Self-organized spatially separated silver 3D dendrites as efficient plasmonic nanostructures for Surface-enhanced Raman spectroscopy applications

Surface-enhanced Raman spectroscopy (SERS) is a promising optical method for analyzing molecular samples of various nature. Most SERS studies are of an applied nature indicating a serious potential for their application in analytical practice. Dendrite-like nanostructures have great potential for SERS, but the lack of a method for their predictable production significantly limits their implementation. In this paper, a method for controllable obtaining spatially separated, self-organized and highly-branched silver dendrites via template synthesis in pores of SiO 2 /Si is proposed. The dendritic branches have nanoscale roughness creating many plasmon-active “hot spots” required for SERS. The first held 3D modeling of the external electromagnetic wave interaction with such a dendrite, as well as experimental data, confirm this theory. Using the example of a reference biological analyte, which is usually used as a label for other biological molecules, the dendrites SERS-sensitivity up to 10 –15 M was demonstrated with Enhancement factor of 10 8 . The comparison of simulation results with SERS experiments allows distinguishing the presence of electromagnetic and chemical contributions, which have a different effect at various analyte concentrations.

Tremendous progress in the area of Raman-based sensors led to the creation of the surfaces allowing Raman signal detection from a few or even single molecules 20 . The SERS enhancement factor (EF), which is usually defined as has been demonstrated to be as large as 10 5 -10 11 (cf. Ref. 21 ). Here, ISERS stands for the Raman intensity obtained for the SERS substrate under a certain analyte concentration CSERS, and IR corresponds to the Raman intensity obtained under non-SERS conditions at analyte concentration CR.
However, the adoption of SERS remains limited, to a great extent due to the fabrication difficulties of effective and affordable nanostructured plasmonic SERS surfaces. So far, SERS substrate fabrication techniques include lithography 7 , laser ablation 22 , templated electrodeposition 23 , wet chemistry 24,25 and self-assembly 8,26 . The self-assembly technique is often considered the most promising because of its simplicity, cost-and time-effectiveness. For example, based on this approach, highly-branched dendritic nanostructures with sub-10-nm gaps between neighboring branches can be fabricated [27][28][29] . However, self-assembly techniques lack control over resulting structures. Thus, despite relatively satisfactory results 30 , regularly reproducible detection of ultra-small concentrations of the analyte (in particular single molecules) using dendrites was not demonstrated. The main problem is related to the formation of bulk agglomerates 27,31-33 , which limit stable detection at ultra-low concentrations of molecules due to the analyte penetration into the interior of such structures. As a result, the excitation laser does not access the analyte localization region and, consequently, the signal from the analyte is poorly recorded.
For that reason, such a problem can be solved by the spatial separation of nanostructures using porous templates: polymer track membranes, anodized aluminum oxide, porous silicon, etc. [34][35][36][37] . The formation of metallic nanostructures in the pores matrix is usually carried out using two methods: electrochemical deposition and electroless galvanic displacement. The first method is used to produce nanostructures filling the pore volume (for example, in the formation of nanorods) 38 , and the second -when it is required to realize individual nanoparticles or thin metallic layers on the pores surface 39 . In this paper, we develop and demonstrate a novel technique for the fabrication of spatially separate silver dendrites, presented schematically in Figure 1, via selforganization of Ag nanostructures in pores of SiO2/Si templates. We investigate the microstructure, morphology, optical features and SERS properties of fabricated nanostructures. Also, we show that observed templates allow us to achieve a SERS enhancement factor of ~10 8 with a single molecule detection possibility (~10 -15 M) of Ellman's reagent, which usually used as a label for others biological molecules.

