Fabrication of polydimethylsiloxane (PDMS)-Based Flexible Surface-Enhanced Raman Scattering (SERS) Substrate for Ultrasensitive Detection

Guanzhou Lin; Jia Zhu; Yang Wang; Bingquan Yang; Shisong Xiong; Jinwen Zhang; Wengang Wu

doi:10.3791/65595

JoVE Journal > Engineering

Ingeniería

Fabrication of polydimethylsiloxane (PDMS)-Based Flexible Surface-Enhanced Raman Scattering (SERS) Substrate for Ultrasensitive Detection

Published: November 17, 2023

doi:

10.3791/65595

Guanzhou Lin¹, Jia Zhu², Yang Wang¹, Bingquan Yang^1,3, Shisong Xiong^1,3, Jinwen Zhang¹, Wengang Wu¹

¹National Key Laboratory of Advanced Micro and Nano Manufacture Technology, School of Integrated Circuits,Peking University, ²School of Electronic Engineering and Computer Science,Peking University, ³School of Software and Microelectronics,Peking University

Summary

This protocol describes a fabrication method for a flexible substrate for surface-enhanced Raman scattering. This method has been used in the successful detection of low concentrations of R6G and Thiram.

Abstract

This article presents a fabrication method for a flexible substrate designed for Surface-Enhanced Raman Scattering (SERS). Silver nanoparticles (AgNPs) were synthesized through a complexation reaction involving silver nitrate (AgNO₃) and ammonia, followed by reduction using glucose. The resulting AgNPs exhibited a uniform size distribution ranging from 20 nm to 50 nm. Subsequently, 3-aminopropyl triethoxysilane (APTES) was employed to modify a PDMS substrate that had been surface-treated with oxygen plasma. This process facilitated the self-assembly of AgNPs onto the substrate. A systematic evaluation of the impact of various experimental conditions on substrate performance led to the development of a SERS substrate with excellent performance and an Enhanced Factor (EF). Utilizing this substrate, impressive detection limits of 10^-10 M for R6G (Rhodamine 6G) and 10^-8 M for Thiram were achieved. The substrate was successfully employed for detecting pesticide residues on apples, yielding highly satisfactory results. The flexible SERS substrate demonstrates great potential for real-world applications, including detection in complex scenarios.

Introduction

Surface-Enhanced Raman Scattering (SERS), as a type of Raman scattering, offers the advantages of high sensitivity and gentle detection conditions, and can even achieve single molecule detection¹^,²^,³^,⁴. Metal nanostructures, such as gold and silver, are typically used as SERS substrates to enable substance detection⁵^,⁶. Electromagnetic coupling enhancement on nanostructured surfaces plays a significant role in SERS applications. Metallic nanostructures with varying sizes, shapes, interparticle distances, and compositions can aggregate to create numerous "hotspots" generating intense electromagnetic fields due to localized surface plasmon resonances⁷^,⁸. Many studies have developed metal nanoparticles with different morphologies as SERS substrates, demonstrating their effectiveness in achieving SERS enhancement⁹^,¹⁰.

Flexible SERS substrates find wide applications, with nanostructures capable of producing SERS effects deposited on flexible substrates to facilitate direct detection on curved surfaces. Flexible SERS substrates are employed for detecting and collecting analytes on irregular, non-planar, or curved surfaces. Common flexible SERS substrates include fibers, polymer films, and graphene oxide films¹¹^,¹²^,¹³^,¹⁴. Among them, polydimethylsiloxane (PDMS) is one of the most widely used polymer materials and offers advantages such as high transparency, high tensile strength, chemical stability, non-toxicity, and adhesion¹⁵^,¹⁶^,¹⁷. PDMS has a low Raman cross-section, making its impact on the Raman signal negligible¹⁸. Since the PDMS prepolymer is in liquid form, it can be cured by heat or light, providing a high degree of controllability and convenience. PDMS-based SERS substrates are relatively common flexible SERS substrates, having been used in previous studies to embed various metal nanoparticles for detecting different biochemical substances with exemplary performance¹⁹^,²⁰.

In the preparation of SERS substrates, the fabrication of nanogap structures is crucial. Physical deposition technology offers advantages like high scalability, uniformity, and reproducibility but typically requires good vacuum conditions and specialized equipment, limiting its practical applications²¹. Additionally, fabricating nanostructures at the few-nanometer scale remains challenging with conventional deposition techniques²². Consequently, nanoparticles synthesized through chemical methods can be adsorbed onto flexible transparent films through various interactions, facilitating the self-assembly of metallic structures at the nanoscale. To ensure successful adsorption, interactions can be adjusted by physically or chemically modifying the film surface to alter its surface hydrophilicity²³. Silver nanoparticles, compared to gold nanoparticles, exhibit better SERS performance, but their instability, particularly their susceptibility to oxidation in air, results in a rapid decrease in the SERS Enhancement Factor (EF), affecting substrate performance²⁴. Hence, it is essential to develop a stable particle method.

