Towards the identification of the gold binding region within trypsin stabilized nanoclusters using microwave synthesis routes

Mark H. Griep a, Michael S. Sellers a, Bijil Subhash b, Alexis M. Fakner a, Abby L. West a and Nicholas M. Bedford *b
aWeapons and Materials Research Directorate, CCDC Army Research Laboratory, Aberdeen Proving Ground, MD 21005, USA
bSchool of Chemical Engineering, University of New South Wales, Sydney, NSW 2052, Australia. E-mail: n.bedford@unsw.edu

Received 2nd October 2020 , Accepted 18th December 2020

First published on 21st December 2020


Elucidating the location of stabilized nanoclusters within their protein hosts is an existing challenge towards the optimized development of functional protein-nanoclusters. While nanoclusters of various metal compositions can be readily synthesized within a wide array of protein hosts and exhibit tailorable properties, the inability to identify the cluster stabilization region prevents controllable property manipulation of both metallic and protein components. Additionally, the ability to synthesize protein-nanoclusters in a consistent and high-throughput fashion is also highly desirable. In this effort, trypsin stabilized gold nanoclusters are synthesized through standard and microwave-enabled methodologies to determine the impact of processing parameters on the materials physical and functional properties. Density functional theory simulations are employed to localize high probability regions within the trypsin enzyme for Au25 cluster stabilization, which reveal that cluster location is likely within close proximity of the trypsin active region. Trypsin activity measurements support our findings from DFT, as trypsin enzymatic activity is eliminated following cluster growth and stabilization. Moreover, studies on the reactivity of Au NCs and synchrotron characterization measurements further reveal that clusters made by microwave-based techniques exhibit slight structural differences to those made via standard methodologies, indicating that microwave-based syntheses largely maintain the native structural attributes despite the faster synthetic conditions. Overall, this work illustrates the importance of understanding the connections between synthetic conditions, atomic-scale structure, and materials properties that can be potentially used to further tune the properties of metal cluster-protein materials for future applications.


Introduction

Composed of metal atoms ranging from fewer than ten to several hundred atoms, metal nanoclusters (NC)s exhibit quantized energy fluorescence similar to quantum dots (QD)s due to their small diameter as opposed to the plasmons associated with the continuous density of states in noble metal nanoparticles.1–3 The discrete energy levels allow for tuneable photonic emission in the visible and IR regions, highly efficient two-photon absorption, and quantum yields exceeding 6%.4 Recent advances in metallic nanoclusters have demonstrated their applicability in diverse applications, such as biolabelling and bioimaging, where nanoclusters can interact with metal ions,5 proteins,4,6–11 nucleic acids,5,9,10,12 and small biomolecules.11 More recently, expanded functionality and materials design strategies have been achievable through protein-stabilized NC (PNC) growth/encapsulation methodologies.13 These include the ability to engineer 2D/3D NC structures14–16 and perform in vivo physiological targeting/monitoring.17 In the majority of these studies the stabilizing protein host largely serves as a scaffold for the nanocluster, without any enhancement/monitoring of the native activity of the protein.

With proper engineering of the hybrid system, opportunities exist to harness the capabilities/functionality of both the nano and bio components in both independent and cooperative systems. In the case of enzyme-nanocluster systems, it has been shown that the growth of nanoclusters within DNASE-I does not inhibit the enzymes native protease activity and, in fact, can directly monitor the enzymatic activity through modulation of the nanocluster emission.18 While the enzyme-nanocluster system proved functional in this specific case, the ability to determine or selectively control the nanocluster stabilization region within a host protein so as to not interfere with the native enzymatic/biological activity has yet to be achieved. In this work, we employ a series of experimental and computational methods to understand structure/function relationships in PNCs using the enzyme trypsin as a model protein host for both traditional and microwave-assisted synthesis methods. While a multitude of enzymes or ion-pumping proteins would be of interest to study potential interactions of associated nanoclusters on their innate biological activity, a trypsin–AuNC model was chosen due its representation of the large family of proteases, a clear understanding of the trypsin enzymatic mechanism,19 and the previous demonstration of the trypsin–AuNC hybrid synthesis for reference.20,21 With a well-defined active region and understanding of the key functional amino acids associated with the catalytic triad, the impact of nanocluster growth on trypsins’ enzymatic active could help infer cluster binding location.

