Mechanically stretching the metallocycle-C60 junction with a scanning tunneling microscopy break junction technique causes the release of the C60 guest from the metallocycle, and consequently the conductance switches back to the free-host level. Metallocycle hosts with different shapes and cavity sizes show different degrees of flexibility to accommodate the C60 guest in response to mechanical stretching. DFT calculations provide further insights into the electronic structures and charge transport properties of the molecular junctions based on metallocycles and the metallocycle-C60 complexes.Host−guest interactions are of central importance in many biological and chemical processes. However, the investigation of the formation and decomplexation of host−guest systems at the single-molecule level has been a challenging task. Here we show that the single-molecule conductance of organoplatinum(II) metallocycle hosts can be enhanced by an order of magnitude by the incorporation of a C 60 molecule significantly enhances the conductance of an organoplatinum metallocycle; mechanical stretching of the junction releases the guest, returning the conductance to free-host level.Studying the single-molecule behavior of host-guest complexes can provide fundamental insights into their supramolecular interactions. Here, the authors use the scanning tunneling microscopy break junction technique to show that encapsulation of a C Host–guest interactions have now been exploited in a wide range of disciplines, including chemosensing10, catalysis11, drug delivery12, nanosciences14, and molecular devices16. These previous works focused on host–guest ensemble behaviors of bulk materials. Studying how host–guest complexes behave at the single-molecule level opens new prospects to understand the fundamentals of supramolecular chemistry by providing critical information about the strength and flexibility of host–guest interactions.The discovery of crown ether and cryptand in 1967 as macrocyclic ligands for alkali metal cations laid the foundation of modern host−guest and supramolecular chemistry27. The technique has been recently extended to supramolecular systems including charge–transfer pairs28, π-stacked dimers29, rotaxanes31, or host–guest interactions32. Mechanically controllable break junction (MCBJ) or STMBJ experiments are well-developed to reveal the electrical and mechanical properties of the molecular structure and junction conductances38.The scanning tunneling microscopy break junction (STMBJ) technique allows measurement of charge transport properties of single-molecules involving covalent bonds60 with STMBJ. The coordination-driven self-assembly yields different metallocycles with precisely controlled shape and cavity size, which allows us to modulate the strength of the host–guest interaction. The conductance of the metallocycles with a suitable cavity size is enhanced by an order of magnitude when complexed with the C60 guest. The molecular conductance properties of the metallocycles and their host–guest complexes are further rationalized by theoretical calculations.The direct monitoring of the size-dependent decomplexation was obtained by mechanically stretching the host-guest complexes at the single-molecule level, which switches the conductance back to the free-host level. This demonstrates the capability of host-guest supramolecular systems to modulate molecular conductance.Herein, we measure the conductance and study the charge transport in three supramolecular metallocycles and their host–guest complexes with C5, 6, and 7 with different shape and cavity size were designed and synthesized for the host–guest interaction studies and conductance measurements . The uncoordinated pyridine groups present in these metallocycles are used to contact the gold metal electrodes to form single-molecule junctions.Three organoplatinum(II) metallocycles, nts Fig. , via the1H NMR, 31P{1H} NMR, 1H−1H COSY NMR spectroscopy, and electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) -optimized structures of 60 under ambient conditions (see the Methods section). The lone pair electrons of the free pyridine nitrogen atoms of metallocycles can bind to the Au tip and substrate to form molecular junctions by repeatedly moving the STM tip into and out of contact with the gold substrate functionalized with the supramolecular metallocycles44. Conductance–distance traces were recorded during the stretching process for each sample, and plateaus in these traces were related to the formation and breakdown of single-molecule junctions. Thousands of conductance traces were used to construct a conductance histogram, where the peak position measures the average conductance of a single molecule  single-crystal substrate to measure the single-molecule conductance of the metallocycles and their host–guest complexes with Cule Fig. .Fig. 2Co5, 6, 7, and those in the presence of C60, denoted as [5 + C60], [6 + C60], and [7 + C60], respectively. A conductance plateau was typically observed for the free metallocycles and the conductance followed the order as 5>6> 7  show no conductance peak in the histogram within the measured conductance range , and after rinsing with copious amounts of solvent, the coverage of C60 on Au surface was too low to be measured by break junction methods50. However, C60 featured two conductance peaks at ~0.2 G0 and 0.5 G0, with a higher concentration of C60 (7 mM), which is in agreement with reported values , with different shapes and cavity sizes, possess different degrees of host–guest interactions with the C60 guest. In contrast to [7 + C60], [5 + C60] and [6 + C60] show higher conductance values, which are greater than those of the free 5 and 6 molecules by about one order of magnitude. This provides direct evidence of large enhancement of charge transport when a C60 guest is incorporated. This finding led us to explore in situ controlling of the host–guest interaction by mechanically stretching the molecular junction. Mechanical switching has been demonstrated in single molecules38, but mechanical control of host–guest interactions is a new and attractive strategy. The 2D conductance histograms and corresponding average plateau lengths of 5, 6, 7, [5 + C60], [6 + C60], and [7 + C60] are shown in Fig. 5, 6) are comparable, but slightly larger than those of the host–guest complexes ([5 + C60] and [6 + C60]). Figure 60 complexes at different stretching distances. For [5 + C60], as the junction is stretched by a longer distance, the relative peak area of the lower conductance becomes increasingly larger, which is consistent with the transition from the high to low plateaus shown in individual traces in Fig. 5 is still present after C60 is released and the conductance switches back to the free host level. For [6 + C60], the relative peak area of the high conductance also decreases with stretching, but the distance dependence is substantially smaller than that for [5 + C60]. The distinct distance dependence gives a higher conductance band that persists for 0.2–0.3 nm and 0.5–0.6 nm for [5 + C60] and [6 + C60], respectively  comparable with the van der Waals diameter of C60 (10.34 Å), a very subtle structural deformation of the host by 0.1~0.2 nm could be destructive for the host–guest interaction. However, for metallocycle 6 with a moderately larger cavity size (14.10 Å), the host could stand a structural deformation as large as 0.5~0.6 nm, while still maintaining the host–guest complex. Such information can not be obtained on conventional studies of bulk materials.The maximum mechanical stretching force of the STM tip for the pyridine contact is ~0.8 nN52. The resulting 1D and 2D histograms of 5, 6, [5 + C60], and [6 + C60] are shown in Supplementary Fig. To provide a more complete view of the overall break junction process over a wider conductance range, we measured the conductance from the quantum point contact formed between the electrodes to the noise level measured when the electrodes are widely separated using a logarithmic amplifier5, [5 + C60], 6, and [6 + C60] are 1.38 nm, 1.38 nm, 1.41 nm, and 1.42 nm, respectively, suggesting similar junction breakdown configurations for the metallocycles before and after encapsulation of C60. From the DFT optimized molecular length of 5 (1.79 nm) and 6 (1.94 nm), the angle between the center axis of the metallocycle and the substrate plane is estimated to be ~50° for both metallocycles at junction breakdown. These results indicate that during the break junction measurements the metallocyclic molecules were lifted and distorted by the retreating STM tip.The distributions of relative displacement from the point of contact to junction breakdown are shown in Supplementary Fig. 5 and 7, no distinct absorbance changes were observed upon adding the metallocycle into the C60 solution. In contrast, the complexation of 6 with C60 resulted in the appearance of a new shoulder band at 366 nm, which was consistent with a charge transfer (CT) absorption band53. The linear Benesi–Hildebrand plot suggests a 1:1 host–guest binding ratio of 6 with C60 and the binding constant was calculated to be 4.1 × 105 M−1. These results suggest that the driving force of forming the target host–guest complexes is derived from the donor–acceptor interactions between the electron-rich metallocylic skeleton and the electron-deficient of C6054. The failure to observe potential CT absorption band from the complexation of 5 with C60 is possibly caused by the relatively weak and shallow interaction between them. This is supported by the DFT calculation results discussed below. Although the absorption spectral analysis implies that the interaction between 5 and C60 is weaker compared with that between 6 and C60, the metallocycle 5 can still hold C60 with a shallower host–guest configuration according to our DFT calculations. This allows the complexed molecular wire, either in the case of [5 + C60] or [6 + C60], to open up the C60-related current path to enhance the conductance .UV−vis absorption spectral titration experiments were carried out to investigate the host–guest interactions in solution  is consistent with that of the powder sample (1.0/2.1/2.7/2.7) within experimental error.In our previous work, Pt(II)-based self-assembled supramolecular