II. Materials and methods
As templates for synthesis of silver dendrites, swift heavy ion-track porous SiO2 matrix on p-type silicon substrate (12 Ohm·cm with orientation <100>) were used with a 170 nm thick silicon oxide layer and pore sizes of 800 nm. The procedure of obtaining porous SiO2/Si templates are described in our previous paper 36 . The parameters of porous  To clarify the crystal structure of silver dendrites, the X-Ray diffraction (XRD) patterns were measured using X'Pert Pro X-ray diffractometer from PANalytical B.V. in the Bragg-Brentano arrangement.
The chemical state of the formed nanostructures was determined by X-ray photoelectron spectroscopy (XPS) in ultrahigh-vacuum (10 -9 mbar) by K-Alpha ThermoScientific system with a monochromatic X-ray source Al-K-alpha 1.486 keV. To neutralize the surface charge, a compensation gun was used. The density of data collection of highresolution spectra was 0.1 eV.
The scattering properties (LSPR -Localized Surface Plasmon Resonance) of the metal nanostructures were recorded with a dark-field micro-spectroscopy similarly as in Ref. 47 .
Briefly, the micro-spectroscopy system consisting of an upright microscope

III. Result and Discussion
All the pores of the ion-track SiO2/p-Si template are filled with dendritic silver nanostructures as shown in Figure 3a. The SEM image of a single pore filled with silver nanostructures is presented in Figure 3b. The image reveals that the nanostructures have the form of dendrites containing the main trunk, lateral branches and many rounded outgrowths on them. Due to the presence of nanoscale inhomogeneities between dendrites branches, the formation of "hot spots" can be expected with the enhanced electric field, which can be used for boosting the SERS effect. The crystallinity of the silver dendrites in the pores of the SiO2/p-Si template was determined using X-ray diffraction method. Figure 3f shows the XRD pattern of the produced structure. Unlike the typical diffraction patterns of silver dendrite 30 The high intensity of (111) reflex indicates the priority direction of crystallite growth along this direction that correlates with EBSD observations in Figure 3 d,e.
The result of XPS is presented in Figure 4. The asymmetric spectra for the Ag 3d line and the presence of low-intensity plasmon loss peaks confirm the metallic nature of the dendrites being formed 52 . To investigate the optical properties of the fabricated dendritic Ag nanostructures numerically, we perform the full-wave simulations using commercial software CST Microwave Studio (Figure 5a, b). The simulation results indicate that the most intense "hot spots" are located between the neighboring dendrite branches, where the electric field enhancement reaches 12 as shown in Figure 5b. It is known that the SERS enhancement factor caused by electric field localization is roughly proportional to the fourth power of the electric field amplitude (~E 4 ). This gives us an estimation for the enhancement factor of ~10 4 . The typical scattering spectrum (for more details see Methods) of the dendritic nanostructures is shown in Figure 5c. The complexness of the structure gives rise to spectral broadening of the measured scattering spectra 43 . Moreover, the scattered lightcollecting area has a radius of 15 µm, and hence each spectrum is averaged over ~50 nanostructures, which causes its additional broadening. As a result, the scattering spectrum in Figure 5c does not exhibit sharp peaks in the range of 500 -750 nm with a broadband resonant behavior nearby excitation wavelength (633 nm). Next, in order to demonstrate the performance of the resulting structures as a sensor, the SERS measurements using DTNB as a model analyte were performed (for details see Methods). Figure 6 summarizes the obtained results. We attributed the observed most intense bands at 1062, 1150, 1331, 1555 cm -1 to the DTNB 53 analyte. The evolution of SERS spectra shows the possibility of detection of ultra-low concentrations of the model DTNB analyte by applying localized silver dendrite nanostructures, which correspond to EF at least 10 8 .
According to modern concepts of Raman signal amplification mechanisms by plasmonic nanostructures, the highest amplification of the Raman signal occurs in "hot spots" due to the increased electromagnetic field strength in these places (figure 5a). The closer the molecule under study is to the "hot spots", the more intense the signal is recorded.
