Ultrafast Laser-Ablated Nanoparticles and Nanostructures for Surface-Enhanced Raman Scattering-Based Sensing Applications

Summary

Ultrafast laser ablation in liquid is a precise and versatile technique for synthesizing nanomaterials (nanoparticles [NPs] and nanostructures [NSs]) in liquid/air environments. The laser-ablated nanomaterials can be functionalized with Raman-active molecules to enhance the Raman signal of analytes placed on or near the NSs/NPs.

Abstract

The technique of ultrafast laser ablation in liquids has evolved and matured over the past decade, with several impending applications in various fields such as sensing, catalysis, and medicine. The exceptional feature of this technique is the formation of nanoparticles (colloids) and nanostructures (solids) in a single experiment with ultrashort laser pulses. We have been working on this technique for the past few years, investigating its potential using the surface-enhanced Raman scattering (SERS) technique in hazardous materials sensing applications. Ultrafast laser-ablated substrates (solids and colloids) could detect several analyte molecules at the trace levels/mixture form, including dyes, explosives, pesticides, and biomolecules. Here, we present some of the results achieved using the targets of Ag, Au, Ag-Au, and Si. We have optimized the nanostructures (NSs) and nanoparticles (NPs) obtained (in liquids and air) using different pulse durations, wavelengths, energies, pulse shapes, and writing geometries. Thus, various NSs and NPs were tested for their efficiency in sensing numerous analyte molecules using a simple, portable Raman spectrometer. This methodology, once optimized, paves the way for on-field sensing applications. We discuss the protocols in (a) synthesizing the NPs/NSs via laser ablation, (b) characterization of NPs/NSs, and (c) their utilization in the SERS-based sensing studies.

Introduction

Ultrafast laser ablation is a rapidly evolving field of laser-material interactions. High-intensity laser pulses with pulse durations in the femtosecond (fs) to picosecond (ps) range are used to generate precise material ablation. Compared to nanosecond (ns) laser pulses, ps laser pulses can ablate materials with higher precision and accuracy due to their shorter pulse duration. They can generate less collateral damage, debris, and contamination of the ablated material due to fewer thermal effects. However, ps lasers are typically more expensive than ns lasers and need specialized expertise for operation and maintenance. The ultrafast laser pulses enable precise control over the energy deposition, which leads to highly localized and minimized thermal damage to the surrounding material. Additionally, ultrafast laser ablation can lead to the generation of unique nanomaterials (i.e., surfactants/capping agents are not obligatory during the production of nanomaterials). Therefore, we can term this a green synthesis/fabrication method^1,2,3. The mechanisms of ultrafast laser ablation are intricate. The technique involves different physical processes, such as (a) electronic excitation, (b) ionization, and (c) the generation of a dense plasma, which results in the ejection of material from the surface⁴. Laser ablation is a simple single-step process to produce nanoparticles (NPs) with high yield, narrow size distribution, and nanostructures (NSs). Naser et al.⁵ conducted a detailed review of the factors influencing the synthesis and production of NPs through the laser ablation method. The review covered various aspects, such as the parameters of a laser pulse, focusing conditions, and the ablation medium. The review also discussed their impact on producing a wide range of NPs using the laser ablation in liquid (LAL) method. The laser-ablated nanomaterials are promising materials, with applications in various fields such as catalysis, electronics, sensing, and biomedical, water splitting applications^{6,7,8,9,10,11,12,13,14}.

Surface-enhanced Raman scattering (SERS) is a powerful analytical sensing technique that significantly enhances the Raman signal from probe/analyte molecules adsorbed onto metallic NSs/NPs. SERS is based on the excitation of surface plasmon resonances in metallic NPs/NSs, which results in a significant rise in the local electromagnetic field near the metallic nano-features. This enhanced field interacts with the molecules adsorbed on the surface, significantly enhancing the Raman signal. This technique has been used to detect various analytes, including dyes, explosives, pesticides, proteins, DNA, and drugs^15,16,17. In recent years, significant progress has been made in the development of SERS substrates, including the use of differently shaped metallic NPs^18,19 (nanorods, nanostars, and nanowires), hybrid NSs^20,21 (a combination of the metal with other materials such as Si^22,23, GaAs²⁴, Ti²⁵, graphene²⁶, MOS₂²⁷, Fe²⁸, etc.), as well as flexible substrates^29,30 (paper, cloth, nanofiber, etc.). Developing these new strategies in the substrates has opened up new possibilities for using SERS in various real-time applications.