rectangle, square, three-dimensional cage and prism have been investigated by scanning tunneling microscopy on Au(111) surfaces, and demonstrated to be intact after adsorption on Au(111) surfaces5 and Au(111)/6 are shown in Supplementary Fig. 57, which are 1.2 nm and 1.3 nm for 5 and 6, respectively, close to the STMBJ values.AFM images of Au(111)/5 and 6 with C60 were optimized by DFT calculations. The C60 molecule is deeply embedded into the metallocycle framework of 6  and the lowest unoccupied molecular orbital (LUMO) plots of the [5 + C60] and [6 + C60] complexes are displayed in Fig. 60 contributes to the LUMO, suggesting the presence of donor–acceptor interactions in the metallocycle-C60 complexes.The host–guest complexes of 60 guest binds to the metallocycle host, the transmission spectra T(E) were obtained by combining the DFT package SIESTA58 and with the quantum transport code Gollum59 (see details in the Methods section). Based on the relaxed geometries of the gold-molecule-gold junctions estimated from experimental nanogap distance in Supplementary Fig. 60 and the smaller angles  is close to that of the undistorted 5 with larger angles (~65°), while the large conductance discrepancy between red ([5 + C60]) and yellow (5′) curves in Supplementary Fig. 6′ with smaller tilt angles is higher than that of 6 with larger angles, while Supplementary Fig. 6′ is much closer to that of 6. This demonstrates that the smaller distortion of this host  tends to appear near EF. This effect is clearly present in the red curves of Fig. 60 LUMO are pinned close to the DFT-predicted Fermi energy. In contrast, the resonances between −1.0 and −0.8 eV for [5 + C60] and between −1.0 and −0.5 eV for [6 + C60] are due to states located on the host. Therefore, these HOMO resonances are sensitive to the gating effect of the negatively charged C60 guest  metallocycles with precisely controlled shape and cavity size were synthesized by coordination-driven self-assembly. These compounds, together with C60 were immobilized on Au (111) single-crystal electrode by two separate steps. (1) The electrochemically cleaned Au (111) single crystal electrode was first annealed with hydrogen flame briefly, immersed in acetone  containing 10 μM 5/6 or methanol  containing 10 μM 7, incubated for 2 h, then rinsed with acetone (for 5 and 6) or methanol (for 7) and dried with nitrogen gas. (2) After the STMBJ measurements were performed on the metallocycle modified Au electrode, the resulting electrode was immersed in 1,1,2,2-tetrachloroethane  containing 10 μM C60, incubated for 1 h, then rinsed with 1,1,2,2-tetrachloroethane and dried with nitrogen gas for further STMBJ measurements.The metallocycles and C21 were carried out by a scanning tunneling microscope  and a STM scanner (Molecular Imaging). The molecules modified on Au (111) single-crystal electrode was measured in the air with a small bias (0.1 V) applied between the STM substrate (Au (111) single-crystal electrode) and the STM tip that was freshly prepared by cutting a gold wire . The STM tip was repeatedly brought into contact and retracted from the substrate, during which thousands of conductance distance traces were collected to construct conductance histograms. For each analyte, the STM conductance measurement was performed three time as repeating experiments.The break junction measurements62. No symmetry constraints were used in the optimization (nosymm keyword was used). Conductance calculations and corresponding geometry optimizations were performed by using the DFT code SIESTA58, with a local density approximation , double-ζ polarized basis set for elements in molecules, double-ζ for gold element, cutoff energy of 200 Ry, 0.02 eV/A force tolerance, and 1 × 1 k points in the transverse directions. To compute the electrical conductance of the molecules, they were each placed between pyramidal gold electrodes. For each structure, the transmission coefficient T(E) describing the propagation of electrons of energy E from the left to the right electrode was calculated by GOLLUM59 code based on the resulting Hamiltonian and overlap matrices from SIESTA and the formulae of transport theory:ΓL,R(E) = i−ΣL,R†(E))/2 is the imaginary part of the self-energies ΓL,R(E). ΓL,R determines the width of transmission resonances and self-energies describe the contact between the molecule and left (L) and right (R) electrodes. G is the retarded Green’s function of the molecule in the presence of the electrodes. The conductance is extracted from the transmission spectrum and evaluated by the following formulae:G0 = 2e2/h is the conductance quantum; h is the Planck’s constant; e is the charge of a proton; EF is the Fermi energy. Finally, the room temperature electrical conductance was computed from the formula (2).DFT calculations were conducted using the B3LYP exchange correlation function and implemented in the Gaussian 09 package. The electronic structures were optimized using a general basis set with the Los Alamos effective core potential LANL2DZ basis set for Pt and 6–31 G* for other atomsSupplementary InformationPeer Review File


Excellent agreement between theory and the experimental findings unambiguously demonstrates a pivotal role in the enhancement of 3PA played by 2D excitons. The theory predicts that the resonant 3PA coefficient should be enhanced further by at least two orders of magnitude with very low temperature. The findings are essential for understanding giant 3PA arising from 2D excitons in layered hybrid perovskites and may open new pathways for highly efficient conversion from infrared light energy to either electrical energy or higher‐frequency light emission/lasing.Multiphoton absorption may find many technological applications, such as enhancing the conversion efficiency of solar cells by the utilization of sub‐band‐energy photons, below‐bandgap photodetection through the simultaneous absorption of several infrared photons for photocurrent generation, or light frequency upconversion for high‐resolution, 3D imaging. To enhance multiphoton absorption in semiconducting materials, one of the strategies is to explore low‐dimensional excitons. Here, a quantum perturbation theory on a giant enhancement in three‐photon absorption (3PA) arising from 2D excitons in multilayered crystals of organic–inorganic hybrid perovskites is presented. The maximal 3PA coefficient is predicted to be in the range of 2–7 cm Our findings open a promising avenue for a significant decrease of the required light irradiances for MPA in the infrared regime, leading 2D perovskites to an advantage in many photonic applications, like multiphoton‐pumped lasing, light frequency upconversion, 3D biomedical imaging, and harvesting solar energy.Figure4H9NH3)2PbBr4) is composed of one inorganic slab (PbBr42−) and two organic layers (C4H9NH3+). Such an organic–inorganic 2D system was chosen because of the recent report for its high photoluminescence (PL) efficiency.FigureFigure3, can be approximated as a function of photon energy, hv, byC is a material‐independent constant which has a value of 1.0 × 1076 in the units such that α3 is in cm3 GW−2; N is the density of active unit cells per cm3; hv, ijE, and Γi are the photon energy, the energy difference between two energy levels: i and j, and the linewidth, respectively, in units of eV; μij is the electric dipole moment between the two energy levels in units of esu; ElocE = 13 (n02+1) is the local‐field correction with n0 being the refractive index; and E1s and Γ1s are the lowest s‐exciton energy and the linewidth, respectively.Based on the 2D excitonic model with the electric dipole approximation and the energy diagram in Figure 3, one may explore the electric dipole moment. For the 3PA, there are three electric dipole moments involved, corresponding to three electronic transitions. In Equation sg corresponds to the 1s‐exciton generation by absorbing a photon. As for the second dipole moment, μps, only transitions between 1s‐excitons and 2p‐ or 3p‐excitons are taken into consideration in our model. And the third dipole moment, μfp corresponds to the transition between one of 2p‐ and 3p‐excitons and one of s‐ and d‐excitons with the primary quantum number greater than 3. Because these highly energetic states are partially overlapping within the linewidths, a summation  is required to account for all their contributions to the 3PA. Moreover, only one conduction band and one valence band involved, Equation 2.In order to increase the 3PA coefficient, as shown by Equation μij=〈ψj|e r⇀⋅u→|ψi〉 where u→  is the unit vector for the electric field direction of light, is proportional to the integral over the product of the two wave functions: one for the lower energy state, ψi and the other for the upper energy state, ψj. To compare the difference between the 2D and 3D excitons, we assume the wave functions of hydrogen atoms in the dipole moment calculation, and they are given byq0=1n−1/2 ,n, l, and m are the principle quantum number, the angular momentum quantum number, and the magnetic quantum number, respectively; aB2D and aB3D  correspond to the effective Bohr radius in the 2D and 3D case, respectively; L(x) is the generalized Laguerre polynomial; ϕ is the rotational angle in the orbital; and Y(x) is the spherical harmonics.The electric dipole moment, 2f, the linewidth of the final state for the 3PA transition. In our calculations, we treat this linewidth as an adjustable parameter. By taking experimentally determined values in Table S1 (Supporting Information) with Γf values being in the range from 0.18 to 0.5 eV, the 3PA coefficients are calculated for the near‐infrared spectral region and are shown by the colored curves in FigureAll the parameters in Equation Tablen > 3, and hence they are neglected justifiably in our model. The enhancement in 3PA by 2D excitons results from the two transitions, that is, from 1s‐ to 2p‐(or 3p‐) exciton; and from 2p‐(or 3p‐) state to highly excited Rydberg states. In Table|μ(np)(1s)2Dμ(n′s)(np)2D|2 to |μ(np)(1s)3Dμ(n′s)(np)3D|2 increases dramatically as n′ increases, where n′ > 3.To validate the above theoretical model, we measured the 3PA‐excited photoluminescence (3PPL) from our 2D perovskite samples. These measurements were conducted with two configurations of