The presence of pesticide residues has garnered significant attention, creating a pressing need for robust methods capable of rapidly detecting and identifying various classes of hazardous chemicals in food in the field²⁵^,²⁶. Flexible SERS substrates offer unique advantages in practical applications, particularly in the realm of food safety. This article introduces a method for preparing a flexible SERS substrate by bonding synthesized glucose-coated silver nanoparticles (AgNPs) onto a PDMS substrate (Figure 1). The presence of glucose protects the AgNPs, mitigating silver oxidation in the air. The substrate demonstrates excellent detection performance, capable of detecting Rhodamine 6G (R6G) as low as 10^-10 M and pesticide Thiram as low as 10^-8 M, with good uniformity. Moreover, the flexible substrate can be employed for detection through bonding and sampling, with numerous potential application scenarios.

Protocol

1. Synthesis of nanoparticles

Preparation of Silver nitrate solution
1. Using a precision weighing balance, measure 0.0017 g of AR-grade silver nitrate (AgNO₃, see Table of Materials) and add it to 10 mL of deionized (DI) water. Stir the mixture to create a 10^-3 mol/L AgNO₃ solution.
Preparation of the silver-ammonia complex
1. Take 1 mL of AR-grade ammonia water (NH₃.H₂O, see Table of Materials) using a syringe, and add it drop by drop into the silver nitrate solution while stirring. Stop the dropwise addition when the solution becomes clear.
Preparation of the glucose solution
1. Using a precise weighing balance, measure 0.36 g of AR-grade glucose powder (see Table of Materials) and add it to 10 mL of DI water. Stir the mixture thoroughly to create a 0.2 M glucose solution.
Synthesis of silver nanoparticles (AgNPs)
1. Employ a pipette gun to add 30 µL of the silver-ammonia complex (prepared in step 1.2) to the glucose solution (prepared in step 1.3) at intervals of 30 min. Repeat this process 4-6 times while stirring until the solution turns yellow.

2. Preparation of flexible substrates

Preparation of PDMS substrate
1. To synthesize the PDMS substrate, take approximately 5 g of PDMS A solution and add B solution (from a commercially available kit, see Table of Materials) at a ratio of 1:10.
2. Stir and thoroughly mix the PDMS A and B solutions.
3. Transfer the mixed PDMS into a square dish and then bake it in an 80 °C oven for 2 h.
4. After curing through the above process, use a scalpel to cut the PDMS along the dark grid of the Petri dish, creating small PDMS cubes with dimensions of approximately 1 cm x 1 cm.
Surface modification
1. Subject the aforementioned small PDMS pieces to plasma treatment. Use a hand-held plasma processor (see Table of Materials) and move it back and forth approximately 5-10 cm above the PDMS surface to perform surface plasma treatment.
2. Utilize the plasma processor to modify the surface, inducing the formation of hydroxyl groups on the PDMS surface, rendering it hydrophilic²⁷.
Modification with APTES
1. Prepare a 10% APTES solution (see Table of Materials).
2. Immerse the surface-modified PDMS obtained in step 2.2 into the APTES solution and allow it to sit for 10 h. This allows APTES to bind with the hydroxyl groups on the PDMS surface.
Self-assembly of AgNPs
1. Immerse the PDMS substrate obtained in step 2.3 into the AgNPs solution synthesized in step 1.4 for 10 h. This self-assembles the AgNPs onto the PDMS substrate, creating the final flexible SERS detection substrate.

Representative Results

In this study, a flexible SERS substrate composed of synthetic AgNPs wrapped in glucose and self-assembled on PDMS using APTES was developed, achieving excellent detection performance for practical pesticide detection applications. The detection limits for R6G and Thiram were both reached at 10^-10 M and 10^-8 M, respectively, with an Enhancement Factor (EF) of 1 x 10⁵. Furthermore, the substrate demonstrated uniformity.

The AgNPs wrapped in glucose were synthesized using an improved Tollens method²⁸^,²⁹. This AgNPs assembly not only generated a strong SERS signal but also effectively shielded the silver in the AgNPs from oxidation, preserving detection performance. From the Environmental Scanning Electron Microscopy (ESEM) images in Figure 2, the synthesized particles appeared relatively uniform, with most having diameters between 40-50 nm. The outer layer of the AgNPs was enveloped by a glucose layer. This structure provided a dielectric layer for the AgNPs' outer layer and shielded the silver particles from oxidation upon exposure to air, preserving SERS performance.

