Tomography Study and Growth Control of Spiky Gold Nanoparticles

A fundamental understanding of the morphological stability and microstructural characteristics of gold nanoparticles (Au NPs) is crucial for their diverse applications. In this study, spiky Au NPs are prepared by a seed‐mediated approach using citrate‐capped Au nanospheres as seeds. The morphological and microstructural characteristics of the spiky Au NPs are studied using 3D tomography transmission electron microscopy (TEM) analysis. Twin boundaries in all spikes of the Au NPs are identified by straight lines in the TEM images and specific crystallographic orientations. In addition, the analysis reveals that a careful approach is required when evaluating the morphological characteristics of spiky Au NPs using 2D images. A series of spiky Au NPs with sizes ranging from 62 to 323 nm is prepared by precisely controlling the amount of seed solution and first growth solution added during seed‐mediated growth. To elucidate the morphological and microstructural characteristics of the spiky Au NPs, a mechanism for the formation and evolution of the spikes is proposed based on the TEM results.


Introduction
Gold nanoparticles (Au NPs) offer unique physical and chemical properties and are often used as a model system for scientists studying the formation of NPs with various shapes, sizes, and surface chemistries.[3][4][5][6] The size and/or shape control of Au NPs is essential for specific applications.[14] Previous studies have reported the size control of Au NPs.[26][27] Spiky branched and/or star-shaped Au NPs consisting of a core and protruding structures can increase the electromagnetic field strength, resulting in an enhanced surface plasmon resonance and Raman scattering due to their high surface areas.The specific electro-optical properties and unique morphological characteristics of spiky-branched Au NPs have been considered for particular applications in drug delivery and photothermal therapy.However, the specific mechanisms related to controlling the size of such Au NP morphologies is not yet well known, which significantly limits their use in specific applications.In addition, the morphological and microstructural characteristics of spiky Au NPs play an important role in determining their physical and chemical properties.Therefore, a fundamental understanding of morphological stability and microstructural characteristics is crucial for their application.
In this study, we investigated the morphological and microstructural characteristics of spiky Au NPs based on 3D tomography transmission electron microscopy (TEM) analysis.We report the successful size control of the spiky Au NPs by tuning the number of Au seeds pre-formed during seed-mediated particle growth.A series of spiky Au NPs with a size range of 62-323 nm was successfully prepared by precisely controlling the amount of seeds.Finally, we discuss the growth mechanisms of spikes on the Au cores for the formation of spiky Au NPs.

Tomography Analysis
To clarify the morphological and microstructural properties of the Au NPs synthesized with 25 μL of seed solution and 50 μL of first growth solution, electron tomography analysis was conducted on the NPs. Figure 1 and Video V1 (Supporting Information) show a tilting series of the bright field (BF) TEM images of a Au NP.Several sharp spikes are observed on the Au NPs in the BFTEM images (Figure 1).Because of the morphology of the Au NPs, they are referred to as spiky Au NPs in this study.However, the morphologies of the spiky Au NPs were dependent on the tilting angle; several images taken of the same spiky Au NP are shown in Figure 1a-e.The number of spikes on the same Au NP that appear on two-dimensional BFTEM images depends on the tilting angle, with 8, 6, 11, 10, and 8 spikes observed in Figure 1b-f, respectively.In addition, the measurable length of the spikes of the Au NP also depended on the tilting angle; the lengths of spikes #2 and #3 are approximately 30 and 60 nm, respectively, from Figure 1d, and approximately 130 and 120 nm, respectively, from Figure 1b.Finally, the shape of the spikes of the Au NP also depends on the tilting angle; spike #8 appears as a straight taper in Figure 1c,d, while it is a curved and tapered in Figure 1b,f.These analysis results indicate that a three-dimensional approach is required to characterize the morphological and microstructural properties of spiky Au NPs in detail.
Specifically, most of the spikes on the Au NPs follow straight lines (Figure 2).Analysis of the tilting series of the tomography images confirmed that all eleven spikes contain a darker straight line (Figure 2a-d), and the appearance of these lines depends on the tilting angle, which indicates that it is related to the crystallographic properties of the NPs.To study the origin of the straight lines, a high-resolution (HR) TEM image was acquired, as shown in Figure 2e, which shows that the straight lines extend along the axis of the spikes from the center of the Au NPs, except for the spike indicated by the asterisk.Figure 2f shows a representative HRTEM image of a typical Σ3{111} coherent twin boundary (CTB) observed within a spike on a spiky Au NP, where the crystal in Region I is the mirror image of that in Region II, where the straight line indicates the twin boundary.
The diffractogram in Figure 2g obtained by the fast Fourier transform (FFT) analysis of the HRTEM image in Figure 2e shows a typical twin relationship of {111} twins in the FCC structure.The twin boundary in Figure 2e is coherent and atomically sharp, without steps at the end of the spike.Moreover, straight lines were also observed inside the curved spikes, although a discontinuous and unclear region was also observed in the spikes.The end of spike #8, a curved and tapered spike in Figure 2b, does not show a straight line, as highlighted in Figure 2d.One of the curved spikes, spike #9 in Figure 2c, had a welldetermined straight line.We conclude that the microtwins in the spiky Au NPs play an important role in the formation of the spikes.