experimental setup. In the first setup, the samples were examined in detail under a confocal microscope. Their lateral shapes and thicknesses are displayed in Figure hv/Eg ≈ 0.37, where Eg is the bandgap.By comparison, in Figure f, the maximal 3PA coefficient is in the range from 2 to 7 cm3 GW−2 at ≈1100 nm. It is three orders of magnitude greater than the experimental measured value (6 × 10−3 cm3 GW−2) for ZnSe at a wavelength of 1310 nm.3 GW−2 are also larger by three orders of magnitude as well. Furthermore, the maximal 3PA for direct band transitions in Ge has been measured to be 0.27 cm3 GW−2 at 4 µm,3 GW−2 at 1064 nm for a bulk crystal of MAPbCl3 perovskite,Depending on the value of the linewidth, Γ2.n0 = 2.5), as compared to n0 = 1.84 in monolayer MoS2. In addition, our experimental results demonstrate that the interaction length can be scaled up to 300 nm, see Figure S5 (Supporting Information). In the thickness range up to 300 nm, the measured 3PPL is approximately proportional to the thickness. It is expected as the excited volume is proportional to the thickness. Beyond 300 nm, the measured 3PPL becomes saturated with the thickness, due to the fact that the one‐photon absorption and 3PPL is partially overlapping as shown in Figure By utilizing the same 2D excitonic model, we find that the maximal 3PA coefficient of 2D perovskites is at least one order of magnitude greater than the maximal 3PA coefficient of monolayer MoS8H24N2PbBr4 can be fitted by using y = A{1 + 2[σ1sin4(θ + ϕ) − σ2sin2(θ + ϕ)]} with σ1 = −2.67 and σ2 = −2.89 and parameters A, σ1, σ2, and ϕ are defined in ref. (5) at room temperature. It is also in agreement with the tetragonal structure of C8H24N2PbBr4 perovskites in plane. Furthermore, with α3 = 7 cm3 GW−2 and peak irradiance I00 = 40 GW cm−2, we estimate that exciton density is ≈2 × 1011 cm−2, at which other excitonic effects  are not so important.As for polarization‐dependent 3PPL measurements, we define θ as the angle between the light polarization vector and the crystallographic axis when the propagation direction is along the [1 0 0] direction, see Figure S6 (Supporting Information). The measured polarization dependence for C−1 (MW cm−2)−2 = 107 cm3 GW−2 for hydrogen‐like donors in silicon in the THz regime at 10 K.3.3‐value was observed when the linewidth of the excited state was very narrow, ≈50 GHz corresponding to 2.1 × 10−4 eV, obtained at 10 K. Our model depends on the linewidth which, in turn, is related to the sample temperature. Figure S1 (Supporting Information) shows the numerical modeling, supporting that the resonant 3PA coefficient is enhanced by two orders of magnitude if the linewidth is decreased from 0.1 to 0.001 eV. The empirical fitting to the numerical modeling reveals that α3 ∝ Γf−0.9. Although our 3PA coefficients in 2D perovskites are significantly less than hydrogen‐like donors in silicon at 10 K, our values are the largest in the infrared spectral region at room temperature, which are of direct relevance to photonic applications including both optical communications and conversion of sub‐band solar energy to electrical energy.It was reported that the 3PA coefficient is as large as 10 cm33 GW−2, the largest values reported so far for any 2D and bulk crystals in the near‐infrared spectral region at room temperature. Excellent agreement between our theory and our experimental findings unambiguously demonstrate a pivotal role in the enhancement of 3PA played by 2D excitons. Our theory predicts that the resonant 3PA coefficient should be enhanced further by at least two orders of magnitude if the temperature is low such that the linewidth of the final excitonic state is below 1 meV. Our findings are essential for understanding giant 3PA arising from 2D excitons in layered perovskites and may open new pathways for highly efficient conversion from infrared light energy to either electrical energy or higher‐frequency light emission/lasing.In summary, we have presented a quantum perturbation theory on a giant enhancement in 3PA arising from 2D excitons in multilayered crystals of organic–inorganic hybrid perovskites, as compared to bulk crystals of perovskites. The maximal 3PA coefficient in 2D perovskites is predicted to be 2–7 cm4Chemical Materials: Butylamine (≥99%), N,N‐dimethylformamide , dichloromethane , hydrobromic acid , and lead (II) bromide (≥98%) were used. All chemicals were purchased and used without further purification.4H9NH3)2PbBr4 Bulk CrystalSynthesis of 2PbBr4 was synthesized by antisolvent vapor assisted crystallization process. Basically, stoichiometric amount of ammonium salt (C4H9NH3Br) and lead bromide (PbBr2) was dissolved in N,N‐dimethylmethanamide (DMF) to form 0.5 m precursor solution and it was sealed in a beaker surrounded by dichloromethane (CH2Cl2) working as an antisolvent. Plate‐like (C4H9NH3)2PbBr4 crystal precipitates from precursor solution since antisolvent vapor lowers down the solubility of hybrid perovskite precursors. Bulk crystal was obtained by suction filtration and then stored in a glove box for later usage.X‐Ray Diffraction Characterization: Single‐crystal X‐ray diffraction to verify the crystal structure was performed on Bruker AXS D8 Venture which was equipped with a Photon 100 CMOS active pixel sensor detector. A molybdenum monochromatized (λ = 0.71073 Å) X‐ray radiation was used. Data reduction and numerical absorption corrections were performed using the APEX3 suite. All structures were solved by direct methods and refined by full‐matrix least‐squares on F2 using the Bruker SHELXTL software package. The crystal is orthorhombic, space group Cmca. The asymmetric unit contains half a molecule of C8H24N2PbBr4. The cation was disordered into two positions with occupancy ratio = 50:50. Restraints in bond lengths and thermal parameters were applied to the disordered atoms. Final R‐values are R1 = 0.0655 and wR2 = 0.1673 for 2θ up to 56°. The results are summarized in Table S2 (Supporting Information).Sample Fabrication: Layered (C4H9NH3)2PbBr4 flakes were mechanically exfoliated from the above‐described bulk crystal, and transferred to a precleaned glass substrate in the cleanroom where then they were also characterized by both Raman spectroscopy (WITec ALPHA 300R), see Figure S2 (Supporting Information) and atomic force microscopy  in air ambient environment. Finally, perovskite samples were encapsulated with poly(methyl methacrylate) (PMMA) thin film  by spin‐coating  and annealing on a hotplate (SD162) at 90 °C for 10 min.One‐Photon Absorption and One‐Photon‐Excited Photoluminescence (1PPL): A confocal microscope was utilized. Its setup is schematically shown in Figure S3 (Supporting Information). For one‐photon absorption measurements, white light from a light source  was illuminated on the perovskite sample and its transmitted light was channeled to a spectrometer (Ocean Optics QEpro). To measure 1PPL, a CW He‐Cd laser (excitation wavelength: 325 nm) was focused by an objective lens through the glass substrate onto the selected area (≈3 µm in diameter) of the perovskite sample and 1PPL was collected by the objective lens and channeled to the spectrometer.Three‐Photon‐Excited Photoluminescence: The same confocal microscope  was employed with a femtosecond pulsed laser : ≈110 fs and polarization: linear). The orientation of the linear polarized laser pulses was varied by a half wavelength plate. The laser pulses were focused by the objective lens through the glass substrate onto the selected area (≈6 µm in diameter) of the perovskite sample. To obtain the 3PA action coefficient (ηα3) of the perovskite sample, the second harmonic generation (SHG) signal from a BBO crystal was used under the same setup as a reference. The reflected SHG signal by the top surface of the BBO crystal was collected by the same objective lens and was channeled to the spectrometer. For the SHG operated in the tightly focused regime, the analytical analysis can be found in ref. ηα3I00 where I00 is the laser pulse peak irradiance and η is the PL efficiency = 26%.Pave) of the laser beam was measured by a power meter . By considering the laser pulses with Gaussian profiles for both its temporal and its spatial domain, the laser pulse peak irradiance was calculated by I00 = 2(1−R)Pave/(fRRτpπ3/2ω2), where R is the Fresnel reflectance of the interface between the perovskite and substrate, fRR is the pulse repetition rate, τp is the pulse duration (HWe−1M), and ω is the minimum beam waist and was measured to be 3 µm by the laser‐induced damage measurements as described in the following.To precisely determine the excitation laser peak irradiance, the perovskite sample was removed and a glass substrate was placed at the focal point of the objective lens. After transmitting through the glass substrate, the average power (−2. No observable change was found within 5 min exposure times (or 3 × 108 laser pulses). All the measurements presented here were obtained within 5 min exposure times. As the exposure time was extended to 1 h, however, an observable decrease in the 3PPL was detected, see an example in Figure S8 (Supporting Information). After the 1 h exposure time, a dark spot was visible, which is attributed to laser‐induced damage on the sample.To investigate both the photostability and the laser‐induced damage, the 3PPL was measured as a function of exposure time to the laser pulses at a peak irradiance of 52 GW cmp ≈ 150 fs) were generated by an optical parametric amplifier  pumped by a regenerative amplified femtosecond Ti:sapphire laser system , which was seeded by a femtosecond Ti:sapphire oscillator . The laser pulses were focused by a lens onto the sample with a spot diameter of 2ω ≈ 10 µm. The 3PPL at an angle of 3° to the reflected direction was filtered by a short‐pass filter and was recorded with the spectrometer . The average power of the laser pulses was measured by the optical power meter  with the appropriate sensor . More details about this setup can be found elsewhere.For excitation wavelength dependence, 3PPL measurements were conducted using the following apparatus. The excitation laser pulses (repetition rate: 1 kHz, wavelength range: 900–1200 nm, pulse width: 2τThe authors declare no conflict of interest.SupplementaryClick here for additional data file.


