Template-Confined Site-Specific Electrodeposition of Nanoparticle

Oct 17, 2018 - After coating these Au nanoparticle cluster-in-bowl arrays with a thin layer of Ag film, they can be used as SERS substrates with an SE...
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Template-Confined Site-Specific Electrodeposition of Nanoparticle Cluster-in-Bowl Arrays as SERS Substrates yanling wang, Yangchun Yu, Yue Liu, and Shikuan Yang ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.8b00711 • Publication Date (Web): 17 Oct 2018 Downloaded from http://pubs.acs.org on October 18, 2018

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Template-Confined Site-Specific Electrodeposition of Nanoparticle Cluster-in-Bowl Arrays as SERS Substrates Yanling Wang, Yangchun Yu, Yue Liu, and Shikuan Yang* Institute for Composites Science Innovation, School of Materials Science and Engineering Zhejiang University, Hangzhou 310027, China KEYWORDS: site-specific growth, template confined growth, electrochemical deposition, nanoparticle cluster, SERS sensing ABSTRACT: Nanoparticle clusters have important applications in plasmonics and optical sensing fields. Various methods have been used to construct nanoparticle clusters, represented by assembling pre-prepared nanoparticles using DNA. However, preparation of nanoparticle clusters using a one-step method is still challenging. Herein, by using pre-patterned microscale bowls as individual reaction containers, clusters of Au nanoparticles with a homogeneous structure are electrodeposited at the bottom of each bowl. The structure of the nanoparticle clusters can be simply manipulated by varying electrodeposition parameters. After coating these Au nanoparticle cluster-in-bowl arrays with a thin layer of Ag film, they can be used as SERS substrates with an SERS enhancement factor of ~ 108. Importantly, the concave bowl structures can facilitate delivery of the analytes into the crevices between the bowls and the nanoparticle clusters where SERS “hot spots” (or sensitive sites) located. The crevices with a gradually changed gap distance between the concave bowl structure and the nanoparticle clusters are excellent traps for catching and SERS sensing of biospecies with varied sizes (e.g., viruses and proteins). We demonstrated sensitive SERS detection of viruses and proteins using the nanoparticlecluster-in-bowl SERS substrates. This technique has the ability to control the resulting structure at specific locations with electrodeposited materials, which enables new opportunities for assembling complex surface patterns with diverse applications in optical and plasmonic fields. Nanoparticle clusters usually exhibit novel collective optical and plasmonic properties when compared to isolated nanoparticles and nanoparticle arrays.1-8 As an example, chiral Au nanoparticle clusters prepared by DNAguided assembly demonstrated defined circular dichroism and optical rotatory dispersion effects at visible wavelengths originating from the collective plasmonplasmon interactions of nanoparticles closely positioned.9 Self- and guided-assembly of nanoparticles created by wet chemical methods have been employed to prepare nanoparticle clusters.10-14 However, the unpredictable aggregation during the self-assembly process often disturbs the formation process of the nanoparticle clusters, leading to disordered assemblies. DNA- or peptide-guided assembly needs complex surface treatment of the preprepared nanoparticles before assembling. Moreover, the organic agents introduced during nanoparticle synthesis using wet chemical approaches or introduced during the subsequent assembly processes may influence the resulting device performance, particularly those applications requiring exposed surfaces (e.g., for sensing and as catalysts). Moreover, it becomes more challenging to prepare an ordered array of nanoparticle clusters which may demonstrate more intriguing optical properties arising from nanoparticle cluster-nanoparticle cluster interactions compared with isolated nanoparticle clusters using existing self- or guided-assembly methods.15-17

Although electrochemical deposition (ECD) enables growth of nanostructures directly on a scaffolding structure or within cylindrical pores without introducing any organic agents,18-22 it is less well-studied for assembling and patterning of nanoparticles due to limitations in in situ control of the location and assembly behavior of the electrodeposited materials. Herein, we have demonstrated a simple yet reliable template-guided, site-specific, ECD method capable of precisely controlling both the structure and, most importantly, the location of the electrodeposited nanoparticle clusters. Large area, highly ordered arrays composed of nanoparticle clusters located at the bottom of the hexagonally-arranged bowls (i.e., a particle cluster-inbowl array, or PCIB array) have been prepared. Even though each bowl behaves as an individual reactor, the nanoparticle clusters in different bowls are homogeneous. The size of the nanoparticles and the resulting clusters, as well as the distance between the adjacent clusters, can be conveniently adjusted by changing the ECD parameters, in order to create customized structures for a broad variety of applications. After evaporating a thin layer of Ag film on the Au PCIB array, SERS substrates are obtained with a high SERS enhancement factor of ~ 108, as verified by finitedifference time-domain simulations and experimental measurements of chemical and biological targets. Most importantly, the crevices between the nanoparticle clusters and the concave bowls with a gradually changed

