We thank the University of Hyderabad for support through the Institute of Eminence (IoE) project UOH/IOE/RC1/RC1-2016. The IoE grant obtained vide notification F11/9/2019-U3(A) from the MHRD, India. DRDO, India is acknowledged for funding support through ACRHEM [[#ERIP/ER/1501138/M/01/319/D(R&#38;D)]. We acknowledge the School of Physics, UoH, for the FESEM characterization and XRD facilities. We would like to extend our sincere gratitude to Prof SVS Nageswara Rao and his group for their valuable collaboration contributions and support. We would like to express our appreciation to past and present lab members Dr. P Gopala Krishna, Dr. Hamad Syed, Dr. Chandu Byram, Mr. S Sampath Kumar, Ms. Ch Bindu Madhuri, Ms. Reshma Beeram, Mr. A Mangababu, and Mr. K Ravi Kumar for their invaluable support and assistance during and after the laser ablation experiments in the lab. We acknowledge the successful collaboration of Dr. Prabhat Kumar Dwivedi, IIT Kanpur.

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
<ol>
	<li>Sample cleaning before laser ablation
	<ol>
		<li>Perform ultrasonic cleaning (40 kHz, 50 W, 30 &#176;C) of the target surface using acetone for 15 min, which removes various organic materials, including oils, greases, and waxes.</li>
		<li>Then, subject the surface to ultrasonic cleaning with ethanol for another 15 min to remove polar contaminants, such as salts and sugars.</li>
		<li>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&#39;s surface. 
		NOTE: These steps will help to eliminate any unwanted impurities that may be present on the surface, ensuring accurate analysis.</li>
	</ol>
	</li>
	<li>Measuring the weight of the sample
	<ol>
		<li>Measure the weight of the sample before ablation.</li>
		<li>Perform the laser ablation experiment on the sample.</li>
		<li>Measure the weight of the sample again after the ablation experiment.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Adjust the laser parameters
	<ol>
		<li>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.</li>
		<li>Combine a polarizer and a half-wave plate to adjust the laser pulse energy. Figure 1B shows the schematic of ultrafast laser ablation.</li>
	</ol>
	</li>
	<li>Laser focus adjustments on to the sample surface
	<ol>
		<li>Focus the laser beam onto the sample using a focusing lens to ablate the material surface.</li>
		<li>Adjust the laser&#39;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).</li>
	</ol>
	</li>
	<li>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.
	<ol>
		<li>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.</li>
		<li>Check and maintain the equipment used to manipulate the laser focus to ensure it functions correctly.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Scanning area of the sample
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Laser ablation in liquid to synthesize metal NPs/NSs
	<ol>
		<li>Conduct a laser ablation experiment after setting up all the desired requirements. Follow the steps mentioned in steps 1.1-1.6.</li>
		<li>Make sure to monitor the laser power and other settings to ensure that they remain consistent throughout the experiment.</li>
		<li>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&#160;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.</li>
	</ol>
	</li>
</ol>
2. Storage of colloidal NPs/NSs
<ol>
	<li>Store the synthesized NPs in clean glass bottles and store NSs in airtight containers. Place both inside a desiccator. 
	&#8203;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.</li>
</ol>
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.
<ol>
	<li>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.
	<ol>
		<li>Sample preparation for UV-visible absorption studies
		<ol>
			<li>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.</li>
			<li>Collect the absorption data (in the spectral range from 200-900 nm) using a typical step size of 1 nm.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>TEM analysis 
	NOTE: Colloidal NP size and shape were examined by a transmission electron microscope and later analyzed using the software.
	<ol>
		<li>TEM grid preparation
		<ol>
