Surface-enhanced Raman scattering (SERS) is widely employed because it offers quick, microscopic, and traceless detection. This study used high-voltage and low-voltage ultrasonic oscillation to embed precious metal nanoparticles into the pores of chemically modified ultra-thin anodic alumina (AAO) films, resulting in a highly sensitive three-dimensional SERS substrate. We improve the substrate's stability and Raman activity by adjusting the particle alloy ratio. For the substrate in this alloy ratio, the Raman signal of probe molecules (Rh6G) adsorbed on the substrate surface is enhanced. the enhancement factor (EF) was as high as 1.40×107, the relative standard deviation (RSD) of 10.6%, and the concentration of Rh6G shows a linear relationship with Raman intensity, with a linear correlation coefficient of 0.961. In addition, we evaluated the substrate's detection effect on thiram molecules, surprisingly, the catalytic effect of the substrate on PATP molecules was discovered during this process. It has been proven that this structure has both good plasmon photocatalytic properties and good practicality and high sensitivity as a Raman enhanced substrate.
Research Article
Assembly nanoparticle arrays decorated with ultrathin AAO nanopores for highly sensitive SERS substrate
https://doi.org/10.21203/rs.3.rs-3100595/v1
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Journal Publication
published 07 Sep, 2023
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Ultrathin AAO nanopores
Assembly
Surface Raman Scattering
When the frequency of incident light matches the oscillation frequency of free electrons on the surface of nanostructured metals, a high-intensity local electric field is created, which increases the Raman signal of the probe molecules adsorbed on its surface. Surface-enhanced Raman scattering (SERS) has been increasingly popular in recent years due to its advantages, such as high sensitivity, fast speed, strong chemical specificity, and non-destructive testing [1–3]. The sensitivity of SERS is mostly determined by the plasmonic "hot spots", which can result in high local field amplification due to localized surface plasmonic excitation resonance (LSPR) generated with a specific wavelength of light [4–7]. It is well known that the three-dimensional periodic distribution of nanoparticles can achieve high enhancement of SERS substrates with the added benefit of reliable Raman signal detection [8–11].
Three-dimensional structures have more distinct optical properties than one-dimensional and two-dimensional structures. On the one hand, the increase in the number and density of "hot spots" in the three-dimensional distribution results in superior electromagnetic field enhancement. On the other hand, the three-dimensional distribution of "hot spots" can better match the focus of the beam in three-dimensional space [12–14]. Thus we chose ultrathin anodic alumina oxide (AAO) film as a periodic array template, to prepare low-cost, easy mass production and highly sensitive substrates [15–16].
Buff discovered that aluminum could be electrochemically oxidized in an aqueous solution, generating a thicker oxide layer than the natural oxide layer in 1857 [17]. In industry, this reaction is known as anodic oxidation. AAO forms a porous structure with regular spacing, uniform distribution, and wide surface area [18]. Most research uses electrodeposition and chemical vapor deposition to assemble gold and silver atoms into AAO pores. Huang used AAO as a mask to deposit Ag-nanorods (AgNRs) inside of it electrochemically and then peeled the AAO off to create an array of AgNRs [19]. It is impressive that many groups have worked to create effective substrates by physically or chemically combining particles into AAO for economic reasons. Lin assembled gold nano bipyramids (Au NBPs) into AAO pores using the "drop dry" approach [20]; Liu assembled PDA on evenly ordered v-type surfaces in order to create arrays of noble metal nanoparticles [21].
We submerged the ultrathin porous alumina film coated with PATP in the alloy colloidal solution for ultrasonic self-assembly. The LSPR characteristics of the substrates were tuned using the nanoparticle fractions, which also improved the substrates' SERS activity. By detecting Rh6G molecules with an enhancement factor (EF) of 1.40×107, the alloy particles with both stability and sensitivity were chosen as the substrate particles for the following analysis, and the practicality of the substrates was confirmed by the detection of thiram molecules, demonstrating the improved Raman characteristics of the substrates and the applicability of the substrates.
2.1 Materials
HAuCl4 (0.01M, Aladdin Biochemical Technology Co., Ltd.), AgNO3 (0.01M, Aladdin Biochemical Technology Co., Ltd.), trisodium citrate (AR, ≥ 99.0%.), perchloric acid (AR, ≥ 99.5%.), ethanol (AR, ≥ 99.7%.), PATP (AR, ≥ 97.0%.), oxalic acid (GR, ≥ 99.8%.), potassium dichromate (AR, ≥ 99.8%.), concentrated sulfuric acid (AR, ≥ 98%), phosphoric acid (AR, ≥ 85.0%.), thiram (GR, ≥ 99.0%), all purchased from Sinopharm Holding Chemical Reagent Co., Ltd (China), aluminum foil (Beijing Institute of Nonferrous Metals and Rare Earths, 99.9995%).
