Synthesis of amphiphilic dumbbell-like Janus nanoparticles through one-step coupling

Abstract In this study, amphiphilic Janus nanoparticles with dumbbell-like structure have been synthesized by connecting two silica nanospheres with differential wettability. In this method, silica nanospheres were modified to be hydrophobic and hydrophilic respectively and then the nanospheres with different wettability were covalently connected together on a one-to-one basis simply by chemical bonds generating between surface groups. The dumbbell-like structure of particles can be observed clearly under scanning electron microscope and transmittance electron microscope. Results showed that 58% of the nanospheres were successfully coupled to build the dumbbell-like structure. Since these anisotropic nanoparticles were based entirely on inorganic materials and owned well-defined surface areas of different wettability, they may have a highly potential for many application fields such as optical and electrical engineering. Graphical Abstract

As a representative category of JNPs, dumbbelllike nanoparticles have attracted much attention because of their typical structure and outstanding performance in many application areas [21]. The dumbbell-like nanoparticles refer to nanoparticles with two spherical lobes which have different chemical properties or morphology. The two connected lobes are relatively independent and synthesis of two parts is also done separately, thus a weaker mutual effect would exist between two areas with different chemical properties and they would be more specifically sectioned comparing with dividing the surfaces of spherical particles into two hemispheres [22]. Previous research [23] has demonstrated that particles with well-defined surface areas of different wettability can be absorbed at oil-water interfaces considerably more strongly than particles of homogeneous wettability. Videlicet, the betterdefined between two areas on surfaces, the more thermodynamically stable Pickering emulsions the JNPs would give. As a result, dumbbell-like JNPs may have much better interfacial properties than the spherical ones. In addition, due to the typical structure of dumbbell-like JNPs, different properties and functions can be combined freely by changing the chemicals loaded onto the two lobes, which can provide more possibilities for new species to be synthesized.
In studies on dumbbell-like JNPs reported so far, dumbbell-like JNPs can be divided into three categories, depending on the materials of the two lobes: organic polymer double spheres, polymer-inorganic double spheres and inorganic materials double spheres. dumbbell-like JNPs of different materials can be synthesized in different ways. For organic polymer double spheres, the most common synthesis methods are two-step emulsion polymerization [9,24] and two-step intramolecular cross-linking reactions [25]. For polymer-inorganic double spheres, for example, E. Passas-Lagos et al. [26,27] introduced a technique to fabricated PS/Fe3O 4 /SiO 2 mushroom-shaped nanoparticles: Fe 3 O 4 particles were immobilized by PS at first and then were spatially selectively coated by SiO 2 . As for inorganic materials double spheres, the most important part in the synthesis process is the multi-step growth of materials. First, synthesize seed particles with material A, and material B was then nucleated on former seeds to form another lobe [28][29][30]. We are particularly interested in dumbbell-like JNPs based on pure inorganic materials for their excellent performance and wide range of applications, such as amphiphilic coatings [9], photonic crystals [11] and supercapacitors [29]. Thus, we believe that a more convenient synthesis method with good universality should be developed. We selected silica nanospheres as the raw material because of its simple preparation process, easy adjustment of size and structure and variable surface properties [31][32][33]. Herein, we present a novel and facile method for preparing amphiphilic dumbbell-like Janus nanoparticles by coupling silica nanospheres with different hydrophilicity through chemical bonds. Different from our previous work [34], in this work, monodispersed silica nanospheres with lager size synthesized through St€ ober method [31] were modified and different hydrophilicity was given. Then, each pair of hydrophilic and hydrophobic silica nanospheres were connected by silane couplers and finally built a dumbbell-like structure with asymmetric hydrophilicity, the whole process is carried out in ethanol. That is, we could easily fabricate the amphiphilic Janus nanoparticles with dumbbelllike structure by one-step coupling. To better understand the coupling process, we analyzed the proportion of nanospheres participating in the reaction through particle size distribution. Compared with other preparation strategies of Janus nanoparticle, such as "two-step growing method" of most dumbbell-like NPs [28][29][30], this method is direct and template-free, preparing and removing templates were not needed during the experiment, and enables the preparation of dumbbell-like particles based on the homogeneous inorganic materials. According to the mechanism, this method can also be applied to many kinds of nanospheres with modifiable surfaces.