Size Control
To control the size of the spiky Au NPs, the volume of the Au seed solution introduced into the first growth solution, V s->1st , and that of the first solution added to the second solution, V 1st->2nd , were adjusted during the synthesis processes.Figure 3 shows representative BFTEM images of the spiky Au NPs prepared by controlling V s->1st during the two-step growth process, while V 1st->2nd was fixed at 50 μL.Although the Au seeds used to grow the Au NPs were spherical (Figure S1, Supporting  Information), the Au NPs had a chestnut-like morphology.Some spikes were observed on the cores of the final structures in Figure 3, and the number of spikes was dependent on the quantity of Au seeds.When V s->1st was 25 μL, many spikes were observed on the core of the Au NPs (Figure 1a).The total diameter of the spiky Au NPs, defined as the distance from the tip of one spike to that of another on the opposite side (S T in the inset in Figure 3d), is in the range of 323 AE 24 nm, and wide cores are observed at the center of the spiky Au NPs (Figure 1a,d).Many spikes (approximately 10) were observed on the cores (Figure 1e).In addition, the length of the spikes, indicated by L S in the inset of Figure 3d, reached approximately 116 AE 20 nm.The widest part of the spike where it meets the core, indicated by W in the inset in Figure 3e, was approximately 36 AE 4 nm.A few bumpy structures, indicated by red arrows, were observed on the spikes of the Au NPs.Although the spiky shape of the Au NPs was conserved, the total sizes of the spiky Au NPs were reduced to 216 AE 19 and 179 AE 11 nm when V s->1st was increased to 50 and 100 μL, respectively.When V s->1st was increased to 50 and 100 μL, the number of spikes on the individual spiky Au NPs was slightly reduced to eight and seven, respectively, L S was reduced to 72 AE 12 and 60 AE 11 nm, respectively, and the initial W was slightly reduced to 30 AE 5 and 27 AE 4 nm, respectively.The surface of the spikes became smoother when V s->1st was increased to 100 μL (Figure 3c).We propose that the reduction in S T of the spiky Au NPs with increasing V s->1st was caused by the reduction in L S , which indicates that the growth of the spiky Au NPs was constrained by increasing the number of Au seeds.Finally, when V s->1st was increased to 200 μL, the shape of the Au NPs dramatically changed to particles with rough surfaces, without long spikes (Figure S2a, Supporting Information).Large smooth Au NPs were synthesized when V s->1st was increased to 400 μL (Figure S2b, Supporting Information).
To prepare smaller spiky Au NPs, V 1st->2nd was controlled because it was difficult to synthesize smaller spiky Au NPs with sizes less than 150 nm by controlling only V s->1st .Figure 4 shows the morphological characteristics of the Au NPs prepared by controlling V 1st->2nd , where V s->1st was fixed to 100 μL because the smallest spiky Au NPs were synthesized under this condition in the previous experiments.The spiky shape of the Au NPs was maintained well when V 1st->2nd was controlled in the range of 100-300 μL during synthesis (Figure 4a-c).When V 1st->2nd was reduced from 100 to 50 μL, S T was increased to approximately 160 AE 13 nm from 179 AE 11 nm, respectively, while the number of spikes was reduced to approximately seven, L S was reduced to approximately 54 AE 11 nm, and W was reduced to approximately 20 AE 3 nm (Figure 4e).The surface roughness of the spikes on the spiky Au NPs was significantly reduced when V 1st->2nd was reduced to 100 μL.
The spiky shape of the Au NPs was conserved, although V 1st->2nd was reduced to control the size of the Au NPs.The S T of the spiky Au NPs was reduced to 128 AE 14 and 62 AE 11 nm when V 1st->2nd was increased to 200 and 300 μL, respectively.The number of spikes on an individual spiky Au NP was six when V 1st->2nd was increased to 200 μL, The shape of the spiky Au NPs was closer to a star shape rather than a chestnut-burr shape when V 1st->2nd was 300 μL.
while it was reduced to five when V 1st->2nd was increased to 300 μL.
The size of the spiky Au NPs was successfully controlled in the range of 62-323 nm by adjusting V s->1st and V 1st->2nd during the two-step growth process (Figure 5a).The S T of the spiky Au NPs was predominantly governed by the growth of spikes, where L S decreased with increasing V s->1st and V 1st->2nd , which caused a reduction in S T of the NPs.In Figure 5a, the size of the cores of the spiky Au NPs, S C , defined by the difference between S T and the double of L S (S C = S T À (2 Â L S )), increased from 31 to 91 nm, while W increased from 16 to 36 nm, indicating that the lateral growth of the spikes occurred during the reaction (Figure 5b).However, axial growth was dominant after the formation of the spikes (Figure 5a).The increase in the number of spikes with decreasing V s->1st and V 1st->2nd indicates that a specific nucleation and growth mechanism was active during the formation of the spiky Au NPs (Figure 5b).