It is evident that a strong enhanced electric field forms between the gaps of the AgNPs, serving as the primary cause of the SERS signal. Therefore, the substrate is densely immobilized with AgNPs on flexible substrates to achieve enhanced performance (Figure 3). The self-assembled SERS flexible substrate developed in this study is simple, of high quality, and free from toxic or harmful substances, making it environmentally friendly.

The SERS flexible substrate prepared in this study exhibited excellent detection performance. To evaluate a SERS substrate, the critical aspect is its detection capability. Here, the Enhancement Factor (EF) was defined to assess the enhancement performance of the substrate, and R6G (see Table of Materials) was used to determine the detection limit. The EF was described by³⁰:

EF = (I_SERS / I_Raman) × (N_Raman / N_SERS)

The peak positions³¹ of R6G and their corresponding values are presented in Table 1.

In this study, the Raman spectrum was obtained using a 633 nm laser with 10x and 50x objectives. The integration time was set at 10 s for spectrum acquisition during measurement, with the incident laser power at 3.7 mW. By adding 30 µL of R6G solutions with varying concentrations onto the substrate and observing the Raman signal through direct detection, Figure 4 illustrates that the substrate exhibited excellent detection capability, reaching a detection limit of 10^-10 M for R6G, indicative of strong detection performance. Subsequently, using 10^-5 M R6G as the test probe, the Enhancement Factor (EF) of the substrate was calculated to be 1 x 10⁵ (the calculation process is detailed in Supplementary File 1), demonstrating a notable enhancement effect (Figure 5).

The flexible SERS substrate enabled the detection of pesticides. Thiram, a widely used dithiocarbamate (DTC) pesticide in fruit and vegetable cultivation, aims to control fungal diseases and prevent deterioration during storage and transport³². However, repeated exposure or ingestion of Thiram residues may result in health issues such as lethargy, muscle tone loss, and severe fetal malformations³³^,³⁴. Therefore, achieving trace-level Thiram detection on the surfaces of fruits and vegetables is crucial. The Raman peaks³⁵ of Thiram and their causes are outlined in Table 2.

Various concentrations of Thiram were applied to the flexible substrate to assess its detection performance. Figure 6 demonstrates that for Thiram detection, its three primary characteristic peaks are clear, and the detection limit is reached at 10^-8 M.

The flexible substrate enabled practical detections. In everyday life, pesticide residues sometimes persist on the surfaces of fruits. Consumption of unwashed fruits may pose health risks. In this study, the flexible SERS substrate was applied using a "paste and peel-off" method, attaching the substrate to the surface of an apple and then removing it for inspection.

Figure 7 illustrates that under this method, the detection of 10^-7 M Thiram was achieved, with relatively clear spectral lines. Thus, the flexible SERS substrate prepared can facilitate the "paste and peel-off" detection method, effectively identifying pesticide residues on fruit surfaces, and offering valuable practical applications.

The flexible SERS substrate presented in this study not only exhibited remarkable detection performance but also offered practical application scenarios.

Figure 1: Schematic design of the PDMS flexible SERS substrate. Illustration depicting the design of the PDMS (Polydimethylsiloxane) flexible substrate used for Surface-Enhanced Raman Scattering (SERS) experiments. Please click here to view a larger version of this figure.

Figure 2: ESEM image of synthesized AgNPs. Environment Scanning Electron Microscopy (ESEM) image showing the synthesized AgNPs (silver nanoparticles). The scale bar in the image is 2 µm, and the diameter of the AgNPs ranges from approximately 20 nm to 50 nm. Please click here to view a larger version of this figure.

Figure 3: Simulation of AgNPs. Simulation showing AgNPs (silver nanoparticles) with significant localized field enhancement occurring in the gap between the particles. Please click here to view a larger version of this figure.

Figure 4: SERS signals of different R6G concentrations. Surface-enhanced Raman Scattering (SERS) signals obtained for various concentrations of R6G (Rhodamine 6G). The peak positions in the figure align with those in Table 1. Please click here to view a larger version of this figure.

Figure 5: SERS signals of R6G on the flexible substrate. Surface-enhanced Raman Scattering (SERS) signals of R6G (Rhodamine 6G) collected from 10 random points on the flexible substrate to demonstrate uniformity. Please click here to view a larger version of this figure.

Figure 6: SERS signals of different Thiram concentrations. Surface-enhanced Raman Scattering (SERS) signals obtained for various concentrations of Thiram. Please click here to view a larger version of this figure.

Figure 7: SERS signals of Thiram on the fruit surface. Surface-enhanced Raman Scattering (SERS) signals of Thiram obtained from the surface of an apple using the "paste and peel-off" method. The detection limit reached 10^-7 M of Thiram. Please click here to view a larger version of this figure.