The efficacy of such vaccines depends on the effector mechanisms triggered by IgG. The complement system and Fc‐gamma receptors (FcγRs) can potentially play a crucial role in IgG‐mediated immunity against bacterial diseases. However, their relative importance in vivo is unclear, and has been the object of controversy and debate. In this brief study, we have used gene‐targeted mice lacking either FcγRI, II, II and IV or the C3 complement component as well as a novel mouse strain lacking both C3 and FcγRs to conclusively show the essential role of complement in antibody‐mediated host resistance to Salmonella enterica systemic infection. By comparing the effect of IgG2a antibodies against Salmonella O‐antigen in gene‐targeted mice, we demonstrate that the complement system is essential for the IgG‐mediated reduction of bacterial numbers in the tissues.Vaccines can serve as essential tools to prevent bacterial diseases  In fact, protection against oral infection in immunized animals, which is IgG dependent,γRs and the C3 component of the complement system to dissect in vivo the impact of FcγR and of complement in the control of a bacterial infection. Here we show unequivocally that C3 plays an essential role in IgG2a‐mediated host resistance to Salmonella in vivo.In the present study, we have used an intraperitoneal challenge model in a novel mouse strain lacking simultaneously all FcγR KO, FcγR/C3 KO, and C3 KO in the text) and wild‐type (WT) control animals, all in C57/BL6 background, have been used in this study.Three sets of gene‐targeted animals (referred to as FcγRI–/– FcγRII–/– FcγRIII–/– FcγRIV–/– mice (FcγR KO mice) lack simultaneously FcγRI, FcγRII, FcγRIII and FcγRIV, and were generated in the transgenic mouse facility of the Leiden University Medical Center.–/– gene‐targeted mice (C3 KO mice) were kindly provided by Dr Mike Carroll ; they lack the C3 component of complement,γRI–/– FcγRII–/– FcγRIII–/– FcγRIV–/– C3–/– (FcγR/C3 KO mice) lack simultaneously FcγRI, FcγRII, FcγRIII, FcγRIV and C3, and were obtained by crossing the FcγR KO animals with the C3 KO mice. All groups were matched for strain, age and sex. Gene‐targeted mice were bred at the laboratory animal facility (PDC) of the Leiden University Medical Center, the Netherlands, and shipped to the animal facility of the University of Cambridge 2 weeks before the start of the experimental procedures. WT C57/BL6 mice were purchased from Envigo UK. The health status of the animals was monitored over time. Animals tested negative for all agents listed in the FELASA  guidelines for SPF mouse colonies.C57BL/6 FcAnti‐O4 IgG2a monoclonal antibodiesSalmonella enterica serovar Typhimurium (S. Typhimurium) SL1344,50 < 20 Colony Forming Units (CFU) for C57BL/6 mice, was used for infections. Bacteria were grown statically in Luria–Bertani (LB) broth at 37° for 16 hr before being diluted in sterile PBS. S. Typhimurium (~1 × 105 CFU) was injected intraperitoneally (i.p.) into each animal. Twenty‐four hours after infection mice were killed by cervical dislocation. Spleens, livers and mesenteric lymph nodes (MLNs) were removed and individually homogenized in sterile water  using a Stomacher 80 (Seward) homogenizer. Homogenates were plated onto LB agar.8 CFU/ml with 6·75 μg/ml of anti‐O4 monoclonal IgG2a, or with isotype control IgG2a, at 37° with shaking for 30 min; non‐bound antibodies were removed by washing with PBS. The optimal concentration of anti‐O4 IgG2a for opsonizing bacteria was determined as the lowest dilution that would not cause bacterial agglutination.Bacteria were grown statically for 16 hr at 37° in LB broth. Opsonization was performed by incubating bacteria at ~106 CFU of opsonized bacteria. Thirty minutes after infection, peritoneal washes were collected via lavage using 5 ml ice‐cold PBS supplemented with 2% heat‐inactivated foetal bovine serum  and 4 mm EDTA (Sigma Aldrich). Bacteria were enumerated by counting appropriate dilutions in LB agar after treatment of the lavage fluid with 0·1% Triton X (Sigma Aldrich) for 15 min to release intracellular bacteria.Mice were injected i.p. with ~5 × 10All animal experiments were performed at the University of Cambridge in accordance with good animal practice as defined by the relevant international  and local (University of Cambridge) animal welfare guidelines. This research has been regulated under the Animals (Scientific Procedures) Act 1986 Amendment Regulations 2012 following ethical review by the University of Cambridge Animal Welfare and Ethical Review Body (AWERB).10(CFU) recovered from the different groups of mice, the Conover post hoc method for multiple comparisons was applied to the data using the Holm–Bonferroni technique to adjust the P‐values. Comparisons between all the experimental groups were also performed by the Mann–Whitney test (two‐tailed) to the 28 possible pairings of the eight groups.To compare the effect of passive transfer of anti‐O4 IgG2a on log10(CFU) values observed between anti‐O4 IgG‐treated and isotype control‐treated animals of one mouse strain (XΔ) with those observed between anti‐O4 IgG‐treated and isotype control‐treated animals of another mouse strain (YΔ), all the possible comparisons of empirical distributions XΔ and YΔ have been tested using the two‐sample Kolmogorov–Smirnov test with respect to the null hypothesis that XΔ and YΔ are randomly drawn from the same probability distribution.To compare the relative differences in terms of logP < 0·05) using R version 3.3.All statistical tests have been performed at a 5% significance level , six mice lacking the complement C3 component (C3 KO), and six mice lacking both FcγR and C3 (FcγR/C3 KO) were passively immunized with anti‐O4 IgG2a monoclonal antibodies.5 CFU of S. Typhimurium SL1344.Groups of six WT mice, six mice lacking either FcγR KO mice by ~2‐log10 CFU compared with animals treated with control IgG2a. In contrast, both in C3 KO and in FcγR/C3 KO animals, although the variance in recovered CFU from organs in these groups is higher compared with mice lacking only FcγR or WT animals, passive immunization did not result in a significant reduction in the bacterial loads in the liver, spleen and MLNs.Viable bacteria were enumerated in homogenates of the liver Fig. a, the spγR, is necessary for IgG2a‐mediated antibacterial function of the mouse immune system following i.p. challenge.This indicates that C3, but not FcS. Typhimurium within the peritoneal cavity was explored by measuring the amount of viable bacteria recovered from peritoneal washes 30 min after i.p. injection of bacteria pre‐opsonized in vitro with anti‐O4 monoclonal IgG2a or with control IgG2a.The inactivation of S. Typhimurium pre‐opsonized with anti‐O4 IgG2a resulted in a reduction in the viable bacterial numbers harvested from the peritoneal cavity of WT mice and FcγR KO mice. Conversely, pre‐opsonization had no significant effect on the amount of bacteria recovered from the peritoneum of C3 KO and FcγR/C3 KO animals , showing that when mice are injected i.p., FcγRs play a detectable, albeit minor, role in host resistance. No significant difference in the IgG2a‐mediated inactivation of bacteria in the peritoneal cavity was seen between FcγR/C3 and C3 KO animals (P = 0·99), showing no additive effect on the reduction of bacterial numbers in the peritoneum due to the simultaneous lack of C3 and FcγR.The reduction in the recovery of anti‐O4 IgG2a‐opsonized viable bacteria in the peritoneal cavity of FcγR plus the C3 component of complement has enabled us to directly compare the relative importance of FcγR and the complement system in IgG‐mediated host defence against Salmonella infections. We used IgG2a in this study as this subclass has strong binding affinity for all FcγR, including as a monomeric IgG and it’s a powerful complement activator.The availability of mice lacking all four FcγR/C3‐deficient animals, anti‐Salmonella IgG2a was unable to induce a reduction in the number of viable bacteria in the spleen, liver, MLNs and peritoneal cavity. In contrast, the absence of FcγR had little effect on the antimicrobial function of IgG.We showed that both in C3‐deficient and in Fcin vivo in the mouse. Complement is required for antibody‐dependent killing of Salmonella by human blood phagocytesSalmonella in mice lends support to the study of infection in mice as a model of invasive Salmonella disease in man, and possibly of other bacterial infections.The results shown in this brief report conclusively establish the so‐far elusive and controversial role of complement in IgG‐mediated antimicrobial resistance Salmonella strains.The mechanisms by which complement mediates IgG‐induced host resistance to i.p. challenge remain to be elucidated. It is likely that cell‐mediated effector mechanisms are involved, given the reported inability of mouse complement to mediate IgG‐induced serum bactericidal activity against wild‐type Salmonella in mice immunized with live attenuated vaccines. Despite the knownγRI, II and III, but not C3, resulted in impaired resistance in the vaccinated animals. These results illustrate that the contribution of the two downstream components of antibody effector mechanisms, FcγR and complement, varies between models. Although the i.p. challenge model is not the natural route of infection, it may be more representative than the oral route when modelling invasive Salmonella disease in man. We focus our attention only on IgG2a in this study because this subclass has strong binding to all FcγRs, including as a monomeric IgG, and it is also a powerful complement activator.Our previous work has shown that complement does not play an essential role in the resistance against oral challenge with virulent γR is lower than that observed in WT animals, indicating a small but significant contribution of FcγR.The differences in the bacterial numbers seen in the spleen, liver and MLNs are likely to be a consequence to the early events in the peritoneal cavity before the bacteria colonize the systemic compartment. The requirement for complement in the IgG‐mediated reduction in bacterial numbers can be observed in the peritoneal washes 30 min after inoculation. Interestingly and differently from what is seen in the systemic compartment, the effect of IgG‐mediated bacterial killing in the peritoneal cavity of animals lacking Fcin vivo role of complement in the antimicrobial function of IgG against virulent Salmonella can be demonstrated using an i.p. challenge model. This mirrors the dependence on complement of killing Salmonella in human blood.In conclusion, the elusive ORo, SJV, PM conceived and designed the experiments; ORo, CC, YSG, JWCC, PM performed the experiments; ORo, PM analysed the data; YSG, SJV, CAM provided reagents and tools; ORo, Cc, YSG, JWCC, CAM, JSV, PM contributed to the writing of the manuscript.The authors have the following conflict of interest to declare: ORo is currently an employee of the GSK Vaccines Institute for Global Health, part of the GSK group of companies. CC is currently an employee of Summit Therapeutics plc. YSG and CAM for part of the time in which the study was conducted were employees of Novartis Vaccines Institutes of Global Health. This does not alter the authors’ adherence to all Journal policies on data and material sharing.


