Au NR Research Articles

End-to-end and side-by-side assemblies of gold nanorods induced by dithiol poly(ethylene glycol)

The assemblies of gold nanorods (Au NRs) exhibit unique properties distinct from the isolated Au NR. We report an effective and simple method for the end-to-end (E-E) and side-by-side (S-S) assemblies of Au NRs with a molecularly defined nanogap (1–2 nm) only in the presence of dithiol poly(ethylene glycol) (HS-PEG-SH). The assembled methods need neither the pH value adjustments nor the addition of other organic solvent. With increasing amount of dithiol molecules, assembled modes of Au NRs experience an interesting procedure, changing from E-E to S-S orientation. The experimental results indicate that when the concentration of HS-PEG-SH is less than 0.25 μM, electrostatic repulsion of positive-charged CTA+ is stronger than the affinity of the Au-S binding, resulting in the E-E oriented assembly. Otherwise, the S-S oriented mode is predominated. The current assembled method will be potentially useful for the optoelectronics and biomedical engineering.

A facile strategy to functionalize gold nanorods with polycation brushes for biomedical applications

The fabrication of highly efficient nonviral gene carriers with low cytotoxicity remains a challenge in gene therapy. This paper reports a facile strategy to combine the advantages of gold nanorods (Au NRs) and polycations through surface functionalization. Different Au NR carriers with a controlled amount of poly(2-(N,N-dimethyl amino)ethyl methacrylate) (PDAEMA) brushes could be readily synthesized via surface-initiated atom transfer radical polymerization to achieve optimized nanohybrids for gene transfection. The obtained gene carriers demonstrate much higher gene transfection efficiency and lower cytotoxicity compared with polyethylenimine (∼25kDa, gold standard of nonviral gene vector) in both COS7 and HepG2 cell lines. In addition, the potential of the PDMAEMA-grafted Au NR carriers to be utilized as a computed tomography contrast agent for the imaging of cancer cells has also been investigated. This strategy may realize the gene therapy and real-time imaging within one nanostructure and facilitate biomedical applications.

Gold nanorod enhanced organic photovoltaics: The importance of morphology effects

Organic photovoltaic devices with a 30% improvement in power conversion efficiency are achieved when gold nanorods (Au NR) are incorporated into the active bulk heterojunction (BHJ) layer. Detailed analysis of the system is provided through microscopy, device characterization, and spectroscopy, demonstrating that the enhancement effects are predominantly caused by induced morphology changes in the BHJ film rather than plasmonic effects. Wide angle X-ray diffraction provides evidence that the nanorods loaded into the BHJ film have an effect on polymer crystal orientation, leading to a systematic performance increase in the devices as a result of both internal and external efficiency improvements.

Plasmonic Property and Stability of Core-Shell Au@SiO2 Nanostructures

Au nanorod (Au NR) is one of the most studied colloidal nanostructures for its tunable longitudinal surface plasmon resonance (SPRL) property in the near infrared region. And surface coating Au NRs into core-shell nanostructures is particularly important for further investigation and possible applications. In this paper, Au NRs colloids were synthesized using an improved seed method. Then as-prepared Au NRs were coated with SiO2 to form a core-shell nanostructure (Au@SiO2) with different shell thickness. And the influence of SiO2 shell on the SPRL of Au NRs was investigated based on the experimental results and FDTD simulations. Under the 808 nm laser irradiating, the stability of Au@SiO2 was studied. Compared with Au NRs, the Au@SiO2 is stable with increasing laser power (up to 8 W), whereas Au NRs undergo a shape deformation from rod to spherical nanoparticle when the laser power is 5 W. The high stability and tunable optical properties of core-shell structured Au@SiO2, along with advantages of SiO2, show that Au@SiO2 composites are promising in designing plasmonic photothermal properties or further applications in nanomedicine.