This protocol discusses the fabrication of Ag NPs using a ps laser at different wavelengths and Ag-Au alloy NPs (with different ratios of Ag and Au targets) fabricated using laser ablation technique in distilled water. Additionally, silicon micro/nanostructures are created using an fs laser on silicon in the air. These NPs and NSs are characterized using ultraviolet (UV)-visible absorption, transmission electron microscopy (TEM), X-ray diffraction (XRD), and field emission scanning electron microscopy (FESEM). Furthermore, the preparation of SERS substrates and analyte molecules are discussed, followed by the collection of Raman and SERS spectra of the analyte molecules. Data analysis is performed to determine the enhancement factor, sensitivity, and reproducibility of the laser-ablated NPs/NSs as potential sensors. Additionally, typical SERS studies are discussed, and the SERS performance of hybrid substrates is evaluated. Specifically, it has been found that the promising gold nanostars' SERS sensitivity can be enhanced approximately 21 times by using laser-structured silicon instead of plain surfaces (such as Si/glass) as a base.

Protocol

A typical protocol flowchart of the application of ultrafast ablated NPs or NSs in the trace detection of molecules via SERS is shown in Figure 1A.

1. Synthesizing metal NPs/NSs

NOTE: Depending on the requirement/application, choose the target material, the surrounding liquid, and the laser ablation parameters.
Here:
Target materials: Ag
Surrounding liquid: 10 mL of DI
Laser parameters: 355/532/1064 nm; 30 ps; 10 Hz; 15 mJ
Focusing lens: Plano-convex lens (focal length: 10 cm)
Stage parameters: 0.1 mm/s along the X and Y directions

1. Sample cleaning before laser ablation
   1. Perform ultrasonic cleaning (40 kHz, 50 W, 30 °C) of the target surface using acetone for 15 min, which removes various organic materials, including oils, greases, and waxes.
   2. Then, subject the surface to ultrasonic cleaning with ethanol for another 15 min to remove polar contaminants, such as salts and sugars.
   3. Finally, clean the surface with deionized water (DW) using ultrasonic cleaning for 15 min to remove any remaining traces of solvents or contaminants from the sample's surface.
      NOTE: These steps will help to eliminate any unwanted impurities that may be present on the surface, ensuring accurate analysis.
2. Measuring the weight of the sample
   1. Measure the weight of the sample before ablation.
   2. Perform the laser ablation experiment on the sample.
   3. Measure the weight of the sample again after the ablation experiment.
   4. By comparing the weight of the sample before and after ablation, estimate the amount of material that was removed during the experiment. This information will be useful in analyzing the properties of the ablated material, such as the concentration and yield of the ablated products.
3. Adjust the laser parameters
   1. Adjust the input laser power such that it is greater than the ablation threshold of the sample. Here, an input power of ~150 mW was used for ps laser ablation of the Ag target.
      NOTE: The threshold refers to the minimum energy per unit area required to heat the target material to the point where it is vaporized and converted into plasma.
   2. Combine a polarizer and a half-wave plate to adjust the laser pulse energy. Figure 1B shows the schematic of ultrafast laser ablation.
4. Laser focus adjustments on to the sample surface
   1. Focus the laser beam onto the sample using a focusing lens to ablate the material surface.
   2. Adjust the laser's focus on the sample manually using a translation stage in the Z-direction by observing the bright plasma produced and the emanating cracking sound.
      NOTE: To visualize the plasma generated during the laser ablation experiments, the photographs of both configurations are provided in Figure 2A: (i) laser ablation in air and (ii) laser ablation in liquid (LAL).
5. Different types of focusing
   NOTE: Focusing optics can help increase the energy density of the laser (plasma formation) beam at the sample surface, leading to more efficient ablation. Various types of focusing optics can be used, such as plano-convex lenses, axicon³¹, cylindrical lenses, etc.
   1. Use focusing optics for focusing the laser beam onto the sample, depending on the specific requirements, like achieving different ablation depths, allowing for better control over the synthesis of NPs/NSs. Figure 2B shows the three focusing conditions used in LAL.
      NOTE: Adjusting the laser focus onto the sample in laser ablation requires certain precautions to ensure safety and accuracy.
   2. Check and maintain the equipment used to manipulate the laser focus to ensure it functions correctly.
   3. Adjust the laser focus safely and accurately to minimize the risk of injury or damage to equipment.
      NOTE: The choice of the focal length of the lenses depends on the material used for laser ablation, the type of laser (pulse duration, beam size), and also the desired spot size at the sample surface.
6. Scanning area of the sample
   1. Position the sample on the X-Y stages that are connected to an ESP motion controller. The sample is moving perpendicular to the laser propagation direction.
      NOTE: The ESP motion controller is used to perform a raster scan of the sample in the X and Y directions to prevent single-point ablation.
   2. Adjust the scan speed (typically 0.1 mm/s for a better yield of metal NPs) and laser processing area to optimize the number of laser pulses that interact with the sample, as this affects the yield of the NPs.
   3. To achieve the desired dimensions and prevent single-point ablation, perform laser patterning while scanning the sample during the laser ablation process.
      NOTE: Figure 3A, B illustrates the fs laser ablation setup photograph by engaging Gaussian and Bessel beams, respectively.
7. Laser ablation in liquid to synthesize metal NPs/NSs
   1. Conduct a laser ablation experiment after setting up all the desired requirements. Follow the steps mentioned in steps 1.1-1.6.
   2. Make sure to monitor the laser power and other settings to ensure that they remain consistent throughout the experiment.
   3. Continuously observe the target material during the laser ablation experiment to ensure that the laser beam remains focused on the desired area.
      NOTE: Figure 3A,B shows the fs laser ablation experimental setups for synthesizing the NPs using a Gaussian beam and an axicon beam, respectively. A plano-convex lens was used for focusing the input pulses. The formation of NPs is evident from the pictures obtained at different times of the experiment. The color of the solution indicates the formation of NPs, and a color change in the solution indicates an increasing yield of the NPs (depicted in Figure 4). Laser safety goggles must be worn when working in the laser lab, using only approved laser safety eyewear for the proper wavelength. Any stray reflection of the high-power laser beam into the eye is extremely dangerous, resulting in irreversible damage. The laser beam should be kept pointing away from all the people in the laser lab. The optical elements in the setup were not disturbed on the optical table. The sample and stages should be monitored while the experiments are being performed.