Spike Formation Mechanism
The BFTEM results highlighted specific morphological features of the spiky Au NPs.For example, the smallest spiky Au NPs (S T %62 nm) had approximately five spikes, while the largest ones (S T %323 nm) had approximately ten spikes.As shown in Figure 3e and 4b, typical spiky Au NPs have less than approximately twelve spikes.Additionally, the spiky Au NPs frequently exhibited overlapping sub-structures.Figure 6a-c show the BFTEM images for the spiky Au NPs with nominally 6, 7, and 10 spikes (although the exact number of spikes should be interpreted based on a tomography analysis).The bright and dark contrast areas of the BFTEM images are related to the scattering differences of incident electrons on the sample.The inside of the spiky Au NPs show a complex contrast distribution; the darker contrast of the core area of the spiky Au NPs is attributed to its larger thickness (Figure 6).By analyzing the boundaries of contrast changes, the overlapping areas were outlined.As a result, specific morphological characteristics were identified; the six-and ten-spike Au NPs were separated into two parts with three and five spikes each, respectively, and the centers of the two three-and five-spike parts overlapped while the seven-spike NP had two parts with four and three spikes (Figure 6).It is clearly confirmed in the ten-spike Au NP that the two five-spike parts are superimposed on each other such that the top and bottom are inverted (Figure 6c).Almost all of the spikes contain a twin boundary.We concluded that spike growth is achieved based on multiple twin particles formed in the first and second solutions.The straight lines indicating typical Σ3{111} CTBs were observed in all spikes on the nominal six-spike Au NP, although the tomography study indicated that the NP was actually comprised of seven spikes (Figure S3 and Video V2, Supporting Information).In the BFTEM images, twin boundaries were observed in all seven spikes, which ran from the center of the NP to the end of the spikes along the axial direction.These twin-related microstructural characteristics were always observed in many spiky Au NPs, which is consistent with the tomography data (Figure 2).In addition, similar morphological features were frequently observed in the spiky Au NPs synthesized under the same conditions; a morphological similarity between the NPs marked Spike 1 and Spike 2 in the BFTEM image in Figure 4b was identified.In addition, a morphological similarity was identified between the two spiky Au NPs in Figure S4 (Supporting Information) by rotating the NP in Figure S4b (Supporting Information) clockwise by 214°, although the NPs were observed on different grids.From these results, we deduced that there is an underlying rule governing the growth of spikes on the Au NPs.[30][31][32] We concluded that the twin boundaries play an important role in the formation of the spikes.The twin boundaries must be generated at the initial stage of the formation of the spiky Au NPs because they start at the center of the particles and were observed in most of the spikes.We deduce that the starting material for the growth of the spiky Au NPs had multiple twinned structures, and the twin structures governed the formation and evolution of the spikes.The number of tetrahedral subunits for the formation of multiple twinned seeds probably plays a key role in determining the spike characteristics; icosahedral Au seeds, a type of convex polyhedron with tetrahedral subunits, is thought to be the starting material for the spiky Au NPs (Figure 6).
The icosahedral seed, with an equal edge length and pentagonal antiprism pyramid caps on both sides, contains twenty tetrahedral subunits, also in contact twin boundaries with each other.On the icosahedral seed, the spikes may grow along the twin boundaries at the corners of the two pentagonal antiprism pyramids, which results in the formation of spikes staggered by 36°( Figure 6c).By excluding the formation of some spikes, the morphological characteristics of the nominal six-and seven-spike Au NPs are well described by adopting the icosahedral seed model (Figure 6a,b).
Based on the experimental results and geometric characteristics of the icosahedral seed structure, we hypothesized that the spikes on the spiky Au NPs form from the center of the icosahedral seeds to the corners, as shown in Figure 7a,b.The observed spikes and their numbers depend on the projection direction; ten spikes are observed in the projected images when the spiky model in Figure 7b is projected along the z(orthographic projection)-and y(vertical projection)-axes of the icosahedron in Figure 7a and eight spikes are observed when it is projected along the x(side projection)-axis.However, the projected images along the zand y-axes of the icosahedron are completely different.Figure 7c,e show the tilting series of the experimental BFTEM images and Figure 7d,f show the corresponding projected images of the reconstructed models for the representative small and large spiky Au NPs.In the reconstruction of the models, the length, width, and number of spikes on the icosahedrons were slightly different to those observed in the experimental BFTEM images.The projected images from the reconstructed models are consistent with the experimental images; the geometric features, angles between the spikes, and relative lengths, are similar in both the projected and experimental images.Overall, all of the BFTEM images obtained by tilting every 5°from À65°to 65°coincide well with the corresponding projected images from the reconstructed models (Figure S5, Supporting Information).The tilting axis for acquiring the tomography data is out of the observing particles because it is related to the rotation axis of the tomography TEM holder.A slight discrepancy exists between the experimental tilting angle and that used for the projected images.The tilting for the projected images of the reconstructed models was obtained by conserving a specific pattern; the z-axis is mainly the upward direction in Figure 7d,f and the xand y-axes were continuously rotated clockwise to match the experimental images.Several tomography datasets for various spiky Au NPs were taken to clarify the proposed model, the icosahedral seed model (Figure S6 and Videos V3-V5, Supporting Information).In Figure S6 (Supporting Information), all of the spiky Au NPs were successfully reconstructed based on the icosahedral models; the corresponding projection images were consistent with the experimental BFTEM images for the NPs.It should be noted that the existence of seven spikes was identified for the small spiky Au NPs in Figure 7c by reconstructing the spiky NP based on the icosahedral seed model although only six spikes were observed in the tilting series of the BFTEM images.
The visibility of the spikes depended on the tilting angle; spike 12 was obscured for tilting angles of þ35°and þ55°while it was visible at tilting angles of 0°, À35°, and À55°(Figure 7d).This highlights the importance of tomography studies to understand the morphological and microstructural properties of complex nanostructures such as spiky Au NPs.
35] In addition, the rough surface morphology of the long spikes may have resulted from the interactions between Brij35 (polyoxyethylene glycol dodecyl ether) and sodium salicylate in the formation of surfactant micelle structures. [36]However, further in-depth studies and theoretical approaches are required to fully understand the formation and growth mechanism of spiky Au NPs.