Ustilago maydis is a model organism for the study of biotrophic plant–pathogen interactions. The sexual and pathogenic development of the fungus are tightly connected since fusion of compatible haploid sporidia is prerequisite for infection of the host plant, maize (Zea mays). After plant penetration, the unfolded protein response (UPR) is activated and required for biotrophic growth. The UPR is continuously active throughout all stages of pathogenic development in planta. However, since development of UPR deletion mutants stops directly after plant penetration, the role of an active UPR at later stages of development remained to be determined. Here, we established a gene expression system for U. maydis that uses endogenous, conditionally active promoters to either induce or repress expression of a gene of interest during different stages of plant infection. Integration of the expression constructs into the native genomic locus and removal of resistance cassettes were required to obtain a wild‐type‐like expression pattern. This indicates that genomic localization and chromatin structure are important for correct promoter activity and gene expression. By conditional expression of the central UPR regulator, Cib1, in U. maydis, we show that a functional UPR is required for continuous plant defence suppression after host infection and that U. maydis relies on a robust control system to prevent deleterious UPR hyperactivation. Ustilago maydis reveals the continuous requirement of the unfolded protein response for biotrophic growth in planta.A new expression system enabling the analysis of stage‐specific gene functions in  Ustilago maydis causes the smut disease on maize (Zea mays) and is a well‐established model organism to study sexual fungal development and biotrophic fungal–plant interactions, but also basic cellular processes such as DNA recombination and vesicular transport  by homologous recombination, conferring carboxin resistance , generating the processed form of the mRNA (e.g. cib1s) that is translated into the active transcription factor. Hence, the effects of genetic UPR activation can be analysed by expression of the cib1s mRNA without drug‐induced side‐effects.et al., et al., et al., et al., et al., et al., U. maydis, the UPR is specifically activated after plant penetration and remains constantly active during all subsequent stages of biotrophic growth inside the host plant  revealed that a functional UPR is not only essential for establishment of biotrophy, but also required for colonization and continuous suppression of the plant defence at later stages of development in planta.Here, we established a system for conditional and stage‐specific gene expression during pathogenic growth of U. maydis mig (maize induced genes) genes that are specifically expressed in planta were used for conditional gene expression during infection  differ in their strength and temporal dynamics of expression. Thus, their promoters represent suitable targets for controlled and plant‐specific expression/overexpression of a gene of interest.In previous studies, promoters of cib1 mRNA (scib1 in the following text), encoding the UPR regulator Cib1, during pathogenic development in planta, we integrated a Pmig2_1:cib1s promoter fusion into the ip locus of the solopathogenic SG200 strain  open reading frame (ORF) that is also present in SG200Pmig2_1:cib1s or from ‘leaky’ Pmig2_1‐driven expression, we used the ∆cib1 background for further analyses. To study if this effect is specific for the ip locus, we generated U. maydis strain FB1∆cib1 ∆mig2_1::cib1s [mig2_1 locus (+NatR)] by replacing the mig2_1 ORF with the cib1s gene. To exclude potential effects of the resistance cassette used for integration, the nourseothricin (NatR) resistance cassette was removed by flippase/flippase recombination target (FLP/FRT) recombination ] were devoid of any detectable cib1s expression integration into (1) the ip locus or the native pit1/2 locus of U. maydis strain SG200∆pit1/2  or (3) hygromycin resistance (HygR) cassettes and (4) after removal of the resistance marker  in comparison to the SG200 (WT) control. Even when expressed from their native genomic locus, transcript levels of both genes were still significantly increased  and only after removal of the resistance marker cassette [pit1/2 locus (−HygR)] was expression of pit1 and pit2 similar to the SG200 (WT) control  or dithiothreitol (DTT), the hypersusceptibility of the FB1∆cib1 progenitor strain was not suppressed, suggesting that Pmig1 and Pmig2_1 are not active in axenic culture  levels. This result suggests that the mechanisms to prevent UPR hyperactivation in planta are robust and efficient in U. maydis, thereby confirming the previous assumption that the UPR is specifically required during biotrophic development in planta  data set published by Lanver et al.  or at later stages during biotrophic growth in planta (UMAG_03597)  or 4 dpi (UMAG_03597)  or if it is also necessary at later stages of pathogenic development, we expressed cib1 under control of the UMAG_12184 and UMAG_03597 promoters . To this end, we replaced UMAG_12184 or UMAG_03597 genes with the cib1 ORF in strain FB2∆cib1  control, demonstrating that cib1 expression driven by either promoter is sufficient to suppress the ER‐stress hypersensitivity of the FB2∆cib1 progenitor strain  or derivatives expressing cib1 under control of conditional promoters (FB1∆cib1 × FB2∆cib1∆UMAG_12184::cib1 or FB1∆cib1 × FB2∆cib1∆UMAG_03597::cib1) were spotted on PD‐CC solid medium  control , virulence was almost completely abolished and no tumours were formed, resembling the ∆cib1 phenotype. By contrast, expression of cib1 under the control of PUMAG_03597 (FB1∆cib1 × FB2∆cib1∆UMAG_03597::cib1) was sufficient to trigger anthocyanin production and the formation of small tumours. This indicates that prolonged expression of cib1 is sufficient to overcome the developmental block of ∆cib1 strains and initiate pathogenic growth in planta.Next, we investigated the effect of plant‐specific repression of rol Fig. C, demonsin planta and assess at which step biotrophic development of the fungus stopped, infected leaves were harvested at 2, 4 and 6 dpi and stained with Chlorazol Black E  infectious dikaryotic filaments penetrated the plant surface via appressoria at 2 dpi, but did not progress further in the plant at later stages (4 and 6 dpi). Consequently, clamp cell formation and extended fungal proliferation was not observed. By contrast, expression of cib1 under control of PUMAG_03597:cib1 (FB2∆cib1∆UMAG_03597::cib1) enabled the fungus to overcome the cell cycle block and induce proliferation, as reflected by hyphal branching and the formation of clamp cells at 4 dpi. However, the subsequent colonization of host tissue by fungal hyphae at 6 dpi appeared strongly reduced in comparison to the controls (FB1 × FB2 and FB1∆cib1 × FB2)  gene expression at 2 dpi ‐related defence responses that are typically suppressed by biotrophic plant pathogens like U. maydis , a compatible interaction appears to be established, allowing further proliferation. This suggests that cib1 is required for plant defence suppression not only at the onset (2 dpi), but also during later (4 and 6 dpi) stages of biotrophic development in planta.Previous studies revealed that plants inoculated with ∆et al., et al., Analysis of gene function typically involves the generation of gene deletion and overexpression strains. To test for functions related to the virulence of plant pathogenic fungi, deletion strains are inoculated into the host plant and scored for development of disease symptoms (Dean U. maydis that enables the study of gene functions at different stages of pathogenic development in the plant. We identified suitable promoters that are active during axenic growth and repressed during pathogenic growth in planta. We demonstrate that promoters (e.g. Pmig2_1 or Ppit1/2), previously used for plant‐specific gene expression, are active during axenic growth and produce considerable amounts of transcripts (up to 800‐fold induced expression for pit2) when integrated into the ip locus or when resistance marker cassettes are located in their vicinity. Proper promoter function required the maintenance of the genomic environment by ‘in locus’ integration (as demonstrated for the mig2_1 or pit1/2 genes) and removal of the resistance marker cassette. RNA‐Seq data suggest that during axenic growth the hsp70 promoter used for the hygR cassette is less active than the gapdh promoter used for the natR marker  or of highly expressed resistance marker genes might affect the chromatin structure and thus derepress silent promoters in their vicinity by providing access for the transcriptional machinery to normally silenced, inaccessible genomic regions.Similar to the cib1s using the mig1 or mig2_1 (max. FPKMs 2393 at 4 dpi) promoter did not result in alterations of disease symptoms. As the mig1 promoter is highly active in planta   light medium  analysis was performed as described  were PCR amplified from genomic DNA, adding a SfiI restriction site to the 5′ of the left border (LB) and a BamHI  or KpnI (for UMAG_03597) restriction site to the 3′ end of the right border (RB). The gene of interest  was PCR amplified from genomic DNA or from plasmid Pcib1:cib1s, respectively, adding SfiI restriction sites to the 5′ and 3′ ends. The HygR cassette was amplified from plasmid pUMa1442 adding a BamHI (for UMAG_12184) or KpnI restriction site (for UMAG_03597) to the 3′ end and a SfiI restriction site to the 5′ end. The resulting DNA fragments were ligated to obtain R‐RBLB‐GOI‐Hyg or R‐RBLB‐GOI‐Nat and integrated into the pCR2.1 TOPO vector (Invitrogen) or the pJet1.2 vector  according to the manufacturer’s instructions to generate plasmids pCR2.1 PUMAG_12184:cib1(NatR), pCR2.1 PUMAG_03597:cib1(HygR), pJet1.2 Pmig2_1:scib1(NatR) and pJet1.2 Pmig1:scib1(NatR).For gene deletions, a PCR‐based approach and the mig2_1:cib1s construct for ip locus integration, the vectors pMig2_1:clp1 and pRU11‐cib1s were cut with NdeI and EcoRI. The resulting 2.0 kb scib1 fragment of pRU11‐cib1s  with a 14 h (28 °C) day and 10 h (22 °C) night cycle. Symptoms were scored according to disease rating criteria reported by Kämper et al.  The gene of interest (GOI) is deleted from its native genomic locus. (2) The GOI is integrated into the genomic locus of the conditionally expressed gene, thereby replacing the native gene. (3) The resistance marker  is removed using the FLP/FRT recombination system.Click here for additional data file.Fig. S2 ∆UMAG_12184 and ∆UMAG_03597 strains do not show increased sensitivity to cell wall or endoplasmic reticulum (ER) stresses. Cell wall and ER stress assays, and tests for filamentous growth of strains SG200, SG200∆cib1, SG200∆UMAG_12184 and SG200∆UMAG_03597. Serial 10‐fold dilutions were spotted on YNB‐Glucose solid medium supplemented with Congo Red (100 µg/mL) or Calcofluor White (50 µM) to induce cell wall stress, and on YNBG solid medium supplemented with tunicamycin (TM) (1 µg/mL) or dithiothreitol (DTT) (1 mM) to induce ER stress. Cells were spotted on charcoal‐containing potato dextrose solid medium to induce filamentous growth. Pictures were taken after 48 h of incubation at 28 °C.Click here for additional data file.Table S1 Strains used in this studyClick here for additional data file.Table S2 Primers used in this studyClick here for additional data file.Table S3 Plasmid used in this studyClick here for additional data file.