			<li>Using a micropipette, gently dispense approximately 2 &#181;L 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 &#181;A 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.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>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.
	<ol>
		<li>SEM sample preparation: For the SEM characterization of NPs, deposit a small droplet of the NP&#39;s suspension onto a cleaned silicon wafer, which serves as the sample holder. Then, dry the sample at RT.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>XRD analysis 
	NOTE: XRD is a commonly used technique for characterizing the crystal structure and crystal quality of NPs.
	<ol>
		<li>XRD sample preparation
		<ol>
			<li>Drop cast 50-100 &#181;L 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&#176;-90&#176; with a step size of 0.01&#176; over a duration of ~1 h. The X-ray wavelength used was 1.54 A&#176;, the generator voltage was 40 kV, and the tube current was 30 mA.</li>
			<li>Subsequently dry the sample at RT to obtain a homogeneous, thin film of NPs.</li>
		</ol>
		</li>
		<li>XRD data analysis
		<ol>
			<li>Analyze the XRD peak positions with Joint Committee on Powder Diffraction Standards (JCPDS) Cards. Each JCPDS card contains information on a specific material&#39;s crystal structure, lattice parameters, and XRD pattern.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
4. Application of the NPs/NSs
<ol>
	<li>Raman analysis
	<ol>
		<li>Initially, collect the desired analyte molecules&#39; Raman spectra in powder form. Analyze the collected Raman data to identify the spectral peaks corresponding to the vibrational modes of the analyte molecule.</li>
	</ol>
	</li>
	<li>Stock solution preparation
	<ol>
		<li>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.</li>
		<li>For example, to prepare a 50 mM stock solution of methylene blue (MB) molecule in 5 mL of ethanol:
		<ol>
			<li>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.</li>
			<li>Weigh out 800 mg of MB powder using a digital balance. Add the powder to a clean glass bottle.</li>
			<li>Add solvent to the bottle and shake vigorously to dissolve the powder. Tightly seal the bottle cap and mix the solution thoroughly.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Raman data collection
	<ol>
		<li>Collect the stock solution Raman spectra by depositing a 10 &#181;L 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.</li>
	</ol>
	</li>
	<li>Analyte molecule preparation
	<ol>
		<li>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.</li>
		<li>Prepare the series of dilutions from a 50 mM stock solution to a final concentration using the formula Cknown x Vknown = Cunknown x Vunknown.</li>
	</ol>
	</li>
	<li>SERS substrate preparation
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>SERS spectra collection
	<ol>
		<li>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).</li>
	</ol>
	</li>
	<li>SERS data analysis
	<ol>
		<li>Process the obtained Raman and SERS spectra for background correction, subtraction of fluorescence signals, smoothing of the signal, and baseline correction.
		<ol>
			<li>Import the text file into ORIGIN software and then follow the steps: analysis &#62; peak and baseline &#62; peak analyser &#62; open dialogue &#62; substract baseline &#62; next &#62; user defined &#62; add base line correction point &#62; done &#62; finish. 
			NOTE: One can write their own Matlab/Python program to achieve this.</li>
		</ol>
		</li>
		<li>Analyze the resulting peaks in terms of their positions and intensities by placing the reader/annotation point on the peak (in ORIGIN).</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Sensitivity calculation
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Limit of detection
	<ol>
		<li>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. 
		&#8203;Limit of detection (LOD) = 3 x (standard deviation of the background noise)/(slope of the calibration curve).</li>
	</ol>
	</li>
	<li>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.
	<ol>
		<li>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</li>
	</ol>
	</li>
</ol>