However, given the fact that each dendrite has a large number of 'hot spots', signal amplification by means of an electromagnetic mechanism is determined by all such places simultaneously. Even taking into account this fact, the obtained result of the amplification of the Raman signal is orders of magnitude higher than the numerical calculation. The EF value of 10 8 exceeds by several orders of magnitude the results of numerical simulations. We believe that this indicates the existence of a strong chemical contribution to SERS enhancement. This contribution can be caused by the following mechanisms: a change in the polarization of the molecule 16 The appearance of new allowed levels in the energy spectrum of the molecule can lead to differences in comparison with the Raman spectrum of a bare analyte. Indeed, our results show (Figure 6a) that the main lines are shifted to the blue side and with decrease in the analyte concentration this effect gets enhanced. This behavior of the SERS spectra can be explained in the following way. With a sufficient amount of analyte molecules, both amplification mechanisms are involved: electromagnetic, which does not affect the spectrum change, and chemical, which causes the shift of the combination bands and is noticeably weaker than the electromagnetic one. With a decrease in the analyte concentration, the role of the electromagnetic mechanism is weakened, because fewer molecules get into "hot spots". Molecules, which still fall into these regions, fit closely to the metal and at several angstroms distances create a molecular-metal complex with silver. Thus, the molecule gets additional allowed energy levels. For that reason, the resonant transitions between these levels can also be enhanced by the electromagnetic mechanism. A decrease in concentration leads to the fact that molecules that fall into "hot spots" at distances of several tens of nanometers from metal do not form a bond with the silver surface. This is a consequence of the dominant effect of the chemical contribution to the SERS spectrum and a more noticeable shift in the combination lines.
spots", but also in other regions of the metal, which also affects the characteristic shape of the SERS spectrum with a decrease of the electromagnetic mechanism role.
There are various ways to create SERS substrates with plasmon-active metal inclusions in modern literature. For example, a highly sensitive SERS substrate was also demonstrated by Kumar et al. 59 and Sharma at al. 60 . They have enough sensitivities for detection ultra-low concentrations of target molecules and they also have a number of limitations associated with the complexity of manufacturing such substrates. The ion-track technology used in this work to create the SERS substrates is well suited for research purposes because it allows controlling template pore diameter very simple. The pore diameter is very important for obtaining spatially separated silver dendrites because this parameter influences the diffusion-limitation processes in the pores on the silver structures growing stage that is necessary for growth of dendrites. We will discuss this more detail in future work where we will consider silver morphology dependence on template pore diameter. In this work the presented results made it possible to determine that it is necessary to use templates with pore diameters of 800 nm for the growth of spatially separated silver dendrites. To obtain such pore diameters, classical lithography is also well suited. Therefore, in the future, this technique can be used to obtain ordered arrays of silver dendrites. Obtaining ordered silver nanostructures will not influence EF because "hot spots" arranged randomly on the dendrites. However, ordered structures are still relevant 61 because it will increase the analytical usefulness of SERS active substrates.

IV. Conclusions
Spatially separated Ag dendritic nanostructures in the pores of the ion-track template SiO2/Si were synthesized using directed self-organization of Ag in a limited volume.
Microstructure and optical features of SiO2(Ag)/Si nanostructures have been studied numerically and experimentally. It has been shown that the Ag nanostructures have the form of dendrites. The SERS measurement results indicate the possibility of ultra-low analyte concentration detection, comparable to the concentrations of single molecules.
The high sensitivity of the fabricated structures is justified by the coexistence of electromagnetic and chemical contributions to the Raman signal amplification. The modeling of the electromagnetic wave interaction with dendrites indicates that the appearance of "hot spots" occurs in places of twinning (intergrowth of the branches of the dendrite). The fabricated dendrites have a large number of "hot spots" with an electric field enhancement factor of 12. The random distribution of "hot spots" on a dendrite leads to its broadened scattering spectrum. We strongly believe that the simplicity, reproducibility, and scalability of the presented method for the formation of spatially separated dendrites make it promising for various vital applications in photonics, chemistry, and biology.