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gap distance are excellent traps and SERS “hot spots” for catching and SERS sensing of biospecies with varied sizes (e.g., viruses and proteins). Sensitive SERS detection of viruses and proteins was demonstrated using the silvercovered nanoparticle-cluster-in-bowl SERS substrates.

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template with a sol-gel method, as previously reported.28,29 Next, Au is electrochemically deposited on the as-prepared bowl template. Due to the concave bowl structure, Au is initially grown on the exposed Au film at the bottom of the bowls. The tertiary structure (e.g., size of the nanoparticles and the nanoparticle clusters, and the distance between adjacent nanoparticle clusters) of the electrodeposited Au can be well controlled by varying the deposition voltage and the deposition time, as well as the size of the PS spheres used. After thermally evaporating a thin layer of Ag film on the electrodeposited Au PCIB arrays, they become ultrasensitive SERS substrates as discussed later.

Figure 2. (a) SEM image of the MCC template. (b) A highlyordered bowl template is replicated from the MCC template. (c) The uniform bowl template under a higher magnification. (d) A tilted-view image of the bowl template. Inset in (d) A cross-sectional image of the bowl template. Figure 1. (a) Illustration of the fabrication process of the Au PCIB structures. Process I. Bowl template is prepared using a sol-gel method using a MCC template. Process II. Electrodeposition at a low deposition voltage (e.g., 2 V) using the bowl template as a working electrode, leading to the formation of nearly spherical Au particle clusters. Process III. Au particle clusters with a concave surface are generated at a medium deposition voltage (e.g., 3 V). SEM images of the Au particle clusters are shown as insets. (b) Side-view illustrating the Au particle cluster growth process under a low (e.g., 2 V, process 1), medium (e.g., 3V, process 2), and high (e.g., 5V, process 3) deposition voltages within a bowl template attached to a Au film-covered glass slide.

Results and Discussion Figure 1a shows the fabrication process for the PCIB arrays. First, a monolayer colloidal crystal (MCC) template composed of hexagonally-arranged polystyrene (PS) spheres is prepared using a self-assembly process.23-27 The ordered silica bowl template on a piece of Au-film-covered glass slide is subsequently prepared by replicating the MCC

A typical scanning electron microscope (SEM) image of a large area, highly ordered MCC template was shown in Figure 2a. After replicating the MCC template using a silica sol-gel approach and removing the PS spheres by dissolving in toluene, an extremely ordered silica bowl template on a thin layer of Au film was generated (Figure 2b). More than 5000 bowls were created, arranged in a regular array with less than 10 vacancies and with no obvious cracks or dislocation lines (Figure 2b and c). Tiltview observations clearly show the thin shell structure of the silica bowls (Figure 2d). The average height of the bowls is ~ 500 nm (inset in Figure 2d). Notably, the area of the bowl template can easily exceed 1 cm2, which is theoretically only confined by the area of the container used to prepare the MCC template. After ECD of Au using the ordered bowl template as an electrode in a pure HAuCl4 solution, each bowl will behave as a separate reactor, leading to the formation of a single Au nanoparticle cluster at the bottom of each bowl. The formed Au particle clusters have similar structures across the different bowls in the template array. For

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example, after electrodeposition at 2 V for 15 min (Figure 3a, b), 540 nm Au particle clusters composed of several Au particles, each with a diameter of about 120 nm were formed in each bowl. When the deposition voltage was raised to 3 V, the size of each particle cluster was increased to 600 nm and the diameter of the individual Au particles within the clusters was decreased to about 106 nm. The filling ratio of the bowl array with Au clusters was above 95% (Figure S1, Supporting Information), further emphasizing the robustness of the nucleation-controlled electrodeposition technique in creating these PCIB arrays. Notably, these Au particle clusters have a concave surface as indicated from the SEM line-scan spectrum shown in Figure 3e.