Preparation of monodispersed SiO 2 nanospheres
Monodispersed SiO 2 nanospheres were synthesized through St€ ober method, which is a hydrolysis-condensation process of TEOS that catalyzed by ammonia. Typically, TEOS (10 mL), EtOH (80.0 g), and NH 3 ÁH 2 O (6.0 g) were mixed and stirred at 30 C for 3 h. The solution was aged at 25 C for 7 days and then a milky SiO 2 sol was formed. Followed by the sol was refluxed at 110 C for 24 h until ammonia was completely removed. Then original SiO 2 nanospheres for following experimental steps were obtained. The sol contained 3.0 wt% SiO 2 .

Preparation of surface-modified SiO 2 nanospheres
The surfaces of silica nanospheres synthesized by St€ ober method is rich in hydroxyl groups, which makes the surface modification simple and diversiform. The as-prepared nanospheres were hydrophilic and hydrophobic modified by 3-Glycidoxypropyltrimethoxysilane (GLYMO) and Hexamethyldisiloxane (HMDS) respectively. Typically, GLYMO and HMDS added into the silica sol were 100% and 50% weight of silica, respectively. After 3-day aging, the chemical modified nanospheres were obtained.

Preparation of dumbbell-like JNPs
Before the coupling between two categories of nanospheres, S 50HMDS should be decorated by a small quantity of APTES (70 lL for 1 g SiO 2 ) and stirred for 6 h at 30 C. At the same time, a small quantity of GLYMO (0.1 g for 1 g SiO 2 ) added to the hydrophilic particles to supply epoxy group which mostly hydrolyzed during 3day aging. Then mixed the hydrophilic and hydrophobic nanoparticles and immediately stirred for 30 min at 30 C and aged for 3 days at 25 C.

Characterization
In order to demonstrate the changes of hydrophilicity of silica nanospheres after modification, films were prepared on glass substrates with sols by dip-coating process. The hydrophilicity of films was characterized by water contact angle (WCA). The size distributions of particles mentioned above were measured by a laser particle size analyzer (Zetasizer nano-ZS, Malvern Panalytical Ltd., the UK) at 25 C using EtOH as dispersant, automatically test 3 times and 11 runs for each time. The size and the morphology of particles were determined by field emission scanning electron microscope (FE-SEM, Hitachi S-4800, Japan) and transmittance electron microscope (TEM, FEI Tecnai F20, America). The samples were coated with a thin layer of gold before FE-SEM measurement.

Properties of silica nanospheres
Original silica nanospheres (o-S) were first synthesized via the St€ ober method. The distribution curve and SEM image of original silica nanospheres were shown in Figure 1. The average size of silica nanospheres was 44 ± 2nm and polydispersity index (PDI) value for the nanospheres was below 0.05, which indicates a good uniformity for the nanospheres. The resulting products after surficial modification can be denoted as S 100GLYMO , S 50HMDS and S 50HMDS-APTES respectively. Also, different ratios of HMDS were added to achieve different hydrophobicity and the modified samples were denoted as S 75HMDS and S 100HMDS . In order to demonstrate the changes of hydrophilicity of silica nanospheres after modification, coatings were prepared on silicon wafers with sols by dip-coating process. The hydrophilicity of films was characterized by water contact angle (WCA) and the results were shown in Figure  2. WCAs of coatings constructed by o-S, S 50HMDS , S 75HMDS and S 100HMDS were 30 , 108 , 121 and 136 , respectively (Figure 2a). The WCA of S 100GLYMO coating decreased from 82 to 5 over aging time due to the increase of hydroxyls from the hydrolysis of epoxy groups and the evolution of the hydrophilicity was shown in Figure 2b. Thus, after modification by HMDS and GLYMO respectively, two kinds of silica nanospheres with significant difference in hydrophilicity were obtained.