We discover that microwave-assisted synthesis methods result in Au PNCs that are similar in structure to those made by established synthetic protocals by using a combination of X-ray absorption spectroscopy (XAS) and high-energy X-ray diffraction (HE-XRD) coupled to atomic pair distribution function analysis (PDF). Moreover, the similarity in structures results in similar optical properties between the two trypsin-based PNCs and also exhibits similar reactivity toward the 4-nitrophenol reduction reaction. Computational modeling efforts indicate Au cluster binding predominately located near the cysteine at the enzymatic binding site, which was further substantiated by a complete loss of enzymatic reactivity. Overall these results showcase the ability to make Au PNCs using microwave-assisted synthetic strategies that do not drastically perturb cluster structure or properties, potentially enabling larger scale production on these emerging materials, while also elucidating the most likely binding site of Au25 within a trypsin host.

Results and discussion

Trypsin-stabilized Au PNCs were synthesized by traditional synthetic routes (12 h) and microwave-assisted synthesis methods (∼5 s), as illustrated in Fig. 1.20 As described in more detail in the Experimental section, the only difference between the two syntheses chosen here is the mode of heating and associated shorter time scale through the use of microwave heating. As such, microwave synthesis can potentially provide functional materials under much faster synthesis times. The impact of these two synthesis methodologies on Au PNC formation within the trypsin protein was probed using a series of experimental methods. While established protocols have shown the ability to achieve high quality NC growth in a wide-array of protein hosts; new avenues for increasing future production rates are still needed for eventual translation into real-world applications. Microwave chemistry has been demonstrated to be an effective methodology to drive and control PNC formation,22–25 however this is the first report of trypsin–AuNC material synthesis through this approach. While microwave-based PNC synthesis approaches have yielded high-quality fluorescent products similar to that of traditional wet-chemistry synthetic methods; tailoring highly functional hybrids is still elusive due to the lack of fundamental knowledge on the precise cluster stabilization region and cluster–ligand interaction within the host protein. Exploring both synthesis routes in this work allows for evaluation of potential differences in the nanocluster formation process and stabilization region with the host protein and provides further clarification if these differing methods provide homologous materials.
image file: d0nr07068h-f1.tif
Fig. 1 Schematic of the two synthetic routes chosen to generate trypsin-based Au-PNCs.

Initial inspection of the fluorescence emission of the prepared nanoclusters reveals similar spectral outputs, as shown in Fig. 2(A). The fluorescence emission peak around λ650 nm remains consistent with both synthesis methodologies, likely indicating similar nanocluster formation and stabilization mechanisms are similar. Previous reports of trypsin–AuNC synthesis have verified that the emission peak around λ650 nm corresponds to an Au25 cluster with a diameter of ∼2 nm.20,21 Furthermore, circular dichroism (CD) spectroscopy measurements reveal that trypsin loses all secondary protein structure upon NC synthesis in both methodologies, instead showcasing a random coil morphology (Fig. 2(B)). This finding indicates that the presence of Au NCs influence protein structure, but not the employed synthetic conditions. The loss of trypsin secondary structure in coordination with the presence of metal ions has been previously shown,26 thus Au ion binding and subsequent nanocluster formation is expected to have a direct impact on α-helix and β-sheet composition. Notably, the synthesis method was found to have no significant impact on other Au PNC hybrids tested as well, including BSA, lysozyme, and pepsin protein hosts (Fig. S1 and S2).


image file: d0nr07068h-f2.tif
Fig. 2 Comparison of trypsin Au PNCs synthesized with standard and microwave methods, evaluating (A) fluorescence emission and (B) CD spectra.