AFHs are synthesized with modified galvanic displacement strategy in nonaqueous solution. To eliminate the restriction of lattice mismatch between Fe and Ag, amorphous Fe@Fe3O4 nanoparticles (NPs) are selected as seeds, meanwhile, reductive Fe can reduce Ag precursor directly even at as low as 20 °C without additional reductant. The thickness of the Fe3O4 shell can influence the amorphous properties of AFHs, and a series of Janus‐ and satellite‐like AFHs are synthesized. A “cut‐off thickness” effect is proposed based on the abnormal phenomenon that with the increase of reaction temperature, the diameter of Ag in AFHs decreases. Because of the interphase interaction and the coupling effect of Ag and Fe@Fe3O4, the AFHs exhibit unique optical and magnetic properties. This strategy for synthesis of monodisperse heterostructures can be extended for other metals, such as Au and Cu.The unique physicochemical properties of silver nanoparticles offer a large potential for biomedical application, however, the serious biotoxicity restricts their usage. Herein, nanogalvanic couple Ag–Fe@Fe Considering that amorphous materials have been demonstrated as an efficient way to nonepitaxial growth heterostructures with large lattice mismatches,3O4 NP was selected both as the reductant and as seeds, in which the zerovalent Fe has strong reduction activity, and the amorphous shell can not only protect the structure, but also eliminate the restriction of large lattice mismatch between Fe and Ag. Moreover, the synthesis of novel metal oleate complex as precursor, silver oleate (AgOA) as an example, allows the complete connection of AgOA and oleylamine‐coated Fe@Fe3O4 NPs in the nonaqueous solution to make sure that the reaction happened homogenously. Both Janus‐ and satellite‐like heterostructures with controlled Ag sizes can be synthesized by carefully tuning the reaction temperature or the shell thickness, resulting in the controlled optical and magnetic properties.Galvanic displacement is a well‐known method to prepare bimetallic heterostructures with core–shell, hollow, and nanocage structures.23O4 heterostructures (AFHs) involves the initial synthesis of AgOA complex as precursors, the synthesis of amorphous Fe@Fe3O4 NPs as seeds, and the subsequent galvanic displacement between Fe@Fe3O4 and AgOA. As illustrated in Scheme3O4 NPs were prepared via the pyrolysis of iron pentacarbonyl (Fe(CO)5) in the 1‐octadecene (ODE) and oleylamine (OAm).3O4 NPs. However, the oleylamine‐coated Fe@Fe3O4 NP is hard to connect with the conventional oleophobic silver compound like silver nitrate (AgNO3). In order to solve this problem, the oleophilic AgOA was prepared through an easy but high‐efficiency two‐phase method, as illustrated in Figure S1 (Supporting Information). According to the periodic law, Ag can be recovered from its ionic states by displacing with active metal, such as Fe. When the fresh prepared AgOA meets the Fe@Fe3O4 in solution, the recovered Ag just attached on the amorphous surface of the Fe@Fe3O4 NPs, resulting in the Janus‐like Ag–Fe@Fe3O4 heterostructures (JAFHs). The size of Ag islands can be tuned depending on the reaction temperature. Note that if the inert atmosphere is introduced and kept throughout the reaction, another structure distinguished from Janus one, i.e., the satellite‐like Ag–Fe@Fe3O4 heterostructures (SAFHs) with several Ag NPs distributed on the surface of Fe@Fe3O4 NPs can be obtained. It is worth noting, in both of the cases, that the amorphous iron oxide shell plays a critical role for grafting of Ag NPs by avoiding the lattice mismatch restriction between Ag and Fe3O4. As, it is well known that the lattice mismatch is the most restriction factor for the fabrication of heterostructures.3O4 NPs with similar sizes and morphologies were synthesized and characterized  for the contrast experiments.The synthetic procedure of Ag–Fe@FeFigure3O4 NPs. The obvious gap between Fe core and Fe3O4 shell could be observed, indicating that the reaction happened between Ag+ and the internal Fe, that the different diffusion rate between the Fe consumption and the Fe3O4 generation process generated the gap according to the Kirkendall effect.3O4 NPs. The fringes with a separation of 2.4 and 2.0 Å corresponded to (111) and (200) reflection, respectively, which is allowed for face center cubic (fcc) lattice of Ag , Ag was also produced but no heterostructures were formed due to the lattice mismatch. As the results described above, it can be concluded that the amorphous character of Fe@Fe3O4 NPs plays a critical role in the fabrication of heterostructures by eliminating the lattice mismatch.The typical morphology of JAFHs was characterized by transmission electron microscopy (TEM). As shown in onded to 11 and , indicating that the Fe3O4 shell cannot recover the Ag from AgOA. When AgOA mixed with OAm only in hexane or in ODE, there were also no Ag recovered even heated to 100 °C. It is generally accepted that the galvanic displacement could be happened when relatively active metal directly contact with the inert metal ion as the electron can transfer directly from the metal to the ion. However, in this study, we found that galvanic displacement also can happen even though the Fe and AgOA were divided by thin Fe3O4 shell. Actually, the Fe3O4 shell was naturally oxidized from Fe core, thus there may be natural transition of elementary composition from Fe to Fe3O4 in the interface with rich oxygen vacancy, and also generate the efficient electron density gradient, as illustrated in Figure S5 (Supporting Information). The thinner the shell is, the higher efficient electron density of the surface has, and easier for the galvanic displacement to happen, vice versa. As a result, the Fe@Fe3O4 as a whole, the efficient surface electron density will influence the displacement ability to the AgOA. However, when the shell thickness reached to the distance, the low efficient surface electron density will cut off the displacement. The pinhole on the oxide shell may also contribute to the electron transfer at the initial stage of the reduction.In order to identify which constitute contributes to the Ag recovery, we synthesized hollow Fe3O4 NPs  in XRD patterns , by carefully controlling the oxidation process. After mixing with AgOA at 40 °C for 3 h, SAFHs with several Ag NPs attached on each NP  were synthesized using the seeds with 1.1 nm shell, while JAFHs  were obtained in the case of the seeds with 2.2 nm shell. However, when the average shell thickness was enlarged to 3.1 nm, the silver NPs became smaller and about 68% of the Fe@Fe3O4 NPs without Ag dots . It is in good agreement with the shell thickness distributions  that more than 65% of Fe@Fe3O4 seeds had the shell thicker than 3 nm. These phenomena indicate that the Fe3O4 shell can inhibit the reaction by increasing the thickness and decreasing the efficient electron density on surface. The thicker of the shell is, the lower the efficient electron density has and the harder for the galvanic displacement to happen. It is easy to understand that there must be a critical thickness of the shell that determines whether galvanic displacement can happen. We define this shell thickness as “cut‐off thickness,” which means that the reaction would be cut off when the shell reaches to that thickness. The “cut‐off thickness” maybe different depending on the reactants, medium, and conditions. And, the “cut‐off thickness” in this reaction system may be about 3.0 nm. Regardless of the shell thickness of the Fe@Fe3O4 seeds, almost all particles had the shell thicker than 3.0 nm after the reaction .In order to examine the influence of shell, we further synthesized the Fe@Fe3O4 NPs . With the reaction going on, Ag islands were grown larger and more Fe@Fe3O4 NPs were decorated with Ag . After 150 min, most of Fe@Fe3O4 NPs were attached with Ag NPs , but not uniform, mainly because the nucleation did not start at the same time considering the various conditions of Fe@Fe3O4 seeds. After 180 min, Ag islands grew uniform , but did not grow larger even for a longer time .To understand the nucleation and growth mechanism, we monitored the synthetic progress at an interval of 30 min from the beginning of the reaction, and the TEM images are shown in Figure S8 (Supporting Information). When the reaction started for 10 min, a handful of small Ag NPs were detected on the Fe@FeScheme3O4 NPs, the nucleation and growth of Ag. First, when dissolved in the low‐polar solvent, the hydrophobic AgOA molecules attached to the oleylamine‐coated Fe@Fe3O4 NPs by van der Waals' force. The displacement first happened on the relatively thinner site of the shell with higher efficient electron density. The irregular arrangement of the iron and oxygen atoms in amorphous iron oxide shell helps the Ag atoms root in the surface, as illustrated in Figure S9 (Supporting Information), however, the crystalline shell with large lattice mismatch would not allow the Ag attach on the surface. Once the Ag nucleated and rooted in the amorphous shell, the subsequently reduced Ag atoms would epitaxially grow around the Ag nucleus layer by layer. Meanwhile, the Fe core lost electron and was oxidized to be Fe3O4 and diffused to the shell, resulting in the gradually thicker Fe3O4 shell and the reduced Fe core. When the thickness of the Fe3O4 shell reached to the “cut‐off thickness,” the reaction would be stopped and the size of Ag would be fixed, resulting in the formation of AFHs.From the process mentioned above, the mechanism for the formation of AFHs through galvanic displacement can be summarized, as illustrated in 3O4 seeds were rapidly used as synthesized in their original solution under Ar atmosphere to avoid oxygen. And, a degassed and Ar‐protected long needle syringe was used for the AgOA injection. As can be seen in Figure3O4 NPs were obtained. The HRTEM image exhibits the high crystalline Ag islands decorated on the amorphous part of the partly crystalline Fe3O4 shell as well as the clear interface, which is in good agreement with the phenomenon in the Janus‐like structures. The lattice spaces of 2.30 and 2.05 Å were corresponding to (111) and (200) planes of Ag, while the 2.53 Å is corresponding to the (311) plane of Fe3O4. The obvious widening diffraction peaks in XRD patterns  revealed the small scale of Ag domain.Accordingly, if the shell can be controlled thinner, there may be