materials is expected to be used for multifarious biomedical applications. Finally, this environmentally friendly BSA-AA combination approach ensures a high level of biocompatibility and is expected to be extended to the preparation of other noble metal NCs.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (Z.C.). *E-mail: [email protected] (H.D.). ORCID

Hongwei Li: 0000-0002-9892-8370 Zhijun Chen: 0000-0001-9131-9460 Author Contributions

Conceived and designed the experiments: H.D., Z.C. Performed the experiments: H.D., H.L, X.W., Y.Z., Z.L. Analyzed the data: H.D., H.L, J.K.H., J.S., Z.C. Wrote the paper: H.D., J.K.H, J.S., Z.C. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (NSF) (No. 21372097) and the Center of International Mobility (CIMO) grant from Finland.

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ABBREVIATIONS BSA, bovine serum albumin; CFM, confocal fluorescent microscopy; EDS, energy dispersive spectrometry; OY, quantum yield; PGHMs, protein-gold hybrid materials; SDSPAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; SEM, scanning electron microscopy; TEM, transmission electron microscopy

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REFERENCES

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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemmater.7b03011. Tables S1−S4, Figures S1−S63, protein sequences 1−2, fluorescence quantum yield and lifetime, EDS analysis, zeta potential, UV−vis and fluorescence spectra, fluorescence photobleaching, XPS spectra and analysis, protein sequence (analysis), metal ion response, SDSPAGE, HPLC analysis, SEM, ESR analysis, time effect, cytotoxicity evaluation, bacterial and yeast imaging (PDF) 8446


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