To better understand the local atomic environment around the Au PNCs, XAS measurements were performed at the Au L3-edge (Fig. 3). The X-ray absorption near-edge structure (XANES) demonstrate that both trypsin PNCs from standard and microwave-assisted syntheses exhibit a drastic departure from that of bulk Au (Fig. 3A). Features in the XANES for both PNCs are broad due to the lack of longer-range order and are consistent with clusters previously reported in the literature.27–30 Slight increases in white line intensity and E0 (∼ +0.5 eV) indicate a small population of oxidized Au atoms with clusters, which is expected given the large surface area of the cluster and their associated interactions with the host protein. Fig. 3B shows the extended X-ray absorption fine-structure (EXAFS) data for the trypsin PNCs along with a reference Au foil, which provide a more immediate and intuitive understanding of the local Au structure. EXAFS were obtained following conversion of the XAS data to k-space (k2-weighted, Fig. S3), and Fourier transformed from 2.0 to 12.0 Å−1. For the Au foil, the split peak arising from Townsend–Ramsauer resonance represent the first coordination sphere of Au–Au atoms in bulk Au. Notably, the first major peak for both trypsin PNCs is located at 1.89 Å (not corrected for phase shift). This distance has been previously identified to be arising from Au cluster–protein interactions, most likely with S-atoms in cysteine residues.27–29 Longer radial distances of 2.42 and 2.75 Å correspond to the Au–Au distances within the clusters, which is significantly truncated when compared to bulk or nanoparticle counterparts.


image file: d0nr07068h-f3.tif
Fig. 3 Au L3-edge (A) XANES and (B) EXAFS for trypsin Au PNCs synthesized using standard method (red line) and microwave-assisted method (green line) along with a reference Au foil.

EXAFS modeling was performed to obtain local average Au coordination numbers (CNs) and nearest neighbor distances as described in the Experimental section. EXAFS fittings results are shown in Fig. S4 in the ESI and summarized in Table 1. The Au–protein CN, representative of all possible protein interactions due to the differences in atomic mass of Au, is approximately ∼2 for both Au trypsin PNCs synthesized via standard and microwave-assisted techniques (2.04 ± 0.28 and 1.92 ± 0.14 respectively). The Au–Au CN is slightly smaller in the PNCs made via standard synthesis techniques (1.15 ± 0.42) than the microwave assisted PNCs (1.46 ± 0.36), suggesting that cluster size is slightly larger in the latter materials, likely due to the rapid heating and nucleation mechanisms that occur in the microwave assisted techniques.31,32 It is important to note that the local coordination in both materials is different to those previously reported on thiolated Au clusters and similar materials, wherein a CN of ∼1.0 is reported for Au–ligand interactions and a Au–Au CN higher than 2.0 is reported for various different cluster syntheses.33–35 These result indicate that Au–trypsin interactions occur from multiple residues in the first coordination sphere of the surface Au atoms in the PNCs vs. the use of thiol-containing organic linkers. The significantly reduced CNs in the PNCs compared to previously reported thiolated clusters strongly indicates a lack of ordering in the cluster, which is somewhat expected given the larger number of possible binding functionalities within trypsin vs. designer thiolated clusters. The average Au–protein NNDs is ∼2.3 Å for both trypsin PNCs while Au–Au NNDs are also similar for both materials. For comparison purposes, EXAFS modeling efforts from previous Au NCs is summarized in Table S1.27–29,34–36 Overall, the XAS data demonstrates that minor structural changes occur when microwave assisted syntheses techniques are used over standard synthetic methods, indicating minimal influence of microwave-bases processes in the creation of Au PNCs.

Table 1 Summary of EXAFS fitting results
Au NC Au–protein CN Au–Au CN Au–protein NND (Å) Au–Au NND (Å) Au–protein σ2 Au–Au σ2 R-Factor
Traditional synthesis 2.04 ± 0.28 1.15 ± 0.42 2.317 ± 0.005 2.79 ± 0.05 0.003 ± 0.001 0.008 ± 0.002 0.00081
Microwave synthesis 1.92 ± 0.14 1.46 ± 0.36 2.309 ± 0.003 2.78 ± 0.03 0.002 ± 0.001 0.002 ± 0.001 0.0026