in the empty bowls until all of the bowls are occupied by Au clusters (Figure 3a). Meanwhile, as the deposition proceeded, the adjacent Au particles within the clusters “fused” together (Figure 3a) through an orientedattachment growth mechanism (Figure 1b), leading to the appearance of Au particle clusters with a reduced diameter after long electrodeposition times.30-32 An ultralong deposition time (e.g., 30 min) at 2 V gave rise to the formation of Au clusters that filled the entire volume of the bowls (Figure S3, Supporting Information). Meantime, the clusters became even denser due to the “fusion” of adjacent individual Au particles through the oriented attachment growth.30-32 In comparison, 5 min of deposition at 3 V led to an almost 100% filling ratio of the bowl array with Au particle clusters (Figure S2, Supporting Information). The diameter of these clusters is about 490 nm. Thus, a high deposition voltage (e.g., 3 V) and a short deposition time (e.g., 5 min) are appropriate when a high filling ratio of the bowl array with small Au clusters is desired. Also, even after a short deposition time, the concave surface of the clusters has already formed due to the relatively faster growth along the inner wall of the bowl template compared with the center area. Further increasing the deposition voltage to 5 V gave rise to the formation of an extremely ordered Au PCIB array over a large area with a filling ratio of nearly 100% (Figure S4 and Figure S5, Supporting Information). The size of the clusters can be controlled by the deposition time. The cluster diameter increased from about 660 nm to 800 nm when the deposition time was increased from 5 min to 10 min. Correspondingly, the diameter of the individual Au particles within the clusters increased from approximately 150 nm to 220 nm (Figure S4, Supporting Information). The oriented attachment growth of the Au particles is not obvious under a high deposition voltage, as observed by the porous structure of the Au particle clusters The size of, and the distance between, neighboring Au clusters can be conveniently and simultaneously controlled by adopting the bowl templates with different sizes. For instance, 450 nm diameter Au clusters with an obvious concave surface were obtained using a 500 nm diameter bowl template on a piece of Au layer-covered

Figure 3. (a) and (b) SEM images of the Au PCIB arrays prepared under 2 V deposition voltage for 15 min. (c) and (d) Au clusters with a concave surface electrodeposited at 3 V for 15 min. (e) A line-scan showing the concave Au cluster arrays.

In order to study the growth process of the Au particle clusters, we stopped the deposition time after 5 min (Figure S2, Supporting Information). Under a 2 V deposition voltage, Au particles about 160 nm in diameter aggregated into clusters in each bowl. The diameter of the clusters is around 700 nm. The filling ratio of the bowl array with clusters is less than 30%. Therefore, at a low deposition voltage (e.g., 2 V), nucleation first takes place randomly in any bowl of the array. After the clusters reach a critical size, their growth stops. Then, nucleation occurs

Figure 4. Au PCIB arrays prepared under 3 V deposition for 5 min using the bowl template with a bowl size of 500 nm on the Au film-coated glass. Some neighboring Au particle clusters have connected each other (see white circle in b). (a) and (b) Different magnifications.

glass slide (Figure 4).

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Based on the above results, the growth process of the Au clusters under different deposition conditions are schematically summarized in Figure 1b. Nucleation of Au first takes place at the bottom of the bowls, where thermally evaporated Au film is exposed. At a low deposition voltage (~ 2 V), separated Au particles begin to aggregate after a short deposition time. Then, adjacent Au particles will merge together through an oriented attachment process,30,31 leading to the formation of a large Au cluster. At a medium deposition voltage (~ 3 V), Au is preferentially grown across the inner wall of the silica bowl, resulting in the formation of Au clusters with a concave surface. Au clusters composed of relatively distinct particles are created under a high deposition voltage (~ 5 V). The structure of the Au clusters can be adjusted by changing the deposition voltage and time. The distance between neighboring clusters can be conveniently manipulated by using bowl templates with different sizes (ranging from 200 nm to more than 10 µm).

Figure 5. FDTD simulation of the electromagnetic field distribution on the Au PCIB structures. (a) and (b) Correspond to the Au PCIB structures shown in Figure 3b and d, respectively. (c) After evaporating a 20 nm thick Ag film on the Au PCIB structures shown in Figure 3d.