Synthesizing Nanoparticles and Nanostructures via Laser Ablation

The authors have nothing to disclose.

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 before the half-wave plate. The polarizer, which is mounted on a rotating mount, allows only light waves of a specific polarization to pass through while reflecting light waves of a perpendicular polarization. The light that passes through the polarizer then enters the half-wave plate, which rotates the polarization of the transmitted light by 90&#176;. When the sample was ablated in the air, only NSs were formed. However, when the sample was securely attached to the bottom of a clean glass beaker, filled with the intended volume of liquid, and ablated in liquid, both NPs and NSs were formed. The portion of the sample that was ablated by the laser contains NSs, while the ablated material dispersed in the surrounding liquid consists of NPs. LAL is a process in which ultrashort laser pulses are directed toward a sample submerged in a liquid, causing localized vaporization of the material. This results in the formation of NPs and NSs in a single step.
LAL has several advantages over other NPs synthesis methods. It is fast, efficient, scalable, and surfactant free. Additionally, the choice of solvent, the concentration of the target material in the solvent, and the presence of any surfactants or stabilizing agents can also impact the NP synthesis process, and therefore should be carefully considered and controlled. The processing and laser parameters (laser fluence, wavelength, pulse duration, repetition rate) can be adjusted to control the size, shape, composition, and surface properties of the produced NPs. Depending on the material, the penetration depth and the material&#39;s ablation threshold depend on the incident laser wavelength. All parameters will affect the yield of NPs/morphology of NSs. This level of control allows for tailoring the properties of nanomaterials to meet the specific requirements of diverse applications. The color of metal NPs is a primary and simple indication of their size and shape, as well as the material they are made of3. When light interacts with metal NPs, the electrons in the metal absorb and re-emit the light at specific wavelengths, leading to the color observed. The LAL technique uses bulk targets, which are cheaper than salts used in the wet-chemical technique. Moreover, hazardous waste is generated during the chemical process. Although the wet-chemical technique has a lower initial investment cost compared to the LAL technique, the latter requires a higher initial investment. However, the cost of LAL gradually decreases over time and ultimately becomes cheaper due to the lower cost of reactants2. Currently, many companies worldwide have launched startups focused on commercializing products synthesized using laser technology. Examples include IMRA (USA), Particular GmbH (Germany), and Zhongke Napu New Materials Co. Ltd. (China)35.
Recently, a lot of studies have been conducted to achieve superior SERS substrates using ultrafast laser techniques. Yu et al.8 have recently reported a hybrid super-hydrophobic/hydrophilic SERS platform by fs laser ablation and detected R6G with an EF of ~1013. Dipanjan et al. have reported the formation of ladder-like laser-induced periodic surface structures (LIPSS) on Ag-Au-Cu using fs Bessel beam ablation and successfully detected two explosive (tetryl and pentaerythritol tetranitrate) traces (200 nM)31. Verma et al. have used the technique of LAL and fabricated Au-Pd core@shell NPs by LAL, and used them in explosive (PA -10-7 and AN- 10-8) trace detection36. Verma et al. have again utilized Au NPs deposited on laser-textured Sn and detected PA at a concentration of 0.37 &#181;M and AN at 2.93 nM37.
During SERS measurements, when a small volume of NPs is dropped onto a substrate and left to dry, a spontaneous hydrodynamic process occurs, creating a local flow field within the drop. This flow carries the NPs to the edge of the drop, resulting in a phenomenon known as the &#34;coffee ring&#34; effect, where a dense array of NPs accumulates at the edge of the drop, rather than being evenly distributed throughout. While this natural process can increase the number of hot spots, it may also affect the reproducibility of the SERS signals8. The deposition of NPs onto the substrate depends on the contact angle between the solvent and the surface. The wetting behavior of the substrate can be altered by tuning the laser processing parameters in the laser ablation technique. Mangababu et al.24 have shown that the contact angle of GaAs laser ablation can vary in different surrounding liquids, such as distilled water, ethanol, and polyvinyl alcohol. Another possible way to avoid the coffee ring effect is to heat the substrate to 70 &#176;C, for instance, and then drop cast the analyte so that it dries up very fast.
The EF is an important factor for characterizing the performance of the SERS active substrate, and it depends on various factors, such as the morphology of the substrate, molecular geometry of the analyte, excitation wavelength, and polarization of the excitation laser. The EF also depends on the orientation of the analyte molecule with respect to the local field, the orientation of the substrate with reference to the incident laser direction, and the thickness of the analyte layer on the substrate. The EF is estimated using the simple relation EF = (ISERS x IR)/(CR x CSERS), where ISERS is the Raman signal intensity with NPs on Si/FP, IR is the Raman intensity on Si/FP (without NPs), CSERS is the concentration of the sample on NP substrates (low concentration), and CR is the concentration of the sample (0.1 M) which produces the Raman signal (IR)30,32,34. A series of standards with known concentrations of the analyte molecule is measured, and the Raman signal intensity of the most prominent peak is plotted against the concentration. The slope of the resulting line represents the sensitivity of the SERS measurement, and the intercept represents the background signal. The limit of detection (LOD), which is the smallest concentration of the target analyte that can be reliably detected, is estimated from the linear calibration curve. From this, we can estimate the sensitivity of the prepared SERS substrate. Multiple SERS measurements were performed on the same substrate in different locations and the intensity values of the most prominent peak were noted. RSD is a commonly used metric to characterize the reproducibility and reliability of SERS signals. It is defined as the standard deviation (SD) ratio of a set of measurements to the mean value expressed as a percentage. The RSD is a measure of the variability of the SERS signals, and it provides information about the precision of the measurements. A low RSD value indicates high precision and reproducibility, while a high RSD value indicates low precision and high variability30,34.
Producing star-shaped NPs using LAL is challenging, but they are proven to be superior SERS substrates due to the multiple hot spots arising from the strong electromagnetic fields at the sharp edges/tips19. Most studies have reported differently shaped metal NP deposition on plain Si/glass38,39. Here, we have shown further improvement in the sensitivity of metal NPs by using laser-textured Si instead of a plain Si surface. The hybrid SERS substrates, consisting of laser-ablated Si NSs and chemically synthesized Au nanostars, exhibited ~21 times the enhancement of the SERS signal compared to plain Si. Even with our laser-synthesized metal NPs, better SERS performance can be achieved by depositing them on laser-textured material. Previously, we demonstrated that laser-ablated Ag NPs coupled with laser-ablated Ag NSs for detecting 2, 4-dinitrotoluene provided one order of increment in the EF40. Here, we aimed to demonstrate that laser-ablated NSs can be used as a platform for any size/shape NPs to achieve better sensitivity and reproducibility. We firmly believe there is tremendous scope for ultrafast laser-ablated NPs and NSs in SERS-based sensing applications2,38,39,41,42,43.