Dumbbell-like silica JNPs
The whole process of preparing the dumbbell-like JNPs, start with monodispersed SiO 2 nanospheres, was shown in Figure 3. Figure 3a illustrates the fabrication procedure of the dumbbell-like silica JNPs, while Figure 3b presents the detailed reactions between groups on the surfaces of different silica nanospheres [35]. Figure 4 shows a TEM image of dumbbell-like structure of S 100GLYMO -S 50HMDS-APTES , in which two silica nanospheres were covalently connected and the dumbbell-like JNPs were monodispersed. The particles had different postures in the TEM images since they were randomly deposited on the background. The shooting angle in the TEM was the top view, different morphologies of the dumbbell-like structure appeared in the pictures as shown Figure 4b and c.
By mixing two sols with different hydrophilicity in equal amounts, the average particle size increased from 44 ± 2nm to 61 ± 1nm, and PDI value still remained below 0.15 (Figure 5a), which indicated that after mixing, silica nanospheres with good monodispersity may be beneficial to studying of the changes in particle sizes before and after coupling. Since the particle size distributions were characterized by dynamic light scattering (DLS), the measurement sizes of non-spherical particles should follow the Equivalent Volume Theory [36][37][38][39][40], which means the measurement result is equal to the diameter of a sphere with equal volume to this particle. Here, we can think of dumbbell-like structure as a cylinder,   as shown in Figure 5b, then the equivalent diameter can be calculated using the equations as follow: Then, we can obtain, Thus, Where, R S , R C and R E represent the radius of silica nanosphere, equivalent cylinder and equivalent sphere. The calculated value of the equivalent sphere's radius R E is equal to 1.4422 R S , that is about 65 nm, which is very close to the measurement value 61 nm.
Combing the TEM images and the change in particle size, it can be determined that the dumbbelllike structure has been successfully fabricated.

Study on coupling reaction of the particles
To further demonstrate that the dumbbell-like structure was the consequence of covalent connection of two nanospheres with diverse hydrophilicity and the detail of the coupling reaction, three control experiments were set up, as shown in Table 1. Particle size distribution curves of each sample in Table 1 were shown in Figure 6. In sample c, hydrophilic nanospheres S 100GLYMO were mixed with hydrophobic nanospheres S 50HMDS , which were without modification of APTES in advance. The reaction conditions remained the same as the previous experiment and as a result, the nanospheres were failed to build the dumbbell-like structure since the particle size of sample c remained 44 ± 1nm. So, the coupling agent APTES played an important role in the process of the formation of the dumbbell-like structure. In sample d and e, all of the modification process were same as the previous experiment, but mixed two kinds of nanospheres S 100GLYMO and S 50HMDS in different ratios 2:1 and 1:2 respectively, the particle sizes were both monodispersed and values were both 56 ± 1nm. Then we studied the evolution of particles sizes within 480 min after mixing, as shown in Figure 7. The particle size of each sample increased immediately within 40 min, and after that, the values tended to be stable and were the same as the final results. Through these control experiments, we can further determine that the dumbbell-like structure was the result of connecting two nanospheres with different hydrophilicity on one-to-one basis by chemical bonds rather than the hydroxyl condensation or hydrophobic association between nanospheres randomly regardless of hydrophilicity. Furthermore, even if one of the modified nanospheres were excessive, the obtained particle sizes were smaller than calculated value in 1:1 ratio and were unimodal, which means that no trimers or multimers were formed and once the coupling reaction occurred between the nanospheres, the dominant structure was dumbbell-like. In addition, in order to further prove that the connection between the nanospheres are chemical bonds, the sample S 100GLYMO -S 50HMDS-APTES (1:1) was treated with two  kinds of sonication instruments, ultrasonic cleaner (SCIENTC, SB25-12DT, China) and ultrasonic cell disruptor (TL92-IID, Tianling Co., Ltd, Jiangsu Province, China). As shown in Figure 8, the particle size of the samples remained stable during the 60 min of sonication treatment by two ultrasonic instruments. The agglomeration between particles in a dispersion system can be divided into two categories: soft agglomeration and hard agglomeration [41,42], where the forces of soft agglomeration include electrostatic force, van der Waals force and hydrogen bonding. Therefore, some mechanical external forces can be used to eliminate such forces; in addition to the above-mentioned several forces that cause soft agglomeration, there are also chemical bonds between the particles that cause hard agglomeration, which makes the force of hard agglomeration more effective, thus hard agglomeration cannot be broken simply by mechanical actions. So it proved that the coupling between the nanospheres is the effect of chemical bonds. The dumbbell-like structure is the dominant conformation in the coupling process of modified nanospheres due to the small amount of coupling active groups as well as the steric hindrance. In the study of Kim et al. [43] on programmable matter, they used an AuNP with one DNA strand as the starting material for the second DNA attachment, and the two for the next. Experiments showed that each step of the attachment, the DNA strand tend to form the largest angle with the previous one, which means the steric hindrance has a conspicuous influence on the arrangement of objects on the particle surface.   Their results are comparable with our experiments that the steric hindrance has a major effect of preventing the mixture from the formation of triple or multiple structures.
In order to better understand more details of coupling process for nanospheres, the proportion of coupled silica nanospheres was calculated. According to the Rayleigh Approximation Theory, the intensity distribution is approximately proportional to the sixth power of particle size for small particles in a relatively stable dispersion system [44], as presented in Equation 6. Equation 7 presents number distribution for a system containing two kinds of particles.
Where, N a and N b are molecules with size a and b respectively. The %I a and %N a present intensityweighted and number-weighted distribution respectively for small particles with size a. Then, regardless of very small amount of multimers in the system, we assume that there were only dumbbell-like dimers and residual single nanospheres after the coupling reaction, and peak-fitting of the particle size distribution curve of the product (the curve of S 100GLYMO -S 50HMDS-APTES (1:1) in Figure 5) was performed. As a result, the obtained intensity ratios of o-S and equivalent diameter of dumbbell-like particles were plotted in Figure 9, which were 13.8% (%I a ) and 86.2% (%I b ), respectively. Finally, combining Equations 7 and 8 and obtained values of %I a and %I b , we can obtain the ratio N a : N b ¼1.45: 1. Thus, the coupled silica nanospheres account for 58.2% of the total number of nanospheres before the reaction. Moreover, the same calculation method was adopted for sample d and e (results were shown in Table 2), the proportions of coupled spheres were 45.3% and 46.4%, respectively. Since the ratios of nanospheres S 100GLYMO and S 50HMDS-APTES in sample d and e were 2:1 and 1:2, 67.9% S 50HMDS-APTES in sampled d and 69.6% S 100GLYMO in sampled e were coupled. In other words, a more thorough transformation from monospheres to dumbbells occurred in a 1:1-mixing system compared with 2:1 or 1:2. As for 2:1-and 1:2-mixing system, the minorities transformed more thoroughly.
During the coupling process, nanospheres move and collide randomly due to Brownian motion, hence the intensity of Brownian motion has a significant impact on the progress of the reaction between nanospheres, which is related to diffusion coefficient. According to the Stokes-Einstein relationship [45], diffusion coefficient of small particles suspended in certain medium can be defined by Equation 8: Where, k is the Boltzmann constant, T is temperature, g is the viscosity of the medium and R is the radius of the particles.
Therefore, if we want to make the particles coupled more thoroughly, we may use smaller nanospheres to build the dumbbell-like structure or in a medium with a lower viscosity.