2. Storage of colloidal NPs/NSs

1. Store the synthesized NPs in clean glass bottles and store NSs in airtight containers. Place both inside a desiccator.
   ​NOTE: Figure 5 shows colloidal NPs of various colors synthesized through LAL by combining different liquids and targets. Here, Figure 5A,B displays the typical photographs of different colloidal NPs, including (i) metal NPs, Ag, Au, and Cu NPs in various solvents, such as DW and NaCl; (ii) metal alloy NPs, Ag-Au NPs with different compositions, Ag-Cu NPs, and Au-Cu NPs; and (iii) metal-semiconductor alloy NPs, titanium-Au and silicon-Au/Ag NPs. These photographs illustrate the variety of NPs that can be synthesized using colloidal methods and showcase the unique optical properties of metal-semiconductor alloy NPs. Storing colloidal NPs properly is crucial to ensure their stability and maintain their properties. Glass bottles are preferred over plastic or metal containers as they do not react with the NPs. NPs/NSs should be stored in a container with a tight-fitting lid to minimize exposure to air and kept in a dark place that protects them from light.

3. Characterization of laser-ablated NPs/NSs

NOTE: Characterizing metal NSs/NPs is vital for comprehending their properties and ensuring their quality, such as size, shape, composition, etc.

1. Absorption spectroscopy
   NOTE: UV-visible absorption spectroscopy is a well-established technique for characterizing metal NPs. It is considered fast, simple, and noninvasive, making it a valuable tool for determining various properties of NPs. The position of the peaks is related to various properties of the NPs, such as their material composition, size distribution, shape, and the surrounding medium.
   1. Sample preparation for UV-visible absorption studies
      1. Prior to recording the spectrum, ensure that the NPs are evenly distributed and suspended in the solution. Fill a sample cuvette with the 3 mL of NP suspension and a reference cuvette filled with the base solvent (in which the NPs are dispersed). Make sure that the cuvettes are clean and free from contaminants.
      2. Collect the absorption data (in the spectral range from 200-900 nm) using a typical step size of 1 nm.
2. TEM analysis
   NOTE: Colloidal NP size and shape were examined by a transmission electron microscope and later analyzed using the software.
   1. TEM grid preparation
      1. Using a micropipette, gently dispense approximately 2 µL of the metal NP suspension onto a TEM grid coated with a thin carbon film on top of a thin copper grid. Let the solvent evaporate naturally at room temperature (RT).
         NOTE: For the collection of TEM images, an accelerating voltage of 200 kV and an electron gun current of ~100 µA were used. The micrographs were collected at different magnifications of 2 nm, 5 nm, 10 nm, 20 nm, 50 nm, 100 nm, and 200 nm. TEM analysis was used to find out the size and shape of the NPs.
3. SEM analysis
   NOTE: The surface morphology of the laser-ablated NSs and deposition/composition of the laser-ablated NPs on the bare Si/NSs was examined using FESEM. A typical photograph of a laser-ablated metal/semiconductor/alloy NS sample is shown in Figure 6.
   1. SEM sample preparation: For the SEM characterization of NPs, deposit a small droplet of the NP's suspension onto a cleaned silicon wafer, which serves as the sample holder. Then, dry the sample at RT.
   2. Use the metal NSs directly for FESEM characterization without further preparation for surface morphology.
      NOTE: For the collection of FESEM images, the electron high voltage was 3-5 kV and the working distance was typically 5-7 mm, at different magnifications of 5,000x, 10,000x, 20,000x, 50,000x, and 100,000x.
4. XRD analysis
   NOTE: XRD is a commonly used technique for characterizing the crystal structure and crystal quality of NPs.
   1. XRD sample preparation
      1. Drop cast 50-100 µL of the NP suspension onto a glass slide. Carefully add the drops to the center of a glass sample drop by drop. Add the drops slowly on the same spot to ensure the NPs are distributed on the glass to obtain good-quality XRD data.
         NOTE: The data was collected from 3°-90° with a step size of 0.01° over a duration of ~1 h. The X-ray wavelength used was 1.54 A°, the generator voltage was 40 kV, and the tube current was 30 mA.
      2. Subsequently dry the sample at RT to obtain a homogeneous, thin film of NPs.
   2. XRD data analysis