The electromagnetic field distribution across the Au PCIB structures was simulated using a commercial finitedifference time-domain (FDTD) method software package (Lumerical FDTD Solution).33 For simplicity in this simulation, the Au PCIB structures were modeled as 100 nm diameter Au particles formed aggregates according to

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the morphology shown in Figure 3b and 3d. The simulation results reveal that the electromagnetic fields around the Au particle clusters shown in Figure 3b are very weak (Figure 5a). Comparatively, there are stronger electromagnetic fields across the Au particle clusters with a concave surface as shown in Figure 3d (Figure 5b). These simulation results are supported by the SERS measurements shown below (Figure 6 and Figure 7). The bowl structure is formed by silica, which has a negligible contribution to the enhancement of the electromagnetic fields. In order to evaluate the contribution of the silver-covered concave bowl structure to the electromagnetic field enhancement, we built a model in FDTD simulations by stacking a 20 nm-thick Ag layer onto the silica bowl structure. As experimentally verified later, extremely strong electromagnetic fields were established at the interface between the Au particle cluster and the Ag film-wrapped silica bowl, as well as at the concave area on the Au particle cluster (Figure 5c). These areas can be used as “hot spots” during SERS sensing, giving rise to dramatically enhanced SERS signals which are at least two orders of magnitude higher intensity than before Ag film deposition (Figure 6). Also, no organic contaminants (e.g., surfactants), which are well known to influence the SERS sensitivity,34 are introduced during the fabrication process. The clean surface of the PCIB structures allows for easier attachment of probe molecules to the “hot spots”. Moreover, as aforementioned, each bowl can be viewed as identical in separate containers, due to the homogeneous structure of Au particle clusters in the bowl array, greatly enhancing the reproducibility of the SERS signals. The SERS performance of the Au PCIB structures was evaluated using 4-aminothiophenol (4-ATP) as a probe molecule (Figure 6). The Au PCIB array synthesized at 2 V for 15 min (Figure 3b) exhibited very weak SERS signals (curve I in Figure 6a), which are comparable with those signals (curve III in Figure 6a) from the Au flower-like particles obtained at 5 V for 10 min (Figure S4d, Supporting Information). Strong SERS signals (curve II in Figure 6a) were observed from Au particle clusters with a concave surface fabricated at 3 V for 15 min (Figure 3d). As predicted by the FDTD simulations, extremely strong SERS signals were observed after coating the Au PCIB structure with a 20 nm-thick film of Ag (Figure S6, Supporting Information). The grain size of the Ag film is about 25 nm (Figure R6, Supporting Information). The SERS signal at 1070 cm-1 is enhanced by 430 (curve IV in Figure 6a), 660 (curve V in Figure 6a), and 950 (curve VI in Figure 6a) times after coating the Ag film onto the Au PCIB structures obtained at 2 V for 15 min (Figure 3b), 3 V for 15 min (Figure 3d), and 5 V for 10 min (Figure S4d, Supporting Information), respectively. The SERS enhancement factor (EF) from these different structures is evaluated using the equation:35-37 EF= (ISERS/Ibulk)/(Nbulk/NSERS) (1) Where ISERS and Ibulk are the intensities of the SERS signal at 1070 cm-1 from the SERS substrate and the 4-ATP powder, respectively. NSERS and Nbulk are the numbers of 4-ATP molecules absorbed on the SERS substrate and in the

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powder exposed to a laser spot, respectively. The Raman spectrum of the 4-ATP thin film obtained by evaporating 100 µL 4-ATP ethanol solution (0.5 M) is shown in Figure 6a (see curve VII). The EF of the Ag-film covered Au particle cluster with a concave surface is calculated to be 7.9 x 107 (Figure 6b). After heating the Ag-filmcovered Au particle cluster with a concave surface at 400 oC for 2 h, the SERS EF decreased by six times (curve VIII in Figure 6a and Figure 6b), which is attributed to the structural deterioration and surface smoothening after heating treatments (Figure S7, Supporting Information). The SERS EFs of different Au PCIB structures prepared at various deposition conditions before and after Ag film evaporation were summarized in Figure 6b.