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 method1,2,3. 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 applications6,7,8,9,10,11,12,13,14.
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 drugs15,16,17. In recent years, significant progress has been made in the development of SERS substrates, including the use of differently shaped metallic NPs18,19 (nanorods, nanostars, and nanowires), hybrid NSs20,21 (a combination of the metal with other materials&#160;such as Si22,23, GaAs24, Ti25, graphene26, MOS227, Fe28, etc.), as well as flexible substrates29,30 (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&#160;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&#39; SERS sensitivity can be enhanced approximately 21 times by using laser-structured silicon instead of plain surfaces (such as Si/glass) as a base.

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

ultrafast laser-ablated nanoparticles and nanostructures for surface-enhanced raman scattering-based sensing applications

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.

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 can cause the desired material removal. If the laser focus is not on the material surface, the laser energy is distributed over a larger area; it may not be sufficient for material removal or surface modifications. It may finally lead to an incomplete or inconsistent ablation. The sample was translated using 0.1 mm/s along the X and Y directions. The Ag target was immersed in 10 mL of DI, and the liquid height above the sample was ~7 mm. In general, the height of the solvent should be sufficient to cover the entire target material and prevent the material from overheating during laser ablation. Also, if the liquid height is too high, it can absorb some of the input laser energy before it reaches the target material, leading to a reduced ablation mechanism and a lower yield of NPs. If the liquid height is too low, at higher input laser energies, it may lead to agglomeration of the NPs. Additionally, it should be chosen to provide sufficient dispersion of the ablated material and prevent agglomeration of the NPs. The weight of the target is measured before and after the ablation process will give an idea of the amount of material that has been removed. Here, the ablated mass was estimated to be ~0.37, ~0.38, and ~0.41 mg at 355, 532, and 1,064 nm, respectively. This is important for estimating the yield of the desired colloidal NPs and ensuring the process is reproducible under the same experimental conditions. Next, the synthesized Ag NPs were characterized by UV-visible absorption spectroscopy. This method measures the amount of light absorbed by the NPs at different wavelengths in the UV-visible near infrared (NIR) regions of the spectrum. The absorption spectra obtained from UV-visible spectroscopy can be used to determine the localized surface plasmon resonance (LSPR) of the NPs. LSPR is a collective oscillation of electrons in the NPs, resulting in an absorption peak in the UV-visible region.
Figure 8A shows the absorption spectra of Ag colloidal NPs achieved by ps laser ablation of Ag in DW at different wavelengths (355 nm, 532 nm, and 1,064 nm). The spectra reveal that the surface plasmon resonance (SPR) peaks of the resulting NPs were located at ~420 nm, ~394 nm, and ~403 nm for the NPs attained at 355 nm, 532 nm, and 1,064 nm, respectively. The absorbance of the NPs increased with decreasing laser wavelength. This may be attributed to the higher levels of self-absorption of the laser pulses at lower wavelengths. Figure 8B illustrates the normalized absorbance spectra of Ag-Au alloy NPs with different compositions. The SPR peak position shifted from 410 nm to 519 nm, with an increase in the Au percentage from 0% to 100%. Figure 8C represents a correlation between the SPR peak position and Au mole fraction in Ag-Au alloy NPs. This relationship provides a useful tool for predicting the SPR peak position of Ag-Au alloy NPs with different compositions, which can aid in the design and synthesis of NPs with specific optical properties. Further, TEM studies were performed to examine the size and shape of Ag NPs. Figure 9A-C presents the TEM images of Ag NPs in DW at 355 nm, 532 nm, and 1,064 nm, respectively. The shape of Ag NPs was spherical, and the size distribution of Ag NPs in DW is shown in Figure 9D-F. The average sizes of the Ag NPs were ~12.4 nm &#177; 0.27 nm, ~23.9 nm &#177; 1.0 nm, and ~25 nm &#177; 0.7 nm, respectively, at 355 nm, 532 nm, and 1,064 nm. The average size of Ag NPs fabricated with 1,064 nm laser light was larger than that of those fabricated with 355 nm and 532 nm laser pulses. It has been reported that the increase in NP sizes with increasing wavelength was