To further explore the longer-range structure of the trypsin-based Au-PNCs, atomic PDF analysis was performed on Au-PNCs and trypsin powder (Fig. 4). Atomic PDFs provide longer range atomic-scale structural information independent of periodicity, which is ideal for studying nanoscale materials.37,38 The PDFs revealed that both the standard and microwave synthesis methods yield PNCs of similar atomic structure at longer length scales, as shown in Fig. 4 and Fig. S5. The longer-range order extends past the size of the Au PNCs (Fig. S5), suggesting the protein–protein and Au–protein atomic pairs are prevalent in their respective PDFs. Indeed, the first major peak in the Au PNCs at 1.38 Å is an aggregation of common bond lengths in proteins and is also found in the PDF for trypsin powder. For reference, the atomic PDFs for peptide-capped Au NPs using the AuBP1 peptide is also shown in Fig. 4 and Fig. S5.39 It is clear that the Au PNCs do not resemble fcc-type Au as compared to AuBP1-capped Au nanoparticles, and more importantly, do not exhibit major peak shifts across the available radial distribution space that would signify significant structural changes. Differences in peak intensities and peak full width half max values can provide some insights into the relative degree of ordering between these materials, wherein the microwave-assistance Au PNCs exhibit slight increases in relative peak intensity. This suggest there is likely some increase in local structural order in this material, consistent with EXAFS results.


image file: d0nr07068h-f4.tif
Fig. 4 PDF analysis comparing control trypsin, a peptide-capped Au nanoparticle (AuBP1), standard trypsin–Au PNCs, and microwave trypsin Au PNCs.

With confirmation that the structural and photophysical properties of the trypsin Au PNCs remain largely unchanged between the microwave-assistance synthesis and traditional PNC synthetic methods, computational energetic and protein displacement data were generated to determine probable gold nanocluster growth locations within the trypsin protein. Results for the top sites identified by greatest free energy difference are listed in Table 2 with corresponding locations highlighted in the protein model in Fig. 5. The simulated cluster in the top ranked stabilization location is shown in Fig. 5B. The models predict, as schematically shown in Fig. 6(A), that the highest probability cluster location is in close proximity to the active site of the trypsin enzyme. As a serine protease, the enzymatic function of trypsin is the result of a coordinated catalytic triad involving the histidine-57, aspartate-102, and serine-195 amino acid residues. As the serine-195 residue is known to be the most directly involved in the catalytic activity,40 coordinating the serine-195 residue with the nanocluster stabilization would reduce its effectiveness at forming the required oxyanion hole, resulting in an increased energy barrier for reaction kinetics. It is thus hypothesized that Au PNCs synthesized by trypsin in a location close to the enzyme's active site could alter the protein's native function. As shown in Fig. 6(B), a significant disruption of enzymatic activity upon cluster formation is verified. In fact, here it is shown that a complete loss of trypsin enzymatic activity is lost upon NC growth/stabilization within the trypsin molecule. The loss of enzymatic activity supports simulation predictions that the cluster stabilization region is in direct proximity to the enzymatic active region. As the loss of activity is measured in both standard and microwave-synthesized trypsin Au PNC systems, the cluster stabilization region and subsequent cluster–ligand interactions are likely similar.


image file: d0nr07068h-f5.tif
Fig. 5 (A) Growth regions highlighted in protein model. (B) Probable AuNC binding pocket in trypsin with associated ligands highlighted.

image file: d0nr07068h-f6.tif
Fig. 6 (A) Schematic of AuNC binding location in trypsin with respect to active site and (B) comparison of trypsin enzymatic activity in control and hybrid conformations.
Table 2 Energetic and protein displacement data at high probability cluster growth regions
Growth site # Free energy (kcal mol−1) NAMD energy (kcal mol−1) Total displacement of protein (Å)
68 −34.32 −104.20 1.95
116 −32.30 −64.25 2.16
58 −31.98 −66.50 1.81
126 −31.93 −97.40 2.14
50 −31.72 −70.79 1.96