Rod-shaped Au@PtCu nanostructures with enhanced peroxidase-like activity and their ELISA application

Pt and its based alloy nanoparticles (NPs) have been reported to demonstrate novel enzyme-like activities. Varying composition is very important to realize the optimization of their functions through the tuning of electronic structure. In this paper, our effort is focused in this direction by tailoring the electronic structure of Pt NPs via alloying with copper. Using gold nanorod (Au NR) as core, a simple method to prepare PtCu alloy shell is developed (termed as Au@PtCu NR). The introduction of copper could result in endcap-preferred growth mode owing to the lattice mismatch between alloy shell and the Au core. The variation in the electronic structure changes the substrate affinity, and enhanced affinity was found for H2O2. Besides, the designed Au@PtCu nanostructures have realized spatial separation of catalytic and recognition sites. Binding of recognition antibodies had negligible effect on their catalytic activity. Based on their peroxidase-like activity, a highly sensitive detection of human immunoglobulin G (IgG) was demonstrated in a direct enzyme-linked immunosorbent assay (ELISA) mode. The detection limit can be as low as 90 pg/mL.

Engineering the Optical Properties of Gold Nanorods: Independent Tuning of Surface Plasmon Energy, Extinction Coefficient, and Scattering Cross Section

The future integration of plasmonic nanoparticles, such as gold nanorods (Au NRs), into applications requires the ability to tune the components of their optical properties to optimize performance for the underlying technology. Verifying techniques that model the resonance energy and associated extinction, scattering, and absorption cross sections necessitate experimental data from series of Au NRs where structural features are independently tuned. Here, the extinction cross section and scattering efficiency are presented for Au NR series with high compositional and structural purity where effective volume, aspect ratio, length, and diameter are independently varied by factors of 25, 3, 2, and 4, respectively. The extinction cross sections quantitatively agree with prior calculations, confirming that the volume of the rod is the dominant factor. Comparisons of the scattering efficiency however are less precise, with both quantitative and qualitative differences between the role of rod volume and aspect ra...

A non-aggregation colorimetric method for trace lead(ii) ions based on the leaching of gold nanorods

A non-aggregation colorimetric sensing method for Pb(ii) ions using S2O32−/Au NR probes.

Gold nanorod-seeded synthesis of Au@Ag/Au nanospheres with broad and intense near-infrared absorption for photothermal cancer therapy.

As a widely-adopted agent for photothermal therapy (PTT), gold nanorods (Au NRs) remain problematic due to the cytotoxicity derived from cetyltrimethylammonium bromide (CTAB) and the comparatively weak and narrow near-infrared (NIR) absorption band. To address this problem, in this study, we propose a shape-controllable and spectrum-adjustable method of synthesis for Au@Ag/Au nanoparticles (NPs) through first coating a Ag nanolayer on the Au NR seed and a subsequent replacement reaction with HAuCl4 to yield a Ag/Au alloy nanoshell. Results from TEM and UV-vis spectra analysis showed that the thickness of the Ag layers directly determined the shape and size of the NPs, and the formation of Ag/Au nanoshells effectively enhanced the NIR absorbance of the NPs. Remarkably, the optimum Au@Ag/Au nanospheres (NSs) with a diameter of ∼40 nm were revealed to have a broad and intense absorption cross section from 400 to 1100 nm, a ∼4.7 times higher hyperthermic effect than Au NRs, and low dark-cytotoxicity. By using A549 lung cancer as the model, a series of in vitro investigations was performed and demonstrated that Au@Ag/Au NSs could efficaciously kill cancer cells under a 980 nm irradiation. The efficacy of PTT could be further improved by increasing the concentration, incubation time or irradiation time of the NSs. Moreover, a preliminary in vivo study also showed that, after injection into the A549 tumor, Au@Ag/Au NSs could cause an obvious necrosis at the irradiation site. Thus, a novel, promising and highly-effective NIR PTT agent has been developed, which might greatly advance the application of PTT in biomedical research.

Manipulating the Collective Surface Plasmon Resonances of Aligned Gold Nanorods in Electrospun Composite Nanofibers

Surface plasmon resonance (SPR) is an interesting optical property that has been intensively studied in recent years. Herein, we report that the SPRs of gold nanorods (Au NRs) embedded in electrospun poly(vinyl alcohol) nanofibrous films can be manipulated via several approaches such as the change of Au NR percentage in the composite nanofibers, swelling-induced refractive index decrease of local environment, and swelling-induced increase of inter-rod distances among the embedded Au NRs. The electrospun composite nanofibrous films exhibit excellent sensing ability to the swelling solvents with short responsible time and remarkable reversibility. This study advances the fundamental understandings of plasmonic properties for electrospun composite nanofibrous films; thus, it can benefit the novel design of smart nanomaterials for broad sensing and nanophotonic applications.