presumably the coexistence of LAL and the self-absorption of laser light by colloid metal particles causing laser fragmentation in liquids (LFL). Further, typical XRD patterns of Ag NPs on glass slides were recorded (Figure 10). The 2theta positions refer to the angles at which a crystalline material diffracts the X-rays. The angle is measured between the incident X-ray beam and the detector and is expressed in degrees. The peak maxima are positioned at 38.4&#176;, 44.6&#176;, 64.7&#176;, and 77.7&#176;, and correspond to the Bragg reflections of Ag from the planes having miller indices (1 1 1), (2 0 0), (2 2 0), (3 1 1), and (2 2 2), respectively. The noticed peaks are matched with JCPDS file number Ag: 03-0921 with a face-centered cubic structure.
Further, the typical FESEM micrographs of Ag NP-deposited Si, single-line laser-ablated Si, cross-patterned on Si, and laser-ablated iron (Fe) in acetone morphologies are provided, which are shown in Figure 11. Depending on the laser-material interaction, the morphology of the substrate structures, such as LIPSS/Groove/ripples, etc., can be formed. Typical FESEM images of star-shaped Au NPs deposited on the bare Si and laser-ablated cross-patterned Si surfaces are depicted in Figure 12. The distribution of the Au NPs on bare Si is shown in Figure 12A. Figure 12B-D presents the distribution of Au nanostars on the laser-ablated Si surface. Figure 12B shows the distribution on the non-interacted surface, while Figure 12C,D illustrates the FESEM images of laser-patterned Si micro/nanostructures with the Au NPs distribution.
Afterward, the application of the laser-ablated NPs/NSs in SERS studies were executed. The Raman and SERS substrate preparation (with and without NPs) and the collection of the corresponding Raman and SERS spectra of MB are shown in Figure 13. The enhancement of the Raman peaks of the MB molecule was clearly observed even at a concentration of 5 &#181;M, which is 20,000 times less than the concentration used for Raman - 100 mM (stock solution). The MB molecule Raman peaks were enhanced in the presence of NPs compared to those without NPs. Figure 14A-C illustrates the obtained SERS intensity of MB, NB, and thiram using the laser-patterned silicon (at different pulse numbers by varying scan speed and pattern): (i) Si_5L, (ii) Si_5C, (iii) Si_0.5L, and (iv) Si_0.5C with Au nanostars. The Raman enhancement of the three molecules is noticed from the Si NSs, and also the reproducibility is verified from 15 different locations from four substrates. The histogram plot in Figure 14D with the RSD for Si_5L, Si_5C, Si_0.5L, and Si_0.5C reveals that the Si NSs with star substrates showed a better SERS signal all over the area.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65450/65450fig01.jpg" /> 
Figure 1: Schematic and flowchart of ultrafast laser ablation. (A) Typical flow chart of trace detection using ultrafast laser-ablated NPs/NSs via SERS. (B) Schematic of ultrafast laser ablation in liquid. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65450/65450fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65450/65450fig02.jpg" /> 
Figure 2: Photographs of various laser ablation experiments. (A) Photographs of (a) LAL of Au in air and (b) LAL of Ag target in a gold salt (HAuCl4) solution (bright spot shown is the plasma). (B) Photographs of laser ablation in different focusing conditions with a (a) plano-convex lens, (b) axicon lens, and (c) cylindrical lens. Here, there is typically a 7 mm liquid height for 10 mL of solution at 500 &#181;J for the plano-convex lens, 3 mm height for 5 mL of solution for the Bessel beam, and 10 mm height for 10 mL for the cylindrical lens. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65450/65450fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65450/65450fig03.jpg" /> 
Figure 3: Photographs of fs laser ablation. (A) Photograph of (a) the fs laser ablation setup and the resulting (b) metal NPs (during laser ablation), and (c) metal NSs (after laser ablation) using the plano-convex lens. (B) Photograph of (a) fs laser ablation using the axicon lens and a (b) zoomed-in image of the photograph. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65450/65450fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65450/65450fig04.jpg" /> 
Figure 4: Photographs of fs laser ablation in liquid at different times using the plano-convex lens. (A) 1 min, (B) 5 min, (C) 20 min. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65450/65450fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65450/65450fig05.jpg" /> 