2.2 Preparation of AAO film
The ultra-thin AAO film was prepared by the "two-step anodic oxidation method". Firstly, the aluminum foil was electropolished (8 v, 8 min) in mixture solution of perchloric acid and ethanol (1:6, V/V) at 0°C; secondly, the polished aluminum foil was subjected to primary oxidation (45 v, 4 h) in oxalic acid solution (0.03 M) at 0°C, which determines the pore growth sites; then the formed irregular aluminum oxide layer was removed in a film removal solution (2.64 g of potassium dichromate mixture, 98 mL of deionized water and 0.96 mL of concentrated sulfuric acid) at 60°C. the solution, 98 mL of deionized water and 0.96 mL of concentrated sulfuric acid); the aluminum foil was oxidized under the same conditions as the primary oxidation for 2 min after de-filming, and the sites on the aluminum foil were further grown; finally, the pores were expanded in 2 wt% phosphoric acid solution (45 min) at 30°C.
2.3 Preparation of nano-colloids
As for the preparation of the colloidal solution, the total sum of HAuCl4 and AgNO3 in the solution system was 0.02 M (the ratio of HAuCl4 to AgNO3 solution was changed to obtain different proportions of alloy colloidal solution). The solution was heated to boiling and stirred for 10 min, followed by adding 1% trisodium citrate solution as a reducing agent, and stirred for 15 min to reduce the gold and silver ions in the system to an atomic state. The final volume of the solution was 87.5 ml, and the solution was cooled to room temperature and washed three times with deionized water by centrifugation (8000 rpm, 10 min). The chloroauric acid was added and stirred for 2 min to prepare gold gum, and the rest was as above.
For AgNPs preparation as follows: NaBH4 (0.01 M) was used as the reducing agent and trisodium citrate (1%) as the protecting agent, and AgNO3 was added drop by drop under boiling conditions (pay attention to the stirring speed as slowly as possible). The AgNPs were obtained by heating and stirring in water bath for 15 min and then removed and cooled to room temperature. The particle size of both the prepared Au-AgNPs and AgNPs was about 30–40 nm.
2.4 Ultrasonic self-assembly method
Thin gold film was deposited on the AAO surface to promote the adsorption of PATP on the AAO surface. Then the samples were immersed in 0.1 M PATP ethanol solution for 12 h. The samples were rinsed with a large amount of ethanol solution and water solution to ensure that the unbonded PATP molecules on the sample surface were removed entirely. The samples were allowed to dry naturally and put into NPs colloidal solution for 2 h at room temperature and keep the samples in the colloidal solution. The structures of colloidal ultrasound self-assembly into nanocavities were obtained in an ultrasound machine (Buck Ultrasound Equipment Co., Ltd. BK-360B) with alternating high and low voltage square wave ultrasound with an oscillation frequency of 40 kHz and repeated twice, as shown in Fig. 1.
2.5 The characterizations of SERS substrates
The morphology of the substrates was characterized using a scanning electron microscope (S4800, Hitachi, Japan). The optical properties of the nanoparticles were characterized by UV-Vis spectrophotometer (UV-5900PC, Metash, China).
2.6 Raman Spectroscopy Acquisition
The samples for SERS measurements were immersed in different concentrations of Rh6G and thiram ethanol solutions for 90 min. Taken out and dried. The Raman scattering spectra were collected on a confocal Raman spectrometer (inVia, Renishaw, UK) with a 532 nm laser, 50× objectives and the numerical aperture (NA) is 0.5. The SERS measurement was conducted at low power (25 mW) and the acquisition time was 10 s for SERS spectra.
We changed the secondary oxidation time and pore expansion time of phosphoric acid, and selected appropriate experimental conditions to produce ultra-thin AAO with small pore gaps [22, 23]. Phosphoric acid was applied to the aluminum foil after secondary oxidation. The reaction between aluminum trioxide and phosphoric acid can lower the hole depth and improve the regularity of the surface shape [24]. In order to achieve a hole wall of an appropriate thickness, we change of phosphoric acid treatment time. The size of AAO after 45 min of phosphoric acid treatment has met the experimental requirements, with a pore diameter of about 90 nm, a pore wall of less than 20 nm, and a pore depth of about 180 nm, which is much thinner than the conventional AAO film, and therefore can be called "ultrathin AAO", and the AAO used for subsequent particle ultrasonic assembly was prepared under these conditions, as shown in Fig. 2a.