Growth Mechanism of Gold Nanorods

Gold nanorods (Au NRs) are the archetype of a nanoantenna, enabling the directional capture, routing, and concentration of electromagnetic fields at the nanoscale. Solution-based synthesis methods afford advantages relative to top-down fabrication but are challenged by insufficient precision of structure, presence of byproducts, limited tunability of architecture, and device integration. This is due in part to an inadequate understanding of the early stages of Au NR growth. Here, using phase transfer via ligand exchange with mono-thiolated polystyrene, we experimentally demonstrate the complete evolution of seed-mediated Au NR growth in hexadecyltrimethylammonium bromide (CTAB) solution. Au NR size and shape progress from slender spherocylinders at short reaction times to rods with a dumbbell profile, flattened end facets, and octagonal prismatic structures at later stages. These evolve from a single mechanism and reflect the majority of reported Au NR morphologies, albeit reflecting different stages. Add...

A versatile approach for the functionalization of gold nanorods and nanoparticles

A robust functionalized procedure for gold nanorods (Au NR) with mercaptoundecanoic acid (MUA), which are assembled with nanoparticles, has been reported in the present work. The self-assembled nanostructures were characterized by UV–Vis-NIR spectrophotometer and transmission electron microscopy (TEM). Self-assembly procedures, governed by surface chemistry, indicated that chemical reactivity of nanomaterial may contribute in construction of potential nanoscale assemblies. These nanostructures have striking applications as single-molecule detection, plasmonics, and sensing.

Preparation and characterization of pH‐responsive and thermoresponsive hybrid microgel particles with gold nanorods

AbstractWe report on pH‐responsive and thermoresponsive hybrid materials based on the assembly of gold nanorods, Au NRs, into multiresponsive, crosslinked copolymer microgel particles. These microgel particles were prepared by the surfactant‐free emulsion polymerization of N‐isopropylacrylamide and acrylic acid using N, N′‐methylene bis‐acrylamide as a crosslinker, which produces particles measuring approximately 160 nm that are interconnected to one other. Cetyltrimethyl ammonium bromide‐stabilized Au NRs were also prepared independently using a seed‐mediated growth method and then loaded into swollen, deprotonated, acrylic acid‐containing microgel particles using the electrostatic interactions between the oppositely charged particles. Transmission electron micrographs of the as‐prepared hybrid Au NR–microgel particles confirmed that the Au NRs were attached to the surface of the microgel particles. The size‐dependent temperature‐responsive characteristics of the hybrid microgel particles were studied by dynamic light scattering, and it was found that as the temperature increased across the phase transition temperature, the particle size decreased to 56% of the original volume. The thermoresponsive and pH‐responsive optical properties of the hybrid microgel particles were also systematically investigated. The thermo‐ and pH‐induced shrinkage of the microgel led to an increase in the UV–vis absorption intensity and caused a significant blue shift in the longitudinal surface plasmon bands of the Au NRs. © 2012 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2013

Abstract 1953: Co-delivery of doxorubicin and siRNA using octreotide-conjugated gold nanorods for targeted neuroendocrine cancer therapy