Figure 5: Photographs of laser-ablated colloidal NPs. (A) Au NPs at different energies (&#181;J): (a) 200, (b) 300, (c) 400, and (d) 500 by fs laser ablation in DW. (B) (a) Ag NPs, (b) Au NPs, and (c) Cu NPs by fs LAL in DW. (C) Aggregated Au NPs (fs laser ablation in DW) in different concentrations of NaCl (mM): (a) 1, (b) 10, (c) 50, (d) 100, (e) 500 mM, and (f) 1 M32. (D) (a) Pure Ag, (b) Ag50Au50,and (c) pure Au by ps laser ablation in NaCl. (E) Alloy NPs: (a) pure Ag, (b) Ag70Au30, (c) Ag50Au50, (d) Ag30Au70, and (e) pure Au by ps laser ablation in distilled water colloidal NPs. (F) Alloy NPs: (a) Ag60Au40, (b) Ag50Au50, (c) Ag40Au60, (d) Ag30Au70, and (e) Ag20Au80 by fs laser ablation in acetone. (G) Metal alloy NPs: (a) Cu_Au, (b) Ag_Au, and (c) Ag_Cu. (H) Metal semiconductor alloy NPs: (a) Au_TiO2, (b) Ag-SiO2, and (c) Au_SiO2 NPs <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65450/65450fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/65450/65450fig06.jpg" /> 
Figure 6: Photograph of fs laser-ablated NPs. (A) Ag, (B) Au, (C) Cu, (D) Si, and (E-H) Ag-Au alloy NPs with different ratios of Ag and Au. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65450/65450fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/65450/65450fig07.jpg" /> 
Figure 7: SERS substrate preparation. (A) Photograph of the portable Raman spectrometer. (B) Schematic of the SERS substrate preparation using (1) colloidal metal NPs, (2) rigid metal NSs, and (3) hybrid substrate (NSs+NPs). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65450/65450fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/65450/65450fig08.jpg" /> 
Figure 8: Absorption spectra. (A) Absorption spectra of the ps laser-ablated Ag NPs in DW using different laser wavelengths. (B) Normalized UV-visible absorption spectra of ps laser (1064 nm)-ablated Au-Ag NPs: (i) pure Ag, (ii) Ag70Au30, (iii) Ag50Au50, (iv) Ag30Au70, and (v) pure Au. (C) Shift in the SPR peak position with increasing Au percentage in Ag-Au alloy NPs. Panels B and C have been reproduced with permission from Byram et al33. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65450/65450fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/65450/65450fig09.jpg" /> 
Figure 9: TEM images and their respective size histograms of Ag NPs fabricated in DW using 30&#160;ps laser pulses. (A,D) 355 nm, (B,E) 532 nm, and (C,F) 1,064 nm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65450/65450fig09large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/65450/65450fig10v2.jpg" /> 
Figure 10: XRD pattern of ps (1,064 nm) laser-ablated Ag NPs in DW. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65450/65450fig10largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 11" class="xfigimg" src="/files/ftp_upload/65450/65450fig11.jpg" /> 
Figure 11: FESEM images. (A) Ag-Au alloy NPs deposited Si. (B) Single line ablation of Si. (C) Cross-patterned ablation on Si. (D) Fe NSs in acetone using fs laser ablation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65450/65450fig11large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 12" class="xfigimg" src="/files/ftp_upload/65450/65450fig12.jpg" /> 
Figure 12: FESEM images. (A) Au nanostars on the bare Si. (B-D) Au NPs decorated on different areas of laser-ablated Si: (B) area of unprocessed Si with redeposited Si NPs, (C) within the channel written using laser pulses, and (D) at the edge of the channel with spikes. This figure has been reproduced with permission from Moram et al.34. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65450/65450fig12large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 13" class="xfigimg" src="/files/ftp_upload/65450/65450fig13.jpg" /> 
Figure 13: Raman and SERS spectra of the MB molecule. Schematic of Raman and SERS spectra collection with preparation and the typical obtained Raman (MB: 100 mM, red color) and SERS (5 &#181;M, green colors) spectra of the MB molecule <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65450/65450fig13large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 14" class="xfigimg" src="/files/ftp_upload/65450/65450fig14.jpg" /> 
Figure 14: SERS spectra. (A) MB: 1.6 ppb, (B) NB: 1.8 ppb, and (C) thiram: 0.1 ppm using star-shaped Au NPs on linear and cross-patterned Si using fs laser ablation in the air at different scan speeds-5 mm/s and 0.5 mm/s: (i) plain Si, (ii) Si_5 mm/s -Linear, (iii) Si_5 mm/s-crossed, (iv) Si_0.5 mm/s-linear, and (v) Si_0.5 mm/s-crossed. MB, NB, and thiram molecular structures are also shown as an inset of the figures. (D) Histogram of prominent peak intensity variation from 15 random sites from all four Si substrates with Au nanostars. This figure has been reproduced with permission from Moram et al.34. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65450/65450fig14large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Advancements and Applications in Nanoparticle Synthesis Through Laser Ablation in Liquids at JoVE.com