multiposition for the Ag nucleation and growth in the displacement reaction. The key point is to avoid oxygen to get in the system. We made efforts to exclude the oxygen in the AgOA solution by vacuuming the solution with a vacuum pump and protecting it under argon (Ar) atmosphere, and the Fe@FeThe reaction temperature also can influence the size of Ag in the satellite‐like structures. As shown in Figure 3O4, JAFHs  measurement), the absorption maxima of the SAFHs shifted to longer wavelengths (415 nm), which may be due to the increase of symmetry of the satellite‐like structure. According to the plasmon mode coupling theory, the symmetric combination results in lower energy (redshift), while the antisymmetric combination results in higher energy (blueshift).] In addition to the major peak, the SAFHs also exhibited a broad shoulder peak in the range of 450–500 nm that may be attributed to the near‐field coupling between the adjacent Ag islands on each heterostructure.3O4 domain through the interface. Furthermore, the influence of Ag sizes to the adsorption also had been scrutinized. The absorption peak shifted to shorter wavelengths with the decrease of Ag sizes in JAFHs . Similar result was also detected in the SAFHs .The slight shift may contribute to the little changes of Ag diameters in the satellite‐like heterostructures. In turn, the obvious shift of the SPR absorption of the heterostructures also confirms the strong connection and interaction of the Ag and Fe@Fe3O4 NPs in the heterostructures.The surface plasmon resonance (SPR) adsorption of Fe@Fe3O4 domain. The magnetic hysteresis loops of the pure Fe@Fe3O4, JAFHs, and SAFHs are compared in Figure Ms) of 32.9, 16.5, and 11.4 emu g−1, respectively, with the coercivity (Hc) of 95, 120, and 60 Oe  of JAFHs is estimated to be 128.3 mm−1 s−1 , zerovalent Fe and Ag in JAFHs and SAFHs in cytoplasm constitute galvanic couples, the accelerated oxidation anodic Fe can protect the cathodic Ag from being oxidized, which will reduce the Ag+ release from JAFHs and SAFHs, so as to reduce the cytotoxicity. This is also being proved by the Ag+ release detection in phosphate buffer saline (PBS) by inductively coupled plasma mass spectrometry (ICP‐MS). As shown in Figure + release compared with pure Ag and Ag–Fe3O4 NPs, this is consistent with the MTT results.In order to test the cytotoxicity of the synthesized samples, 4T1 mouse breast tumor cell line, RAW264.7 mouse macrophage cell line and LO2 normal human liver cell line were selected for 3‐‐2,5‐diphenyltetrazolium (MTT) experiment. As shown in 3) as well as much less active copper oleate complex (Cu(OA)2), compared with AgOA, were selected for the experiment. Au–Fe@Fe3O4 heterostructures were easily obtained at room temperature , while Cu–Fe@Fe3O4 heterostructures can hardly be generated until the temperature increased to 120 °C for 5 h . However, the pure Cu(OA)2 cannot be decomposed by oleylamine at the same condition , indicating that the Cu was recovered by Fe@Fe3O4.To examine the general applicability of the method reported here, the more active gold oleate complex (Au(OA)33O4 plasmonic–magnetic heterostructures using amorphous Fe@Fe3O4 NPs as seeds as well as reductive agent. Under this strategy, the replacement could be carried out at as low as 20 °C without additional reductant. The “cut‐off thickness” effect was proposed based on the abnormal phenomenon that with the increase of the reaction temperature, the sizes of Ag domain in heterostructures were decreased. Under the guidance of “cut‐off thickness” effect, a family of uniform Janus‐ and satellite‐like AFHs with tunable Ag sizes were obtained. Owing to the strong interphase interaction and the coupling effect of Ag and Fe@Fe3O4, the heterostructures exhibited tunable optical properties and favorable magnetic properties, which were further reflected in the good performance of plasmon‐enhanced TPF and MR dual‐modal imaging. The galvanic couples of Ag and Fe effectively reduce the cytotoxicity of JAFHs and SAFHs by reducing Ag+ release from cathodic Ag, which would open the door for Ag‐based application in biomedical studies.In summary, a facile‐modified galvanic displacement method was generated to synthesize monodisperse Janus‐ and satellite‐like Ag–Fe@Fe4Materials: AgNO3 (≥99.9%), ODE (90%), ammonium bromide , borane‐tert‐butylamine complex , and 4‐nitrobenzoic acid  were purchased from Alfa Aesar. OAm (70%), iron pentacarbonyl (Fe(CO)5, 99.9%), and dimethyl sulfoxide (DMSO) were purchased from Sigma‐Aldrich. Gold chloride trihydrate  was purchased from STREM chemicals. Sodium oleate  was purchased from TCI. Deionized water  was refined from a Milli‐Q Ultrapure Water System. Other solvents including normal hexane , ethanol (AR grade), and chloroform  were purchased from Beijing Chemical Works. All chemicals were directly used without further purification.Measurements: XRD patterns were obtained using a Rigaku DMAX‐2400 X‐ray diffractometer with Cu Kα radiation (λ = 1.5405 Å). TEM images were taken on an FEI Tecnai T20 microscope with an acceleration voltage of 200 kV, and HRTEM images were obtained on an FEI Tecnai G20 F30 microscope and operated on an acceleration voltage of 300 kV. UV–visible spectra were measured using a UV‐2550 Shimadzu UV–vis spectrophotometer. Magnetic study was carried out on a Quantum Design PPMS XL with fields up to 30 kOe at room temperature. The concentrations of Ag and Fe in the samples were quantified using an ICP‐AES .Methods—Synthesis of Metal Oleate or Oleylamine  Precursor: Typically, 0.25 mmol of AgNO3 was dissolved in the mixture of DI water (10 mL) and ethanol (20 mL), followed by adding hexane (30 mL) to form a two‐phase solution . 3 mL fresh NaOA (0.1 m) aqueous solution was added dropwise under vigorous stirring for 30 min. Then, the mixture was stood for the stratification, the AgOA was dissolved in upper layer with white color . Finally, the AgOA solution was separated and washed with DI water for two times in a separating funnel  for further use. Copper oleate precursor (Cu(OA)2) was synthesized with Cu(NO3)2 instead of AgNO3 in the above process. The green color upper solution indicated the generation of Cu(OA)2. Gold oleylamine precursor (Au(OAm)3) was obtained with the similar approach using HAuCl4·3H2O and OAm instead of AgNO3 and NaOA. The molar rate of Au3+ and OAm was 1:3, and saffron yellow upper solution indicated the formation of Au(OAm)3.3O4 NPsMethods—Synthesis of Fe@Fe: The amorphous Fe@Fe3O4 NPs (A‐FeNPs) were synthesized by the modified method reported by Peng et al.[[qv: 16a]] Briefly, in a four‐necked flask, ODE (20 mL) and OAm (0.5 mL) were mixed under vigorous magnetic stirring and degassed with a vacuum pump at 100 °C for 1 h and then ventilated argon (Ar). As soon as the mixture was heated to 180 °C, iron pentacarbonyl (Fe(CO)5, 0.7 mL) was quickly injected into the flask. After 30 min, the products were cooled down and washed with hexane and ethanol for three times and finally redispersed and stored in hexane. The Fe@Fe3O4 nanocrystals (FeNCs) were synthesized with the similar progress, except NH4Br (8 mg) added at the beginning.[[qv: 16b]] In order to examine the influence of the thickness of iron oxide shell to the JAFHs, the as‐prepared A‐Fe was exposed to air and heated at 80 °C for 0, 6, and 12 h, resulting in the average shell thickness of about 1.1, 2.2, and 3.1 nm, respectively. The hollow Fe3O4 NPs (h‐Fe3O4) were synthesized by heating the Fe@Fe3O4 NPs at 100 °C for 4 h and then heated to 250 °C for 10 h.Methods—Synthesis of JAFHs: Typically, AgOA solution  was added in a four‐necked flask with a magnetic stir bar. After 20 min stirring under Ar at room temperature, 2 mL of amorphous Fe@Fe3O4 NPs (0.25 m) was injected. The color of the mixture was changed to dark brown gradually, indicating the generation of Ag. Three hours later, the products were collected and washed for three times and finally redispersed in 10 mL hexane and protected under Ar atmosphere.Methods—Synthesis of SAFHs: First, ODE (5 mL) was mixed with the as‐prepared AgOA solution  and then degassed under Ar at 30 °C for 30 min in a four‐necked flask. Next, Fe(CO)5 (100 µL) was quickly injected into the adequately degassed and Ar‐protected solution (15 mL ODE and 0.5 mL OAm) at 180 °C and stirred for 30 min. As soon as the mixture was cooled down to 40 °C, the Ar‐protected AgOA solution was quickly injected into the as‐prepared amorphous Fe@Fe3O4 solution under Ar and reacted for 3 h.+ Release MeasurementMethods—Ag: The Ag+ release from the samples was monitored by ICP‐AES. 1 mL of samples at Ag concentration of 2 mg mL−1 was sealed into a dialysis bag with the cutoff molecular weight of 3000 Da. These dialysis bags were immersed into 40 mL PBS (pH = 7.0) in a shaking incubator at 37 °C with a shaking speed of 150 rpm. At given intervals, 1 mL of the solution was collected for Ag concentration (µg mL−1 (ppb)) analysis in ICP‐AES. And then, 1 mL of fresh PBS was added.Methods—Cell Culture: All the cell lines were purchased from Shanghai Institute of Cells, Chinese Academy of Science. 4T1 mouse breast tumor cell line and LO2 normal human liver cell line were cultured in Roswell Park Memorial Institute (RPMI) 1640  supplemented with 10% heat‐inactivated fetal bovine serum (FBS), 1% streptomycin/penicillin at 37 °C in humidified atmosphere with 5% CO2. RAW264.7 mouse macrophage cell line was cultured in the same condition in high‐glucose Dulbecco's modified Eagle medium (DMEM) .Methods—Cytotoxicity Measurement: 4T1 mouse breast tumor cell line, RAW264.7 mouse macrophage cell line, and LO2 normal human liver cell line were seeded in 96‐well plates (5000 cells in 100 µL of corresponding culture medium per well). After incubated for 24 h, the mediums were replaced with fresh corresponding culture medium containing A‐Fe, Ag, Ag–Fe3O4, JAFHs, or SAFHs at Fe concentration (for A‐Fe) or Ag concentration  of 6.25, 12.5, 25, 50, 100, 200, 400, 500, and 600 µg mL−1, respectively. After incubated for another 24 h, the cells were mildly washed with PBS for three times, then coincubated with MTT solution (0.5 mg mL−1 in 100 µL of PBS) at 37 °C for 4 h. Then, the MTT solution was replaced by 150 µL of DMSO, and the absorbance was tested on a microplate reader  at the wavelength of 490 nm.The authors declare no conflict of interest.SupplementaryClick here for additional data file.