Conclusion
The morphological and microstructural characteristics of spiky Au NPs were studied using 3D tomography TEM analysis.The presence of twin boundaries between specific crystallographic orientations was observed in all the spikes as straight lines in the TEM images and was identified.In addition, the tomography results revealed that a careful approach is required when evaluating the morphological characteristics of spiky Au NPs using 2D images.A series of spiky Au NPs with a size range of 62-323 nm was successfully prepared by precisely controlling the amount of the seed solution and the first growth solution added during the synthesis via seed-mediated growth.The total size of the spiky Au NPs decreased when increasing the number of particles in the first and second growth solutions, where the overall growth of the spiky Au NPs was governed by the growth of the spikes.In addition, the number of spikes also decreased with an increasing number of particles in the growth solution.We deduced that the growth of spikes and the number of spikes were controlled by the number of tetrahedral subunits and the formation of the twin boundaries between tetrahedral subunits in the seed particles.As a result, an icosahedron Au seed, composed of twenty tetrahedral subunits with twin boundaries, was considered the starting material for the spiky Au NPs.Synthesis of Spiky Au NPs: The spiky Au NPs were synthesized using a seed-mediated method.First, the Au seed NPs were synthesized by mixing 0.5 mL of HAuCl 4 (0.1 M), 0.5 mL of sodium citrate (0.1 M), 0.5 mL of NaBH 4 (0.01 M), and 13 mL of deionized (DI) water for 40 s.The solution was incubated for 2 h and refreshed for each synthesis experiment.Two growth solutions were prepared, as follows.The first growth solution was prepared by adding 0.5 mL of Brij35 (0.2 M (aq)), 0.5 mL of CTAB (0.1 M (aq)), and 0.1 mL of sodium salicylate (0.05 M) to a vial, which was then shaken for 1 min.Then, 0.005 mL of AgNO 3 (0.005 M), 0.04 mL of HAuCl 4 (0.01 M), and 0.01 mL of ascorbic acid (0.1 M) were added to the solution and shaken for 2 min.The second growth solution was prepared in the same way in a flask with ten times the amount of the first growth solution.Both growth solutions had a light-yellow appearance.