To further probe the structural similarity between the two trypsin Au PNCs, catalytic activity was tested using 4-nitrophenol (4-NP) reduction reaction, a thermodynamically feasible but kinetically restricted reaction in the absence of a catalyst. In the presence of catalyst, the reduction of 4-NP can be understood using a Langmuir–Henshelwood model, where reactants, 4-NP and surface-hydrogen species (derived from NaBH4) are adsorbed on the surface of the catalyst to facilitate the formation of 4-aminophenol.41 The kinetics of 4-NP reduction was probed using time-resolved UV-Vis spectroscopy, which demonstrated a gradual decay of the characteristic 4-NP peak (400 nm) with a concomitant increase in 4-AP peak (300 nm), as shown in Fig. 7A for the microwave synthesized Au PNCs. The presence of isosbestic points in the UV-Vis spectra indicates that the reaction proceeds without the formation of any unwanted by-products.42 Owing to the fast reaction and the high concentration of BH4 ions, the absorbance data were fit using a pseudo-first order model (Fig. 7B), where the slope of the fit is the apparent rate constant of the reaction. As per the previously reported studies of similar reaction systems,43 the reaction rate constants exhibited a linear dependency on the temperature (Fig. 7C). The temperature dependency of rate constant for standard Au PNCs is shown in Fig. S6. Arrhenius plots of Au PNCs are shown in Fig. 7D and allow for the calculation of the catalytically-driven activation energies (Ea, Table 3). The microwave synthesized Au PNCs exhibits a lower Ea (54.68 ± 5.71 kJ mol−1) than the standard synthesized Au PNCs (63.85 ± 1.55 kJ mol−1), indicating that reactivity is slightly improved through the use of microwave-based synthetic techniques. Given the predicted Au NC location within trypsin (Fig. 6A) and the similarities in optical properties (Fig. 2), protein-based influences on reactivity (impeded substrate diffusion, protein assessed reactions, etc.) can likely be ruled out. The slight increase in overall size Au structure using microwave assisted synthesis methods, as observed by EXAFS analysis (Fig. 4 and Table 1) and PDF observations (Fig. 5), is a more likely cause given the closer resemblance to fcc Au, which have exceeding lower Ea for 4-NP reduction reactions.39,44


image file: d0nr07068h-f7.tif
Fig. 7 (A) UV-Vis Spectra of 4-NP reduction, (B) absorbance at 400 nm as a function of time, (C) apparent rate constant plotted against reaction temperature, and (D) Arrhenius plots of 4-NP reduction using Au PNC samples.
Table 3 Arrhenius parameters of 4-NP reduction on Au PNCs
Sample Activation energy (kJ mol−1) Frequency factor (s−1)
Standard Au PNCs 63.85 ± 1.55 1.74 × 107
Microwave Au PNCs 54.68 ± 5.71 3.21 × 108


Conclusion

In this work, a direct comparison of trypsin Au PNC synthesis methodologies, including traditional wet chemistry and microwave approaches, are compared and demonstrated to yield near identical products as evidenced by fluorescence, CD, and synchrotron X-ray characterization. To identify potential cluster locations with the trypsin Au PNC complex, a DFT approach is utilized, isolating the highest probability cluster stabilization region to be in direct proximity to the trypsin's active region responsible for catalytic activity. Experimental support of this cluster location is evidenced through the complete elimination of trypsin enzymatic activity within the Au PNC, suggesting the close proximity of the cluster to the active region inhibits enzymatic function. As the benefits of dual-functional PNC hybrids could serve as an enabling tool in emerging technologies, this work presents a foundation to determine cluster location within the host protein which will enable protein engineering pathways to selectively place clusters at specified regions within the protein.

Experimental

Traditional synthesis Au PNCs

The control protein[thin space (1/6-em)]:[thin space (1/6-em)]AuNCs were synthesized with slight modifications according to previously reported methods.20 In brief, hybrids were prepared by mixing aqueous protein solution (5 mL, 20 mM) with an aqueous solution of HAuCl4 (5 mL, 10 mM) under stirring. Once mixed, the solution was adjusted to pH 12 through addition of NaOH (0.5 mL, 1 M). The solution is incubated at 37 °C for 12 hours under vigorous stirring. The final solution is purified of excess gold salt with a 5 kDa molecular weight cut-off (MWCO) spin filter.

Microwave synthesis of Au PNCs

The methodology to synthesize protein[thin space (1/6-em)]:[thin space (1/6-em)]AuNCs via a microwave reactor utilizes the same starting materials as with the traditional methods. Initial solutions were prepared by mixing aqueous protein solution (5 mL, 20 mM) with an aqueous solution of HAuCl4 (5 mL, 10 mM) under stirring. Once mixed, the solution was adjusted to pH 12 through addition of NaOH (0.5 mL, 1 M). A 5 mL volume of the prepared solution was transferred to a microwave reaction tube and placed into a CEM Discover microwave synthesizer. The samples were reacted for 3–10 seconds at 250 W. The final solution is purified of excess gold salt with a 5 kDa MWCO spin filter.