The goal of creating ultrathin AAO is to reduce the capillary force in the pore as much as possible so that the particles can be deposited into the pore more easily during ultrasonic self-assembly [25]. The ultrasonic device uses piezoelectric materials to transform electrical energy into electrical mechanical vibrations, operating at 40 kHz. The mechanical waves that are compressed and expanded as a result of the sound waves' energy transfer to the medium can be contained within a tiny region of space. The self-assembling tendency is caused by the compression of the acoustic waves, which contain energy [26]. Due to the spatial variation of pressure, a combined force is generated in any material region, driving the local particles' directional motion.
In a liquid medium, the local pressure (N), density ρ (Kg/m3), and particle displacement ξ (m) vary with time and position, and there is a specific relationship between these variables. As shown in the following equation named Athanasiadis [27]:
Athanassiadis calculated using equations (1) and (2) that the oscillations induced by acoustic waves in an aqueous medium can displace particles in the range of 1 nm-1 µm. Thus, the ultrasonic self-assembly technique is sufficient to move nanoparticles at the nanoscale, enabling the assembly of arrays of nanoparticles distributed periodically in AAO nanopores.
As a result, we pretreated AAO with PATP. PATP has bifunctional groups, and the -SH in the molecule is easily broken to create bonds with noble metals [28–30]. Moreover, the Au-AgNPs prepared by sodium citrate reduction have a negative surface charge and are adsorbed by the amino electrostatic force in PATP. The particles adsorbed by PATP around the pores are vibrated into the pores through a square wave oscillating alternately from high to low. The prepared substrates were homogeneous in shape, the alloy nanoparticle arrays appeared to be distributed periodically, and the filling ratio reached 100%. The particle sizes were essentially the same for different alloy ratios, aside from the slightly different number of fillings in the pore size due to different particle sizes, as shown in Fig. 2b.
The ultrathin AAO treated with PATP was placed in different ratios of alloy nanoparticles (Au:Ag = 3:1, Au: Ag = 1:1, Au:Ag = 1:3, Au:Ag = 1:5) and AgNPs colloidal suspensions for ultrasonic self-assembly to prepare periodically distributed alloy nanoparticle arrays SERS substrates, respectively. When the Ag ratio increases, the absorbance peak of the colloidal suspension of nanoparticles is blue-shifted in Fig. 3a, and it denotes the change in the produced substrate's dielectric constant. Using immersion adsorption of Rh6G molecules, the produced substrate's spectral enhancement properties were examined. The Raman scattering spectra of Rh6G molecules adsorbed on substrates with various alloy ratios at 10− 6 M concentrations are displayed in Fig. 3a. The blank sample is an unassembled AAO with adsorption of Rh6G molecules at a concentration of 10− 3 M in Fig. 3b, indicating that the inherent SERS activity of AAO is weak at this concentration. Compared with blank sample, the prepared SERS substrate can effectively enhance the Raman signal of Rh6G molecules and can detect the Raman characteristic peaks of Rh6G molecules at 611 cm− 1, 774 cm− 1, 1182 cm− 1, 1313 cm− 1, 1361 cm− 1, 1508 cm− 1, 1573 cm− 1 and 1648 cm− 1 for clear identification. Compared to the unself-assembled AAO, the intensity of the Raman signal was noticeably higher. By altering the alloy ratio of nanoparticles in the AAO pores, the SERS activity of the substrate was also improved. It was discovered that the SERS activity of the substrate improved as the proportion of Ag in the alloy rose [31].
In actuality, alloy nanoparticles have unique benefits: AgNPs perform better in terms of SERS enhancement performance, but AgNPs are prone to oxidation, which results in morphological changes and affects the stability of the substrate, so the addition of AuNPs does not only has good sensitivity but also improves stability. Gold nanoparticles are stable and biocompatible, but their sensitivity is at least one order of magnitude lower than silver nanoparticles. Thus, the addition of Au-AgNPs ensures the stability of the system as well as excellent sensitivity. Therefore, we chose AAO@Au1Ag5NPs-Ultrasonic as the substrate for the subsequent study.
We investigated the homogeneity of Rh6G molecule adsorption at a concentration of 10− 7 M. Any 10 places on the sample surface had highly identical intensities of the probe molecules’ characteristic peak at 611 cm− 1, and this peak's intensity at these points was compared with an RSD of 10.6%, as shown in Fig. 3c and 3d, demonstrating the substrate's superior uniformity.