      1. Analyze the XRD peak positions with Joint Committee on Powder Diffraction Standards (JCPDS) Cards. Each JCPDS card contains information on a specific material's crystal structure, lattice parameters, and XRD pattern.

4. Application of the NPs/NSs

1. Raman analysis
   1. Initially, collect the desired analyte molecules' Raman spectra in powder form. Analyze the collected Raman data to identify the spectral peaks corresponding to the vibrational modes of the analyte molecule.
2. Stock solution preparation
   1. Confirm the solubility of the analyte molecules in the chosen solvent. Then, prepare stock solutions of the analyte molecules with accurately weighed or measured amounts.
   2. For example, to prepare a 50 mM stock solution of methylene blue (MB) molecule in 5 mL of ethanol:
      1. Calculate the amount of MB powder needed using the formula: mass = concentration (in mM) x volume (in L) x molecular weight (in g/mol). In this case, mass = 50 mM x 0.005 L x 319 g/mol = 0.7995 g or approximately 800 mg.
      2. Weigh out 800 mg of MB powder using a digital balance. Add the powder to a clean glass bottle.
      3. Add solvent to the bottle and shake vigorously to dissolve the powder. Tightly seal the bottle cap and mix the solution thoroughly.
3. Raman data collection
   1. Collect the stock solution Raman spectra by depositing a 10 µL drop of stock solution on a piece of clean silicon wafer. Figure 7A shows the photograph of a portable Raman spectrometer with a 785 nm laser excitation.
4. Analyte molecule preparation
   1. Using a micropipette, dilute the stock solution to different concentrations by adding an appropriate volume of solvent to a series of glass vials depending on the concentration range of interest.
   2. Prepare the series of dilutions from a 50 mM stock solution to a final concentration using the formula C_known x V_known = C_unknown x V_unknown.
5. SERS substrate preparation
   1. To prepare a SERS substrate using NPs, deposit a small drop of NPs on a clean silicon surface and allow it to dry. Then, place a tiny drop of the desired analyte molecule on the NP-coated silicon substrate. A schematic of the preparation of SERS substrates using NPs, hybrid, and metal NSs is shown in Figure 7B.
6. SERS spectra collection
   1. Collect the SERS data using a portable Raman spectrometer with a 785 nm laser excitation source. Compare the Raman peaks of the analyte molecule to the spectra with those of reference standards (powder and stock solution).
7. SERS data analysis
   1. Process the obtained Raman and SERS spectra for background correction, subtraction of fluorescence signals, smoothing of the signal, and baseline correction.
      1. Import the text file into ORIGIN software and then follow the steps: analysis > peak and baseline > peak analyser > open dialogue > substract baseline > next > user defined > add base line correction point > done > finish.
         NOTE: One can write their own Matlab/Python program to achieve this.
   2. Analyze the resulting peaks in terms of their positions and intensities by placing the reader/annotation point on the peak (in ORIGIN).
   3. Assign the peaks to their corresponding Raman vibrational mode assignments based on their spectral characteristics by collecting the bulk Raman spectrum, literature survey, and/or density functional theory (DFT) calculations.
8. Sensitivity calculation
   1. Calculate the enhancement factor (EF) scale, defined as the ratio of the Raman signal intensity obtained from the SERS active substrate to that obtained from the non-plasmonic substrate for a specific Raman mode of the analyte molecule.
9. Limit of detection
   1. Perform quantitative SERS analysis using a linear calibration curve, which represents the relationship between the concentration of the target analyte and its measured Raman signal intensity.
      ​Limit of detection (LOD) = 3 x (standard deviation of the background noise)/(slope of the calibration curve).
10. Reproducibility
    NOTE: The ability of the substrate to consistently produce the same or similar SERS signals for a given analyte molecule under the same experimental conditions is referred to as the reproducibility of the SERS substrate.
    1. Calculate the relative standard deviation (RSD) as follows: RSD = (standard deviation/mean) x 100%
       NOTE: In general, RSD values in the 5%-20% range are considered acceptable for most SERS experiments, but lower RSD values are often desirable for more quantitative and reliable SERS measurements