Characterization

Fluorescent emission of the protein-nanocluster hybrids was measured on a PerkinElmer UV/Vis/NIR fluorospectrometer operating over a range of 500 to 900 nm, with a path length of 1 cm. Circular dichroism (CD) was performed according to previously published methodologies.45 All samples were run at 25 °C in a 25 mM NaH2PO4, 100 mM NaCl buffer at pH 7.5. Secondary structure content was evaluated on CD Pro software. X-ray absorption spectroscopy (XAS) experiments were performed at 10-BM beamline at the Advanced Photon Source (APS), Argonne National Laboratory (ANL). Aqueous PNC samples were loaded into 1 mm OD Kapton capillaries and measured in a fluorescence geometry from 200 eV below the Au L3-edge (11.918 keV) to 1000 eV above the edge. Raw data manipulation, data reduction, and subsequent modeling were performed with the Demeter software package.46 EXAFS model was performed using Au–S and Au–Au backscattering paths were generated using the reported structure of a thiolate protected Au25 cluster, and a S02 value of 0.844 obtained from modeling the EXAFS of a Au reference foil.33

High energy X-ray diffraction (HE-XRD) experiments were performed at 11-ID-C beamline at the APS. Lyophilized powders of trypsin PNCs made by standard and microwave-assisted synthetic techniques were loaded into 1 mm OD Kapton capillaries along with trypsin powder for HE-XRD experiments at 105.7 keV. HE-XRD patterns were background subtracted, converted into reduced structure functions (F(Q)), and Fourier transformed into atomic pair distribution functions using the program RAD.47

Catalytic reactions

The catalytic activity of both standard and microwave stabilized Au PNC samples were studied using the model 4-nitrophenol reduction, with slight modifications to previously reported procedure.43 Briefly, 1 mL of as-synthesized Au PNC sample was mixed with 1 mL of 150 μM 4-nitrophenol solution in a 3.5 mL quartz cuvette with 1 cm path length and left undisturbed for 5 minutes. To this solution, 1 mL of freshly prepared aqueous NaBH4 (60 mM) was added to initiate the reaction. The extinction of 4-nitrophenol was monitored at 400 nm using Agilent Cary 8454 UV-Vis Spectrometer at 6 seconds interval for 30 minutes. Reactions were performed in triplicate at temperatures of 20, 30, 40 and 50 °C in order to obtain activation energies (Ea) for each catalyst.

Molecular dynamics simulations

A raw Au25 cluster was optimized using first principle ab initio quantum-chemistry package, the General Atomic and Molecular Electronic Structure System, using Density Functional Theory (DFT) with a basis set of LanL2DZ3. The coordinates of the optimized Au25 nanocluster were introduced within the lowest energy sites obtained using Au10 clusters. Molecular dynamics simulations at each of the grid points were run in parallel. Energies and the atomistic trajectories from these simulations were analyzed using the XPairIt simulator to determine: (1) the free energy required to “grow-in” a sphere representing a nanocluster; (2) the interaction energy between the sphere and the surrounding protein amino acids; (3) the total displacement of atoms from their original positions upon “growing” the sphere. The data at each site was subsequently ranked based on free energy and total displacement to determine the most probable locations for nanocluster growth.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was supported in part by an internal Directors Research Initiative at the Army Research Laboratory. B. S. would like to acknowledge scholarship support from the Research Training Program administered by the Department of Education of the Australian Government. HE-XRD and XAS experiments were carried out at the 11-ID-C and 10-BM beamlines of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. The 10-BM beamline was further supported by the Materials Research Collaborative Access Team and its member institutions. The authors would like to thank Dr Yang Ren and John Katsoudas for assistance at 12-ID-C and 10-BM, respectively. The authors would also like to thank Govind Mallick (ARL) for his input and efforts on protein nanocluster simulations.