Abstract Introduction: Gastrointestinal (GI) carcinoids are neuroendocrine (NE) tumors that secrete hormones causing carcinoid syndrome. Metastatic carcinoids are not amenable to curative surgery. Therefore, siRNA-mediated gene silencing, in combination with targeted chemotherapy, may be a potential therapeutic strategy for patients with carcinoid neoplasms. We have developed and assessed antitumor effects of a gold nanorod (Au NR)-based nanocarrier conjugated with an anticancer drug (doxorubicin (DOX)) and small interfering RNA (siRNA against carcinoid tumor marker ASCL1, a protein required for NE cancer cell proliferation) specifically targeting the NE cancer cells with overexpressed somatostatin receptors (SSTRs) using octreotide (Oct) as an active - tumor targeting ligand. Methods: Au NR-based nanoparticles (NPs) were synthesized via a multiple step reaction. DOX was conjugated onto the Au NR via hydrazone bonds. siRNA was complexed onto the cationic poly(L-arginine) polymer. Oct targeting ligand was selectively conjugated onto the distal end of PEG arms. A human GI carcinoid cell line (BON) was treated with either non-targeted (Au-DOX) or targeted (Au-DOX-Oct) NPs with or without siRNA, at a concentration of 1 μg/ml for 30′ and 120′ or 24, 48, 72 hours. Based on the doxorubicin fluorescent intensity, the cellular uptake of NPs was analyzed by flow cytometry and confocal laser scanning microscope (CLSM). MTT assay and Western blot were used to determine cell proliferation and assess ASCL1 expression, respectively. Results: DOX release profiles from NPs were dependent on the environmental pH values, which can minimize systemic spread of toxic drugs during circulation. BON cells incubated with Au-DOX-Oct exhibited higher cellular uptake than Au-DOX (14% and 44% increase after 30 min and 120 min, respectively) measured by flow cytometry. The CLSM observations confirmed that the intracellular transport of Au-DOX-Oct is faster than Au-DOX. MTT assay showed that Au-DOX-Oct-ASCL1siRNA has the strongest inhibitory effect on carcinoid cell proliferation (more than 50% of growth reduction). Immunoblot analysis demonstrated that Au-DOX-Oct-ASCL1siRNA reduced the expression of ASCL1 much more than other NPs. Conclusions: We have developed a multifunctional, pH-responsive and tumor-targeting Au NR-based nanoplatform for anticancer drug therapy. We showed that the presence of the surface ligand Oct increased the cellular uptake of the NPs (Au-DOX-Oct) substantially in a BON cell line compared to the non-targeted NPs (Au-DOX). Targeted NPs with ASCL1siRNA were the most efficient in altering the neuroendocrine phenotype of BON cells demonstrating the strongest anti-proliferative effect. Thus, targeted delivery of anticancer drug (DOX) and genetic material (ASCL1siRNA) specifically to the tumor tissue could achieve therapeutic outcomes in treating carcinoid disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1953. doi:1538-7445.AM2012-1953

Reversible Plasmonic Circular Dichroism of Au Nanorod and DNA Assemblies

Reversible plasmonic circular dichroism (CD) responses are realized for the first time based on temperature-dependent assembly and disassembly of Au nanorod (Au NR) and DNA hybrids. Compared with the conventional UV-vis absorption spectra, the changes in both intensity and line shape of plasmonic CD signals are much more pronounced, leading to a preliminary detection limit of DNA as low as 75 nM. The mechanism and influence factors of reversible plasmonic CD responses are explored.

High‐Yield Assembly of Soluble and Stable Gold Nanorod Pairs for High‐Temperature Plasmonics

Colloidal synthetic approaches to discrete, soluble plasmonic architectures, such as nanorod pairs, offer numerous advantages relative to lithographic techniques, including compositionally asymmetric structures, atomically smooth surfaces, and continuous fabrication. Density-driven colloidal assembly, such as by solvent evaporation, produces some intriguing structures, e.g., particle chains; however, controllability and post-processibility of the final architecture is inadequate. Also the limited quantity of product nominally comprises a broad distribution of assembly size and type. Herein, the high-yield formation of soluble, stable, and compositionally discrete gold nanorod (Au NR) architectures by inducing-then arresting-flocculation is demonstrated using bifunctional nanorods and reversible modulation of solvent quality to deplete and reassemble an electrostatic stabilization layer, thereby eliminating the need for an additional encapsulant. Analogous to dimer formation during step-growth polymerization, the initial yield of Au nanorod side-by-side pairs can be greater than 50%. The high solubility and stability of the assembly enable purification, scale-up of nanomolarity solutions, and subsequent chemical modification of the assembled product. As an example, in situ silica deposition via Stöber synthesis onto the assembled pair produces highly processable nanostructures with a single pair of embedded Au NRs at their center, which exhibit thermal stability at temperatures in excess of 700 °C.

Nanorod Assemblies in Polymer Films and Their Dispersion-Dependent Optical Properties.