Results

Silver NPs were synthesized via ps laser ablation in liquid technique. Here, a ps laser system with a pulse duration of ~30 ps operating at a 10 Hz repetition rate and with a wavelength of one of 355, 532, or 1,064 nm was used. The input pulse energy was adjusted to 15 mJ. The laser pulses were focused using a plano-convex lens with a focal length of 10 cm. The laser focus should be exactly on the material surface during laser ablation because the laser energy is most concentrated at the focal point, where it ca...

Discussion

In ultrasonication cleaning, the material to be cleaned is immersed in a liquid and high-frequency sound waves are applied to the liquid using an ultrasonic cleaner. The sound waves cause the formation and implosion of tiny bubbles in the liquid, generating intense local energy and pressure that dislodge and remove dirt and other contaminants from the surface of the material. In laser ablation, a Brewster polarizer and a half-wave plate combination were used to tune the laser energy; the polarizer is typically placed bef...

Materials

Name	Company	Catalog Number	Comments
Alloys	Local goldsmith	N/A	99% pure
Axicon	Thorlabs	N/A	100, IR range, AR coated, AX1210-B
Ethanol	Supelco, India	CAS No. 64-17-5
Femtosecond laser	femtosecond  (fs)  laser amplifier  Libra HE, Coherent	N/A	Pulse duraction 50 fs; wavelenngth 800 nm; Rep rate 1 KHz; Pulse Energy: 4 mJ
FESEM	Carl ZEISS, Ultra 55	N/A
Gatan DM3	www.gatan.com	Gatan Microscopy Suite 3.x
Gold target	Sigma-Aldrich, India		99% pure
HAuCl4.3H2O	Sigma-Aldrich, India	CAS No. 16961-25-4
High resolution translational stages	Newport SPECTRA PHYSICS GMBI	N/A	M-443 High-Performance Low-Profile Ball Bearing Linear Stage; The stage is only 1 inch high, and has 2 inches of travel.
Micro Raman	Horiba LabRAM	N/A	Grating-1,800 and 600 grooves/mm; Wavelength of excitation-785 nm,632 nm, 532 nm, 325 nm; Objectives 10x, 20x, 50 x, 100x; CCD detector
Mirrors	Edmund Optics	N/A	Suitable mirrors for specific wavelength of laser
Motion controller	NEWPORT SPECTRA PIYSICS GMBI	N/A	ESP300 Controller-3 axes control
Origin	www.originlab.com	Origin 2018
Picosecond laser	EKSPLA 2251	N/A	Pulse duraction 30ps; wavelenngth 1064 nm, 532 nm, 355 nm; Rep rate 10 Hz; Pulse Energy: 1.5 to 30 mJ
Planoconvex lens		N/A	focal length 10 cm
Raman portable	i-Raman plus,  B&W Tek, USA	N/A	785 nm, ~ 100 µm laser spot  fiber optic probe excitation and collection
Silicon wafer	Macwin India Ltd.		1–10 Ω-cm, p (100)-type
Silver salt (AgNO3)	Finar, India	CAS No. 7783-90-6
Silver target	Sigma-Aldrich, India	CAS NO 7440-22-4	99% pure
TEM	Tecnai TEM	N/A
TEM grids	Sigma-Aldrich, India	TEM-CF200CU	Copper Grid Carbon Coated  200 mesh
Thiram	Sigma-Aldrich, India	CAS No. 137-26-8
UV	Jasco V-670	N/A
XRD	Bruker D8 advance	N/A