References

  1. L. Shang, S. J. Dong and G. U. Nienhaus, Nano Today, 2011, 6, 401–418 CrossRef CAS.
  2. J. Zheng, P. R. Nicovich and R. M. Dickson, Annu. Rev. Phys. Chem., 2007, 58, 409–431 CrossRef CAS.
  3. J. Zheng, C. Zhang and R. M. Dickson, Phys. Rev. Lett., 2004, 93, 077402 CrossRef.
  4. H. Kawasaki, K. Hamaguchi, I. Osaka and R. Arakawa, Adv. Funct. Mater., 2011, 21, 3508–3515 CrossRef CAS.
  5. W. Y. Chen, G. Y. Lan and H. T. Chang, Anal. Chem., 2011, 83, 9450–9455 CrossRef CAS.
  6. H. Wei, Z. Wang, L. Yang, S. Tian, C. Hou and Y. Lu, Analyst, 2010, 135, 1406–1410 RSC.
  7. X. Le Guével, N. Daum and M. Schneider, Nanotechnology, 2011, 22, 275103 CrossRef.
  8. J. Xie, Y. Zheng and J. Y. Ying, J. Am. Chem. Soc., 2009, 131, 888–889 CrossRef CAS.
  9. G. Liu, Y. Shao, K. Ma, Q. Cui, F. Wu and S. Xu, Gold Bull., 2012, 45, 69–74 CrossRef CAS.
  10. G. Liu, Y. Shao, F. Wu, S. Xu, J. Peng and L. Liu, Nanotechnology, 2013, 24, 015503 CrossRef.
  11. C. T. Chen, W. J. Chen, C. Z. Liu, L. Y. Chang and Y. C. Chen, Chem. Commun., 2009, 7515–7517 RSC.
  12. J. T. Petty, J. Zheng, N. V. Hud and R. M. Dickson, J. Am. Chem. Soc., 2004, 126, 5207–5212 CrossRef CAS.
  13. M. H. Griep, A. L. West, M. S. P. Sellers, M. Karna, E. Zhan and N. Hoque, in Handbook of Nanoparticles, ed. M. Aliofkhazraei, Springer International Publishing, Cham, 2015, pp. 1–24,  DOI:10.1007/978-3-319-13188-7_33-1.
  14. H. Ding, H. Li, X. Wang, Y. Zhou, Z. Li, J. K. Hiltunen, J. Shen and Z. Chen, Chem. Mater., 2017, 29, 8440–8448 CrossRef CAS.
  15. M. H. Griep, J. D. Demaree, D. P. Cole, T. C. Henry and S. P. Karna, Plasmonics, 2020, 15, 101–107 CrossRef.
  16. M. H. Griep and N. M. Bedford, J. Phys.: Mater., 2020, 3, 045002 CAS.
  17. A. L. West, N. M. Schaeublin, M. H. Griep, E. I. Maurer-Gardner, D. P. Cole, A. M. Fakner, S. M. Hussain and S. P. Karna, ACS Appl. Mater. Interfaces, 2016, 8, 21221–21227 CrossRef CAS.
  18. A. L. West, M. H. Griep, D. P. Cole and S. P. Karna, Anal. Chem., 2014, 86, 7377–7382 CrossRef CAS.
  19. L. Polgár, Cell. Mol. Life Sci., 2005, 62, 2161–2172 CrossRef.
  20. H. Kawasaki, K. Yoshimura, K. Hamaguchi and R. Arakawa, Anal. Sci., 2011, 27, 591–591 CrossRef CAS.
  21. Y. Xu, J. Sherwood, Y. Qin, D. Crowley, M. Bonizzoni and Y. Bao, Nanoscale, 2014, 6, 1515–1524 RSC.
  22. Y. Yue, T.-Y. Liu, H.-W. Li, Z. Liu and Y. Wu, Nanoscale, 2012, 4, 2251–2254 RSC.
  23. L. Yan, Y. Cai, B. Zheng, H. Yuan, Y. Guo, D. Xiao and M. M. F. Choi, J. Mater. Chem., 2012, 22, 1000–1005 RSC.
  24. Y. Liu, G.-F. Tian, X.-W. He, W.-Y. Li and Y.-K. Zhang, J. Mater. Chem. B, 2016, 4, 1276–1283 RSC.