Experimental Section
The spiky Au NPs were synthesized by adding 25 μL of the Au seed NPs into the vial of the first growth solution which was prepared as described above and shaken for 30 s.The color of the first growth solution rapidly changed from light yellow to light purple.Then, 50 μL of the reaction solution was added to the flask of the second growth solution, and the reaction mixture was shaken vigorously for 2 min.The reaction solution became colorless and then was kept undisturbed for 8 h.The mixture was then purified via centrifugation (10 min; 12 000 rpm) and redispersed twice in DI water.
To investigate the quantitative effect of the gold seeds on the formation of the spiky Au NPs, the amount of Au seeds in the first growth solution was controlled in the range of 25-100 μL, while the amount of the first solution introduced into the second growth solution was fixed at 50 μL.To understand the quantitative effect of the first growth solution on the formation of the spiky Au NPs, the Au seed solution volume was fixed at 100 μL and the amount of the first growth solution added to the second growth solution was varied in the range of 100-300 μL.All other experimental conditions were fixed during the growth processes.
Microstructural Characterization: The morphological and microstructural properties of the spiky Au NPs were characterized using TEM.A FEI Tecnai G2 F30 microscope equipped with a field-emission gun operating at an accelerating voltage of 300 kV was used for BFTEM and HRTEM imaging.A total of 1.7 μL of the spiky Au NPs in DI water was dropped onto a holey carbon-coated copper grid (Agar Scientific).The grid was then dried in a desiccator at room temperature for at least 2 h before the TEM observations.TEM tomography was conducted using a JEM-F200 TEM with a cold field-emission gun operating at an accelerating voltage of 200 kV and a high tilt holder (maximum tilt angle = 80°on the x-axis).A 200-mesh copper grid was used for the TEM tomography study by dropping 1.7 μL of spiky Au NPs in DI water without other specific treatments, followed by drying in air at room temperature.