Figure 7. (a) SERS spectra of 4-NBT molecules at different concentrations absorbed on the Ag-film covered Au PCIB arrays prepared at 3 V deposition for 15 min. (b) The relationship between the intension of the 1075 cm-1 SERS peak and the concentration of the 4-NBT ethanol solutions. The error bars are obtained based on more than 20 SERS spectra. (c) Microscopic photo of the area where SERS mapping were performed. (d) SERS mapping result obtained based on the 1075 cm-1 SERS peak. The concentration of the 4-NBT solution was 10-7 M. (e) 160 typical SERS spectra withdrawn from the SERS mapping result. (f) RSD obtained from the 160 SERS spectra shown in (e).

Figure 6. (a) SERS spectra of 4-ATP molecules absorbed on the Au PCIB arrays prepared at different ECD conditions. Curves I, II, and III correspond to Au particle clusters shown in Figure 3b, Figure 3d and Figure S4d in the Supporting Information, respectively. Curves IV, V, and VI are measured from Ag film-covered Au PCIB arrays shown in Figure 3b, Figure 3d and Figure S4d in the Supporting Informaiton, respectively. Curve VII is from the 4-ATP film formed by drying 100 µL 0.5 M 4-ATP ethanol solution. Curve VIII is measured after heating the Ag film-covered Au PCIB array shown in Figure 3d at 400 oC for 2 h. (b) SERS enhancement factor of the different structures.

The SERS sensitivity of the Ag-film covered Au PCIB arrays fabricated at 3 V deposition for 15 min (Figure 3d) was further studied (Figure 7a). SERS signals of pnitrobenzenethiol (4-NBT) molecules were still observable when the concentration is 10 nM. The relationship between the intensity (I) of the 1075 cm-1 SERS peak and the concentration (C) of 4-NBT could be described by the following equation: Log C = 2.85 × Log I – 15.58 (Figure 7b). To evaluate the SERS signal reproducibility, SERS mapping measurements over an area of 20 µm × 20 µm on the Agfilm covered Au PCIB arrays were performed (Figure 7c). The relatively uniform color of the mapping image demonstrated a good SERS signal reproducibility (Figure 7d). 160 typical SERS spectra withdrawn from the SERS mapping image were shown in Figure 7e. The relative standard deviation (RSD) was determined to be about 12.9% based on the SERS mapping results (Figure 7f).38 Notably, the RSD should be overestimated based on the SERS mapping results, because the laser beam was not accurately focused at the center area of the Au PCIB structure at some of the points during SERS mapping

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measurements. The RSD should be able to be significantly improved by focusing the laser beam exactly into the

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concave surface and the interface area between the Au particle cluster and the Ag bowl, where the “hot spots” are located. Particularly, when the target species are large in size (e.g., bioorganisms), which are unlikely to stick onto the walls of the Ag bowls and therefore can be delivered to the “hot spot” locations. As a proof of concept, sensitive SERS detection of a poliovirus was achieved using the Agfilm covered Au PCIB array. Wild-type poliovirus (Mahoney) with a mean length of about 30 nm was used as a target model.44 The viruses were stuck within the crevices between the Au nanoparticle clusters and the Ag bowls, where SERS “hot spots” are located (Figure 8a). Therefore, strong SERS signals were observed from the Mahoney virus even at very low concentrations (e.g., nM). Due to the low concentration and the random orientation of the viruses, the SERS peak positions varied slightly at different sites on the SERS substrate (Figure 8b). In the future, surface functionalization of the SERS substrate to anchor the viruses in the same orientation might be able to resolve this poor signal reproducibility problem. Further, sensitive SERS detection of proteins (e.g., lysosome, bovine hemoglobin and bovine serium albumin)45-47 with different sizes was realized using the Ag-film covered Au PCIB structures (Figure 8c). Conclusion

Figure 8. (a) Demonstration of the Ag film-covered Au PCIB structure enabled delivery of analyate molecules (particularly large bio-organisms) during solvent evaporation into the Au cluster/Ag bowl interface area. (b) SERS spectra of poliovirus. (c) SERS spectra of different proteins at a concentration of 100 ug/mL. Curve I, II, and III correspond to lysosome, bovine hemoglobin and bovine serium albumin, respectively.