  25. K.-T. Chuang and Y.-W. Lin, J. Phys. Chem. C, 2017, 121, 26997–27003 CrossRef CAS.
  26. L. Yang, Z. Gao, Y. Cao, R. Xing and X. Zhang, ChemBioChem, 2005, 6, 1191–1195 CrossRef CAS.
  27. M. A. MacDonald, D. M. Chevrier, P. Zhang, H. Qian and R. Jin, J. Phys. Chem. C, 2011, 115, 15282–15287 CrossRef CAS.
  28. G. A. Simms, J. D. Padmos and P. Zhang, J. Chem. Phys., 2009, 131, 214703 CrossRef.
  29. M. A. MacDonald, P. Zhang, H. Qian and R. Jin, J. Phys. Chem. Lett., 2010, 1, 1821–1825 CrossRef CAS.
  30. M. H. Griep and N. M. Bedford, J. Phys. Mater., 2020, 3, 04500 Search PubMed.
  31. Z. Duan, J. Timoshenko, P. Kunal, S. D. House, H. Wan, K. Jarvis, C. Bonifacio, J. C. Yang, R. M. Crooks, A. I. Frenkel, S. M. Humphrey and G. Henkelman, Nanoscale, 2018, 10, 22520–22532 RSC.
  32. I. Bilecka, P. Elser and M. Niederberger, ACS Nano, 2009, 3, 467–477 CrossRef CAS.
  33. T. Dainese, S. Antonello, J. A. Gascón, F. Pan, N. V. Perera, M. Ruzzi, A. Venzo, A. Zoleo, K. Rissanen and F. Maran, ACS Nano, 2014, 8, 3904–3912 CrossRef CAS.
  34. A. Shivhare, K. E. Lee, Y. Hu and R. W. J. Scott, J. Phys. Chem. C, 2015, 119, 23279–23284 CrossRef CAS.
  35. S. Yamazoe, S. Takano, W. Kurashige, T. Yokoyama, K. Nitta, Y. Negishi and T. Tsukuda, Nat. Commun., 2016, 7, 10414 CrossRef CAS.
  36. D. J. Morris, R. Yang, T. Higaki, M. J. Ward, R. Jin and P. Zhang, J. Phys. Chem. C, 2018, 122, 23414–23419 CrossRef CAS.
  37. V. Petkov, Mater. Today, 2008, 11, 28–38 CrossRef CAS.
  38. S. J. L. Billinge, Philos. Trans. R. Soc., A, 2019, 377, 20180413 CrossRef CAS.
  39. N. M. Bedford, Z. E. Hughes, Z. Tang, Y. Li, B. D. Briggs, Y. Ren, M. T. Swihart, V. G. Petkov, R. R. Naik, M. R. Knecht and T. R. Walsh, J. Am. Chem. Soc., 2016, 138, 540–548 CrossRef CAS.
  40. D. R. Corey and C. S. Craik, J. Am. Chem. Soc., 1992, 114, 1784–1790 CrossRef CAS.
  41. P. Zhao, X. Feng, D. Huang, G. Yang and D. Astruc, Coord. Chem. Rev., 2015, 287, 114–136 CrossRef CAS.
  42. S. Gu, S. Wunder, Y. Lu, M. Ballauff, R. Fenger, K. Rademann, B. Jaquet and A. Zaccone, J. Phys. Chem. C, 2014, 118, 18618–18625 CrossRef CAS.
  43. Y. Li, Z. Tang, P. N. Prasad, M. R. Knecht and M. T. Swihart, Nanoscale, 2014, 6, 3165–3172 RSC.
  44. H. Yamamoto, H. Yano, H. Kouchi, Y. Obora, R. Arakawa and H. Kawasaki, Nanoscale, 2012, 4, 4148–4154 RSC.
  45. N. J. Greenfield, Nat. Protoc., 2006, 1, 2876–2890 CrossRef CAS.
  46. B. Ravel and M. Newville, J. Synchrotron Radiat., 2005, 12, 537–541 CrossRef CAS.
  47. V. Petkov, J. Appl. Crystallogr., 1989, 22, 387–389 CrossRef CAS.

Footnote

Electronic supplementary information (ESI) available. See DOI: 10.1039/d0nr07068h

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