Conclusion
In summary, a feasible and facile way was developed to prepare amphiphilic dumbbell-like Janus nanoparticles by coupling two different modified silica nanospheres through the coupling reaction between the amino groups and the epoxy groups. In this process, hydrophilic and hydrophobic lobes of the JNPs are separately prepared in advance, JNPs with well-defined anisotropic properties can be obtained after coupling. TEM images clearly showed the existence of the dumbbell-like structure, the particle size of the reaction system increased from 44 nm to 61 nm and more than 58% nanospheres were transformed successfully to dumbbell-like structure. This method can be applied to the synthesis of many other dumbbell-like JNPs with asymmetric chemical properties in large scale and also give inspiration to design and synthesis more peculiar nanoparticles with Janus property and nonspherical morphology.

Author contributions
Bo Jiang and Jianhui Luo conceptualized and designed the research; Meiying He performed the synthesis and characterization of the materials; Pingmei Wang, Peiwen Xiao, Xinli Jia and Meiying He analyzed and interpreted the experimental data; Meiying He and Bo Jiang finished writing the manuscript; Bo Xiao supervised the work.

Disclosure statement
No potential conflict of interest was reported by the authors.

Notes on contributors
Meiying He received the B.Sc. degree from the Chemistry College of Sichuan University, Chengdu, China, and now is a Ph.D. candidate in polymer chemistry and physics in Sichuan University. Her current research interests include functional polymers, Janus nanocomposites and optical film technology.