center area of the Au PCIB structures. SERS detection of chemicals in a solution is frequently required in many fields, including waste water analysis, food safety, anti-smuggling, national security, and in clinical medical laboratories.39-43 When the analyte solution is pipetted onto these PCIB structures, the bowls can behave as containers (Figure 8a). As the solvent evaporates, most of the molecules are delivered to the

In summary, template-defined, site-specific, electrochemical growth has been achieved on the bowl template, resulting in the formation of Au PCIB arrays. The structure of the Au particle clusters can be simply adjusted by varying the deposition time and applied voltage. The controllability of the Au PCIB array structures creates opportunities for many novel applications. Strong electromagnetic fields are formed on the Ag-film covered Au PCIB arrays, enabling SERS applications. Dense SERS hot spots and the uniform structure of the Agfilm covered Au PCIB arrays make them good SERS substrates with an enhancement factor of around 108 and good reproducibility with less than 15% intensity deviation. The unique PCIB structure facilitates trapping and delivery of viruses and proteins to the SERS “hot spots”. Sensitive SERS detection of a poliovirus and several proteins (e.g., lysosome, bovine hemoglobin and bovine serium albumin) is achieved. This template-defined, site-specific, electrodeposition method developed here may be extended into other material systems, further widening its applications in catalytic fields, in plasmonics and solar cells, as well as in biochemical sensing applications.

Experimental Section Bowl template fabrication. A spin-coating method was used to prepare the MCC template as previously reported.28,29 Briefly, glass slides (1 cm x 4 cm) were treated in a plasma cleaner (Harrick Plasma Cleaner at 10 W) for 5 min to obtain superhydrophilic surfaces. The freshly cleaned glass slides were fixed onto a spin coater and 20 µL of 1 µm PS beads (purchased from Alfa Aesar) was dispensed. After rotating at 500 rpm for 5 min, a colorful MCC template was obtained. In order to increase the uniformity of the MCC template, the dried substrate was slowly immersed into deionized water at an angle of 45o

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normal to the surface. The MCC template was detached from the substrate and floated on top of the water surface. A 50 nm Au-covered glass slide, after a 5 min plasma treatment, was used to pick up the floating MCC template. Then the transferred MCC template was placed onto a hot plate set at 110 oC for 1 min to strengthen the adhesion between the MCC template and the substrate. This heating treatment is very important, since it enables the exposure of the Au film after the formation of the bowl structure. The bowl structure was fabricated using a sol-gel method. 40 µL of silica sol solution composed of tetraethylorthosilicate, HCl (0.1 M), and ethanol with a volume ratio of 1:1:10 was dropped uniformly onto the MCC template. After natural convective drying, the MCC template was removed using toluene, producing a highly ordered bowl array. Fabrication of Au PCIB structure. The bowl structure was used as the working electrode to perform the electrodeposition of Au. The electrolyte was pure HAuCl4 aqueous solution at a concentration of 10 mM. SERS characterization. 4-ATP was used as a probe molecule to evaluate the SERS enhancement factor of the Au PCIB structures. 10 µL of 4-ATP ethanol solution (10-5 M) was pipetted onto the SERS substrate covering an approximate 1 cm2 area. We assume that the 4-ATP molecules were uniformly distributed over the substrate surface to facilitate the subsequent evaluation of the SERS enhancement factors. A 632 nm laser (about 0.5 mW) and a WITec CRM 200 Confocal MicroRaman system with a 100x objective was used to collect the SERS signal from the 4-ATP molecules absorbed on the substrate. The laser beam could be focused into a spot with a diameter of around 1.5 µm. The accumulation time was 5 s. Ethanol solutions composed of 4-NBT molecules at different concentrations were used to evaluate the SERS sensitivity of the Ag-film covered PCIB structures. The SERS spectra of poliovirus were measured using the same equipment and a similar dispensing procedure as the 4-ATP.

ASSOCIATED CONTENT Supporting Information. SEM images of large area gold nanoparticle cluster-in-bowl arrays, SEM images of silver-film covered gold nanoparticle cluster-in-bowl array after thermal treatment. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *Email: [email protected].

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

ACKNOWLEDGMENT This work is supported by the Fundamental Research Funds for the Central Universities (2017QNA4009) and National Science Foundation of China (51702283). Y. Wang and Y. Yu contributed equally to this work.

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