Peak position (cm^-1)	Assignment
612	C-C-C in-plane bending vibration
774	C-H stretching
1127	C-H in-plane bending vibration
1180	C-H and N-H bending vibration
1310	C=C stretching
1364	Stretching vibration of the C-C bond
1509	Stretching vibration of the C-C bond
1574	Stretching vibration of the C=O bond
1647	Stretching vibration of the C-C bond

Table 1: Raman shift and frequency mode assignment in R6G SERS spectrum. Table listing Raman shift values and their corresponding frequency mode assignments in the Surface-Enhanced Raman Scattering (SERS) spectrum of R6G (Rhodamine 6G).

Peak position (cm^-1)	Assignment
440	CH3-N-C deformation (δ (CH3-N-C)), C=S stretching (υ(C=S))
549	S-S symmetric stretching (υs (S-S))
928	C=S stretching (υ (C=S)), C-N stretching (υ (CH3-N))
1136	C-N stretching (υ (C-N)), the rocking CH3 modes (ρ(CH3))
1388	C-N stretching (υ (C-N)), CH3 symmetric deformation (υ(C=S))

Table 2: Raman shift and frequency mode assignment in Thiram SERS spectrum. Table listing Raman shift values and their corresponding frequency mode assignments in the Surface-Enhanced Raman Scattering (SERS) spectrum of Thiram.

Supplementary File 1: Calculation of Enhancement Factor (ER). Please click here to download this File.

Discussion

In this study, a flexible SERS substrate was introduced, which bonded AgNPs to PDMS through chemical modification and achieved excellent performance. During particle synthesis, specifically in the silver ammonia complex synthesis (step 1.2), the color of the solution plays a crucial role. Adding too much ammonia water dropwise can adversely affect AgNPs synthesis quality, potentially leading to unsuccessful detection results. Attention should be paid to substrate modification (step 2.2) during the synthesis process; otherwise, AgNPs may not bond properly to PDMS, resulting in weakened detection performance.

In practical preparations, the SERS substrate's detection performance may exhibit instability²². This can be optimized by changing the solvent of the substance. For example, using acetonitrile as a solvent for Thiram yields better results than using ethanol. Additionally, the quality of Thiram can impact the detected SERS signal, emphasizing the importance of ensuring that the reagents used are within their expiration dates during detection.

Compared to other studies³⁶^,³⁷^,³⁸, the SERS flexible substrate's detection method proposed in this study is straightforward. AgNPs can be easily synthesized through a simple method, avoiding the need for complex experimental conditions and environments, as well as intricate fabrication processes. The substrate is environmentally friendly and does not introduce harmful pollutants. However, it should be noted that due to the glucose layer around AgNPs, it can weaken the enhancement effect of silver particles, suggesting that further improvement in the Enhancement Factor (EF) of the SERS substrate is necessary. The SERS flexible substrate, prepared using the method in this study, also requires further exploration in the detection of biomolecules.

The flexible SERS substrate proposed in this study demonstrates applicability in real-life scenarios, enriching the methods for pesticide residue detection and carrying significant implications. Furthermore, in future applications, the flexible SERS substrate holds great potential for biomedical applications.

Divulgaciones

The authors have nothing to disclose.

Acknowledgements

The research is supported by the National Natural Science Foundation of China (Grant No. 61974004 and 61931018), as well as the National Key R&D Program of China (Grant No. 2021YFB3200100). The study acknowledges the Electron Microscopy Laboratory of Peking University for providing access to electron microscopes. Additionally, the research extends thanks to Ying Cui and the School of Earth and Space Science of Peking University for their assistance in Raman measurements.

Materials

Ammonia (NH_3.H₂O, 25%)	Beijing Chemical Works
APTES (98%)	Beyotime	ST1087
BD-20AC Laboratory Chrona Treater	Electro-Technic Products Inc.	12051A
D-glucose	Beijing Chemical Works
Environmental Scanning electron microscope (ESEM)	FEI	QUANTA 250
Raman microscope	Horiba JY	LabRAM HR Evolution
Rhodamine 6G	Beijing Chemical Works
Silicone Elastomer Base and Silicone Elastomer Curing Agent	Dow Corning Corporation	SYLGARD 184
Silver nitrate	Beijing Chemical Works
Thiram (C₆H₁₂N₂S₂, 99.9%)	Beijing Chemical Works

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Lin, G., Zhu, J., Wang, Y., Yang, B., Xiong, S., Zhang, J., Wu, W. Fabrication of polydimethylsiloxane (PDMS)-Based Flexible Surface-Enhanced Raman Scattering (SERS) Substrate for Ultrasensitive Detection. J. Vis. Exp. (201), e65595, doi:10.3791/65595 (2023).