Author Spotlight: Advancements and Applications in Nanoparticle Synthesis Through Laser Ablation in Liquids 

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.

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

The technique of ultrafast laser ablation in liquids has evolved and matured over the past decade, with several impending applications in various fields ...

ultrafast-laser-ablated-nanoparticles-nanostructures-for-surface

Nanyang Technological University

Research

JoVE Journal

Engineering

1.7K Views.  University of Hyderabad. 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.

Video: Author Spotlight: Advancements and Applications in Nanoparticle Synthesis Through Laser Ablation in Liquids 

Hyperpolarized Xenon for NMR and MRI Applications

Nuclear magnetic resonance (NMR) spectroscopy and imaging (MRI) suffer from intrinsic low sensitivity because even strong external magnetic fields of ~10 T generate only a small detectable net-magnetization of the sample at room temperature 1. Hence, most NMR and MRI applications rely on the detection of molecules at relative high concentration (e.g., water for imaging of biological tissue) or require excessive acquisition times. This limits our ability to exploit the very useful molecular specificity of NMR signals for many biochemical and medical applications. However, novel approaches have emerged in the past few years: Manipulation of the detected spin species prior to detection inside the NMR/MRI magnet can dramatically increase the magnetization and therefore allows detection of molecules at much lower concentration 2.
Here, we present a method for polarization of a xenon gas mixture (2-5% Xe, 10% N2, He balance) in a compact setup with a ca. 16000-fold signal enhancement. Modern line-narrowed diode lasers allow efficient polarization 7 and immediate use of gas mixture even if the noble gas is not separated from the other components. The SEOP apparatus is explained and determination of the achieved spin polarization is demonstrated for performance control of the method.
The hyperpolarized gas can be used for void space imaging, including gas flow imaging or diffusion studies at the interfaces with other materials 8,9. Moreover, the Xe NMR signal is extremely sensitive to its molecular environment 6. This enables the option to use it as an NMR/MRI contrast agent when dissolved in aqueous solution with functionalized molecular hosts that temporarily trap the gas 10,11. Direct detection and high-sensitivity indirect detection of such constructs is demonstrated in both spectroscopic and imaging mode.