Optical absorption due to surface plasmon resonances in ensembles of gold nanorods (Au NRs) depends strongly on the nanorod separation and orientation. Here, we study the dispersion of polystyrene-functionalized Au NRs in polystyrene (PS) thin films using UV-visible (UV-vis) spectroscopy and transmission electron microscopy (TEM) and find that Au NRs are dispersed for brush chain lengths that exceed the PS matrix chain length and are aggregated otherwise. Monte Carlo simulations using parameters from classical density functional theory (DFT) calculations indicate that this behavior is due to substantial depletion-attraction forces for brush chain lengths that are much smaller than the PS matrix chain length. Both UV-vis measurements and discrete dipole approximation (DDA) calculations confirm that optical absorption is a facile method to determine nanorod morphology in nanocomposite films (i.e., aggregation or dispersion). Futhermore, a dispersion map is constructed showing the conditions required for nanorod dispersion and, correspondingly, the optical absorption properties of Au NR:PS nanocomposites. Using this information, optically active materials with tunable morphologies can be fabricated and routinely characterized using optical spectroscopic methods.

Formation of PdPt Alloy Nanodots on Gold Nanorods: Tuning Oxidase-like Activities via Composition

The island growth mode of Pt was employed to guide the forma-tion of PdPt alloy nanodots on gold nanorods (Au@PdPt NRs). Well-defined alloy nanodots, with tunable Pd/Pt ratios from 0.2 to 5, distribute homogeneously on the surface of the Au NR. Formation of nanodots shell leads to the red-shift and broadening of the longitudinal surface plasmon resonance (LSPR) band of the Au NRs. The Au@PdPt alloy NRs exhibit catalytic activity toward oxidation of often-used chromogenic substrates by dissolved oxygen under mild conditions, suggesting a new type of oxidase mimics. Composition dependence catalytic activity is observed for the oxidation of ascorbic acid (AA) and 3,3',5,5'-tetramethylbenzidine (TMB) and for the reduction of p-nitrophenol. For AA and TMB, catalytic activity enhances quickly at lower Pd/Pt ratios and tends to saturate at higher Pd/Pt ratios. For p-nitrophenol reduction, catalytic activity shows a nice linear relationship with Pd/Pt ratio owing to much higher catalytic activity of Pd. In conclusion, proper alloying of Pd and Pt presents an effective route to tailor the catalytic activity. Interesting, alloy nanodots can also catalyze the oxidation of Fe (II) to Fe (III) by dissolved oxygen. Thus, based on the competitive oxidation of TMB and Fe (II), selective detection of the latter can be achieved.

Surface-Enhanced Infrared Absorption and Raman Scattering of Adsorbate Molecules on Self-assembled Au Nanorods

The localized surface plasmon resonance (LSPR) is a collective oscillation of conduction electrons confined in metal nanostructures and is largely responsible for surface enhanced spectroscopies. Surface enhanced Raman scattering (SERS) is the best known and most widely applied example of such a surface enhanced spectroscopy. Although closely related and complementary to SERS, surface enhanced infrared absorption (SEIRA) spectroscopy requires more careful engineering of the LSPR of metal nanostructures so that the resonance is within the mid-infrared region. In this study, we demonstrate the use of gold nanorods (Au NR) as a suitable substrate capable of sustaining strong SEIRA spectroscopy. Adsorbate saturated Au NR typically exhibit Fano-type resonances in their SEIRA spectra obtained using reflectance FTIR. Such line asymmetry occurs due to the coupling of the relatively sharp molecular vibrations to the broad continuum of the LSPR resonance of the aggregated Au NR.

Nanorod Self-Assembly for Tuning Optical Absorption

Metallic nanoparticles that absorb and concentrate light are leading to greater efficiencies in nanophotonic devices. By confining gold nanorods (Au NRs) in a polymer film, we can control their spacing and orientation and, in turn, the absorption and polarization characteristics of the nanocomposite. In this study, we systematically increase the volume fraction of Au NRs (φrod) (aspect ratio v=3.3) while maintaining a uniform dispersion. As φrod increases from 1 to 16 vol %, the spacing between rods decreases from 120 to 20 nm and scales as φrod φ0.4. Simultaneously, the local 2D orientational order parameter increases linearly with φrod, although the rods are globally isotropic. The Au NR dispersion is found to depend on the enthalpic interactions between poly(ethylene glycol) brush grafted to the Au NRs and the poly(methyl methacrylate) matrix chains. Furthermore, the plasmon resonance exhibits a red shift with increasing φrod, and coupling is observed for separations up to 70 nm. Because NR spacing and orientation can be finely controlled using polymer matrix, these films are ideally suited for understanding fundamental behavior (e.g., plasmon coupling) as well as practical devices (e.g., solar cells).