The substrate's sensitivity and enhancement factor at low-concentration detection was used to assess the SERS activity of AAO@Au1Ag5NPs-Ultrasonic. Rh6G is a cationic dye that is electrostatically adsorbed onto the surface of negatively charged NPs. The Raman scattering spectra of Rh6G molecules at various concentrations are shown in Fig. 4a and 4b to assess the SERS substrate sensitivity. The probe molecules' concentration ranged from 10− 6 to 10− 11 M. It was discovered that when the concentration of the probe molecules was below 10− 6 M, the substrate could detect the Raman signals of PATP molecules and dimethylamine borane (DMAB) molecules activated by PATP molecules, and it’s positions include: 546 cm− 1, 720 cm− 1, 917 cm− 1, 1003 cm− 1, 1071 cm− 1, 1142 cm− 1, 1193 cm− 1, 1304 cm− 1, 1388 cm− 1, 1476 cm− 1, 1436 cm− 1 and 1578 cm− 1 (shown in Fig. 4f). This phenomenon indicates that the substrate exhibits plasmonic catalytic effect and the substrate is rich in plasmonic “hot spots” [32–34].
However, these peaks were insufficient to mask the characteristic peaks of Rh6G molecules. Figure 4c shows the Raman signal at 611 cm− 1 versus Rh6G concentration. The concentration showed a significant linear correlation with the Raman signal (R2 = 0.961), which provided a basis for the substrate calibration for detecting low concentrations of Rh6G molecules. EF values were calculated according to Eq. (3):
Where ISERS and Inorm are the intensity of the selected characteristic peak (611 cm− 1) of Rh6G molecules in the SERS spectrum and typical Raman spectrum, respectively, CSERS and Cnorm are the Rh6G concentrations of 10− 11 M and 10− 3 M. The EF value of the substrate was estimated to be 1.40×107. These results showed that the AAO@Au1Ag5NPs-Ultrasonic had excellent SERS activity.
It has been established that thiram, which is frequently used as a plant fungicide and accelerator in the rubber sector, is toxic to humans at concentrations exceeding 7 ppm [35]. With a molecular probe concentration of 10− 4-10− 7 M, thiram was detected on the chosen AAO@Au1Ag5NPs-Ultrasonic substrate. The research object is the characteristic peak of thiram at 1514 cm− 1, which is still clearly distinguishable at a concentration of 10− 7 M, and the minimum detection concentration is far below 7 ppm, Fig. 4d and 4e. It was found that when the concentration was lower than 10− 4 M, the substrate could clearly detect the Raman signals of PATP molecules and dimethylamine-borane (DMAB) molecules catalyzed by PATP. Although the PATP and DMAB peak could be seen in the sample structure, it did not affect how the thiram peak could be observed clearly.
We conducted electromagnetic field simulation analysis on the transverse and longitudinal cross-sections of the substrate under 532 nm laser irradiation to better understand the near-field enhancement effect of AAO@Au1Ag5NPs-Ultrasonic substrates and the polarization direction is in the y-axis direction. We fixed the alloy nanoparticles' average size to 30 nm and randomly distributed them in order to simplify the model further. In Fig. 5a and 5b the plasmonic “hot spots” may be seen to be dispersed throughout the nanoparticles' three-dimensional interstices.
By modifying the secondary oxidation time and phosphoric acid treatment time, ultrathin AAO films were created, and the periodically distributed 3D Au-AgNPs arrays were created using the ultrasonic self-assembly method. Using 10− 6 M Rh6G molecules as probe molecules adsorbed on the sample's surface, as the amount of silver in the colloid increases, the dielectric constant of the substrate changes, and the Raman activity increases, thereby improving the LSPR performance. The most active Au1Ag5NPs in the alloy ratio were selected as the substrate particles to explore the spectrum properties. The Rh6G molecules were evaluated at various concentrations, confirming the substrate enhancement factor of 1.40×107 and the substrate exhibits good uniformity in enhancing Raman signals. The detectable thiram level being much lower than the human hazard threshold. In addition, due to the presence of PATP molecules in this substrate structure, its superior plasmonic catalysis effect has been observed. The study of plasmonic catalysis is of great significance for exploring the mechanism of charge transfer in SERS chemical enhancement and therefore it may be used for future research in surface catalysis, biosensing, and other fields.
Author Contribution Jun Dong and Mengtao Sun put forward ideas, design the experiment and revised the manuscript, Chenlu Li and Yan Wang performed the experiment and wrote the main manuscript text, Yi Cao and Yimeng Fan help to perform the experiment, Qingyan Han, Wei Gao and Jianxia Qi help to revised the manuscript, and Yongkai Wang performed the simulation work.
Funding This work was financially supported by the National Science Foundation of China (12274341, 12004304), the Shaanxi Province Natural Science Foundation of China(2022JZ-05), the Shaanxi Provincial Research Plan for Young Scientific and Technological New Stars (2021KJXX-45), and the Key R & D project of Shaanxi Province (2022SF-333).
Data Availability Not applicable.
Conflict of Interest The authors declare no competing financial interest.
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No competing interests reported.