The Michaelis–Menten analysis for kinetics of macromolecular catalysis is also suitable for hinokitiol-supported iron import. To compare hinokitiol to DMT1 relative to other metal ions that DMT1 can transport, we employed an organic extraction procedure with which we initially matched the results obtained for Fe2+, Fe3+ and Mn2+, and then showed that multiple other cations were unlikely to enter via hinokitiol. The small chelator thus shares some functional properties with DMT1, but distinct difference were also noted.Hinokitiol, a natural lipophilic chelator, appears capable of replacing several iron transporters after they have been genetically ablated. Divalent metal-ion transporter (DMT1) is the major iron importer in enterocytes and erythroblasts. We have compared DMT1 and hinokitiol in multiple fashions to learn if the smaller molecule is a suitable substitute using two HEK293 cell lines engineered to overexpress different isoforms of DMT1. Both the macromolecule and the lipophilic chelator enable import of ferrous ions into HEK293 cells. Hinokitiol also mediates ferric ion import but DMT1 cannot do so. While DMT1 can also import MnThe online version of this article (10.1007/s10534-019-00207-2) contains supplementary material, which is available to authorized users. A recent, ingenious proposal  could be applied to hinokitiol and what other metal ions could rely on hinokitiol to gain entry.Over-expression of DMT1 in HEK293 cells . This study asks how well hinokitiol recapitulates iron transporter(s) (here DMT1) for which it could be substituted. So issues are for which metal ion gradients can the smaller molecule allow entry into cells and how well can the Km concept apply to it.If one advocates using hinokitiol to replace defective DMT1, then a direct comparison of functioning DMT1 and hinokitiol needs to be made. We set out to do so for the most physiologically relevant metal ion, FeFigure 3+ unless supported by a ferrireductase to convert it to Fe2+ /kf. This result assumes that [S] > [E] so that one sees a saturable process but the left hand side of Eq. Equation mentclass2pt{minim2+ for a channel like hinokitiol could also follow Michaelis–Menten kinetics. We therefore examined this response for hinokitiol with time dependency graphs like those in Fig. This property of transporters encouraged us to compare hinokitiol head-to-head to DMT1 in such an assay of transport or catalysis as one can argue that the concentration response to Fe2+] was varied  and Vmax (3.5 vs 4.3) are mostly attributable to the inverse weighting in a reciprocal plot so we chose to use the latter two values in Table 2 vs 455 ry Table . Noting 2+ entry into cells provided that there is a concentration gradient of the substrate across the plasma membrane. They also show that saturable kinetics apply to using hinokitiol in place of DMT1 with Fe2+ entry into cells. Hinokitiol can also do this for Fe3+ entry unaided whereas DMT1 requires reduction of the substrate prior to entry and again a Km analysis is applicable. Remarkably, one can also hold the initial concentration of ferrous or ferric ions constant, vary the hinokitiol concentration and fit the results to Michaelis–Menten kinetics. The saturable behavior could be due to the symmetry of E and S on the left hand side of Eq. The results with hinokitiol extend those obtained by Grillo et al. , demonst59Fe2+, 59Fe3+ and 54Mn2+ analyses.It is also important to learn how hinokitiol interacts with other metal ions, given their physiological and toxicological implications. Rather than develop assays for radioactively tagged metal ions, we adapted another assay. Grillo et al.  also sho2+ without an excess of the chelator so that it will not appear in the organic phase. This expectation was tested and met . First, however, we examined the concentration dependence in the assay above to assure that the higher concentrations needed for ICP-OES should produce interpretable results. Supplementary Fig. S2 portrays these analyses with the data indicating that C2d binds neither ferrous (S2a) nor ferric (S2b) ions consistent with Figs. 2+ analysis. The bar graph like Fig. 2+ concentrations. We surveyed some of the metals ultimately tested at this level (10 µEq/L with 20 µM hinokitiol) and were usually unable to detect partitioning (data not shown). Because the Burke group found that they had to use higher, non-physiological concentrations of most other metals, we also tested similar high levels; data for Cd2+ are representative . The results show that statistically significant amounts of this toxic ion appear in the organic phase when hinokitiol is present; but the actual quantity is close to minimal. These data are summarized in Table 2+; they indicate that accumulation of toxic levels of either metal ion due to hinokitiol substituting for DMT1 is not a likely problem.The next experiments tested whether the results for ICP-OES were equivalent to those with radioisotope tags for the three metal ions already examined. Supplementary Fig. S3 illustrates results for Fe59Fe2+ that is extracted with C2d is 0; while with hinokitiol it is 34.7 ± 0.5% . To the extent that one can rely on this partition assay, our data do not support physiological transport of the other metal ions tested in Table 3+ could pose an issue relative to iron overload but clearly Fe2+ import by hinokitiol is more effective than for Fe3+ so a little caution in monitoring iron status while using hinokitiol appears to be what this property calls for rather than excluding the use of hinokitiol.There have been many studies showing that DMT1 is relatively promiscuous in terms of which metal ions it can transport  and 54MnCl2 (NEZ040) were purchased from Perkin Elmer  supplied in 0.5 M HCl. Hinokitiol and C2doxyhinokitiol were synthesized in the Burke laboratory as described  or just its solvent  then grown fully to confluence (24–48 h) all as described before  unless otherwise specified; while hinokitiol and C2d were 500 nM. As iron binding of hinokitiol is tridentate, the concentrations chosen provided a modest excess of Fe. When Fe2+ was present, this status was achieved by having a  × 10 excess of FeSO4 relative to the 59Fe and a  × 10 excess of ascorbic acid relative to the FeSO4. When Fe3+was present, this status was achieved by having a  × 10 excess of ferric ammonium citrate (FAC) present relative to the 59Fe. For 54Mn experiments, [Mn] = 350 nEq/L was achieved by adding MnCl2. All extractions involved 1 mL for the aqueous phase and an equal volume of hexane. Tubes were vortexed at low speed, allowed to settle and aliquots of the two phases removed and allowed to dry in a fume hood then counted in a LKB Compugamma 1282 γ counter.For 4 (with a  × 10 excess of ascorbate), FAC, MnCl2, Pb(CH3CO2)2, Cd(CH3CO2)2, CoCl2, NiCl2, VOSO4, Cu(CH3CO2)2 with a  × 10 excess of histidine, Zn(CH3CO2)2 and CuSO4 with both histidine and ascorbate in  × 10 excess. Extractions were performed as above but metals were determined using ICP-OES  after drying the two phases in a fume hood and dissolving an aliquot of each in 3 N HCl/10% trichloracetic acid.For nonradioactive experiments, the metals were at 10 or 100 μEq/L in HBSS; while hinokitiol and C2d were 20 or 200 μM, respectively, leading to a slightly larger excess of metal ion than in the radioactive extractions. Metals were provided as FeSOP < 0.05 was treated as significant, we report P where possible. We used no correction for multiple statistical tests when making them because we tested only experimental hypotheses but readers should be aware that this approach was employed.Statistical analysis were done with Stata version 15.1 . Although Supplementary material 1 (DOCX 42 kb)Below is the link to the electronic supplementary material.