Side‐by‐Side and End‐to‐End Gold Nanorod Assemblies for Environmental Toxin Sensing

Controllable assembly of nanoscale building blocks (monomers) is a necessary part of practical realization of the unique optical, electrical, magnetic, and chemical properties of nanoscale matter in macroscale materials. Such assemblies also contain much fundamental information about collective behavior of nanocolloids, which we are just beginning to understand. The key decisive factors for the successful assembly of nanocolloids is the anisotropy of nanoscale interactions, which stems from both the shape of nanocolloids and unequal distribution of organic molecules on their surface. Gold nanorods (Au NRs) have both geometrical and chemical anisotropy components and demonstrate strong optical extinction in the range of visible and near-infrared (NIR) wavelengths convenient for both research and practical purposes. Au NRs can be assembled by interactions with organic molecules, polymers, an antibody–antigen reaction, biotin–strepavidin connectors, and DNA, leading to superstructures with different degree of organization and complexity of collective behavior. Besides the utilization of NR monomers in non-linear optics, cellular imaging, and cancer therapy, optical effects corresponding to monomer!superstructure transitions allowed preparation of excellent biosensors because of large changes in oscillation frequencies of plasmons when NR pairs are formed. These studies mostly targeted biomedical applications. Simultaneously, their unique sensing capabilities have been virtually unexplored for the needs of environmental detection and monitoring. These challenges and impact can equal or exceed those encountered in detection of cancer. A better understanding of methods for the realization of speed/selectivity/sensitivity detection of common environmental pollutants is thus of great importance. Therefore, we decided to explore the potential of NR assemblies taking a pervasive environmental toxin, namely microcystin-LR (MC-LR), as the model while also addressing the general questions about the choice of different assembly motif for different sensing tasks. MC-LR is common in both developed and developing countries, with recorded cases of mass poisoning. MC-LR originates from common bluegreen algae and causes rapid liver failure; prolonged exposure to small concentrations of MC-LR in drinking water causes liver cancer. Herein, we describe the successful use of Au NRs for detection of MC-LR, which is significantly more sensitive than the traditional techniques, such as ELISA, yielding detection limit of 5 pgmL . It is also much simpler and faster than any other methods. These two factors are critical for environmental monitoring and have been a long-standing challenge. The pattern of the assembly strongly affects the sensitivity parameters for MC-LR detection. To realize different modes of assembly, such as side-byside and end-to-end motifs, with a degree of control sufficient for conclusive evaluation of sensing implications, two kinds of protein-carrying Au NRs were synthesized (Figure 1). One type of NR carried MC-LR antibodies (ABs) preferentially on the sides, while the other type carried antibodies located almost exclusively in the ends. These motifs were formed by using either electrostatic binding or covalent attachment of the antibodies mediated by a bifinctional linker, thioctic acid (TA).When electrostatic forces govern the placement of ABs, they attach primarily to the sides of NRs due to the larger area of contact and thus stronger electrostatic interactions. When a TA anchor covalently binds by a S Au bond, the conjugation of the ABs occurs predominantly in the ends of the rods due to better accessibility of the gold surface to the reactive thiol end. Variation of pH also allows varying repulsion or attraction of NRs and MC-LR, modality of attachment, and geometrical characteristics of assemblies (see the Supporting Information). The number of AB molecules on the surface of one Au NR was estimated to be 31 and 10 for the side-by-side [*] L. Wang, Y. Zhu, L. Xu, Dr. W. Chen, H. Kuang, L. Liu, Prof. C. Xu School of Food Science and Technology, State Key Laboratory of Food Science and Technology Jiangnan University, Wuxi, 214122 (China) E-mail: xcl@jiangnan.edu.cn Dr. W. Chen, A. Agarwal, Prof. N. A. Kotov Department of Chemical Engineering, Department of Biomedical Engineering Department of Materials Science and Engineering University of Michigan, Ann Arbor, MI 48109 (USA) E-mail: kotov@umich.edu [] These authors contributed equally to this paper.