Figure 1 .
Figure 1.a) Schematic illustration showing the tilt series during tomography imaging.bÀf ) Tilt series of BFTEM images of a spiky Au NP.

Figure 2 .
Figure 2. aÀd) Tilt series of BFTEM images of a spiky Au NP.The insets in (b), (c), and (d) show the magnified BFTEM images.The rectangles in the magnified images show the areas including the straight lines.e) Magnified BFTEM image.The asterisk indicates the spike free from a straight line in the NP.f ) HR TEM image taken from the spike on an Au NP.The white straight line is the twin boundary, and the red and yellow straight lines indicate other {111} planes in different two grains in Regions I and II.The white dotted lines indicate {002} planes in Regions I and II.g) Diffractogram taken from the twin boundary in (f ).The diffractogram is indexed along the <110> direction of the face-centered cubic (FCC) structure of Au.The red-dotted and yellowstraight rectangles show that there exist two grains with a twin boundary.Alternated long and short lines indicate the straight lines observed in the spikes.

Figure 3 .
Figure 3. BFTEM images of spiky Au NPs synthesized by controlling the amount of the seed solution at a fixed 50 μL in the amount of the first solution.a) Seed solution: 25 μL.b) Seed solution: 50 μL.c) Seed solution: 100 μL (scale bars: 200 nm).d) Plot of the total size and the length of spikes as a function of the amount of seed solution.S T and L S indicate the total size of the Au NP and the length of the spike in the inset, respectively.e) Plot of the width and the number of spikes as a function of the amount of seed solution.W in the inset indicates the definition of the initial width of a spike.

Figure 4 .
Figure 4. BFTEM images of spiky Au NPs synthesized by controlling the amount of the first solution at a fixed 100 μL in the amount of the seed solution.a) First solution: 100 μL.b) First solution: 200 μL.c) First solution: 300 μL (scale bars: 100 nm).d) Plot of the total size and the length of spikes as a function of the amount of the first solution.e) Plot of the width and the number of spikes as a function of the amount of the first solution.Furthermore, L S decreased to 44 AE 10 and 15 AE 5 nm for V 1st->2nd values of 200 and 300 μL, respectively, and the W values also reduced to 19 AE 3 and 16 AE 3 nm, respectively.The shape of the spiky Au NPs was closer to a star shape rather than a chestnut-burr shape when V 1st->2nd was 300 μL.

Figure 5 .
Figure 5. a) Plot of the total size, length, and core size of spikes as a function of the total size of spiky Au NPs.b) Plot of the width and the number of spikes as a function of the total size of spiky Au NPs.

Figure 6 .
Figure 6.a-c) Representative BFTEM images, their outlines, and their schematic drawings of the spiky Au NPs with nominal six, seven, and ten spikes, respectively.The dash and dot lines indicate the twins in the spiky Au NPs.

Figure 7 .
Figure 7. a) Schematic drawing and the projection images along the z(orthographic projection)-, y(vertical projection)-, and x(side projection)-axes of the icosahedron structure.Blue and green lines indicate the top and bottom pentagonal pyramid areas, respectively.b) Schematic drawing and the projection images along the z(orthographic projection)-, y(vertical projection)-, and x(side projection)-axes of the spiky Au model with 12 spikes formed along the direction from the center to the corners.c,d) Experimental BFTEM images of the spiky Au NP with nominal six spikes and the corresponding projection images from its reconstructed model.e,f ) Experimental BFTEM images of the spiky Au NP with eleven spikes and the corresponding projection images from its reconstructed model.