The production of hyperpolarized xenon by means of spin exchange optical pumping (SEOP) is described. This method yields a ~10000-fold enhancement of the nuclear spin polarization of Xe-129 and has applications in nuclear magnetic resonance spectroscopy and imaging. Examples of gas phase and solution state experiments are given.

Nuclear magnetic resonance (NMR) spectroscopy and imaging (MRI) suffer from intrinsic low sensitivity because even strong external magnetic fields of ~10 ...

Synthesis and Operation of Fluorescent-core Microcavities for Refractometric Sensing

This paper discusses fluorescent core microcavity-based sensors that can operate in a microfluidic analysis setup. These structures are based on the formation of a fluorescent quantum-dot (QD) coating on the channel surface of a conventional microcapillary. Silicon QDs are especially attractive for this application, owing in part to their negligible toxicity compared to the II-VI and II-VI compound QDs, which are legislatively controlled substances in many countries. While the ensemble emission spectrum is broad and featureless, an Si-QD film on the channel wall of a capillary features a set of sharp, narrow peaks in the fluorescence spectrum, corresponding to the electromagnetic resonances for light trapped within the film. The peak wavelength of these resonances is sensitive to the external medium, thus permitting the device to function as a refractometric sensor in which the QDs never come into physical contact with the analyte. The experimental methods associated with the fabrication of the fluorescent-core microcapillaries are discussed in detail, as well as the analysis methods. Finally, a comparison is made between these structures and the more widely investigated liquid-core optical ring resonators, in terms of microfluidic sensing capabilities.

Fluorescent-core microcavity sensors employ a high-index quantum-dot coating in the channel of silica microcapillaries. Changes in the refractive index of fluids pumped into the capillary channel cause shifts in the microcavity fluorescence spectrum that can be used to analyze the channel medium.

This paper discusses  fluorescent core microcavity-based sensors that can operate in a microfluidic  analysis setup. These structures are based on the ...

Fabricating a UV-Vis and Raman Spectroscopy Immunoassay Platform

Immunoassays are used to detect proteins based on the presence of associated antibodies. Because of their extensive use in research and clinical settings, a large infrastructure of immunoassay instruments and materials can be found. For example, 96- and 384-well polystyrene plates are available commercially and have a standard design to accommodate ultraviolet-visible (UV-Vis) spectroscopy machines from various manufacturers. In addition, a wide variety of immunoglobulins, detection tags, and blocking agents for customized immunoassay designs such as enzyme-linked immunosorbent assays (ELISA) are available.
Despite the existing infrastructure, standard ELISA kits do not meet all research needs, requiring individualized immunoassay development, which can be expensive and time-consuming. For example, ELISA kits have low multiplexing (detection of more than one analyte at a time) capabilities as they usually depend on fluorescence or colorimetric methods for detection. Colorimetric and fluorescent-based analyses have limited multiplexing capabilities due to broad spectral peaks. In contrast, Raman spectroscopy-based methods have a much greater capability for multiplexing due to narrow emission peaks. Another advantage of Raman spectroscopy is that Raman reporters experience significantly less photobleaching than fluorescent tags1. Despite the advantages that Raman reporters have over fluorescent and colorimetric tags, protocols to fabricate Raman-based immunoassays are limited. The purpose of this paper is to provide a protocol to prepare functionalized probes to use in conjunction with polystyrene plates for direct detection of analytes by UV-Vis analysis and Raman spectroscopy. This protocol will allow researchers to take a do-it-yourself approach for future multi-analyte detection while capitalizing on pre-established infrastructure.

Nanoparticle-based optical probes have been designed as a vehicle for detecting antigens using Raman and UV-Vis spectroscopy. Here we describe a protocol for preparing such probes for a UV-Vis/Raman spectroscopy immunoassay in such a way to incorporate future multiplexing capabilities.

Immunoassays are used to detect proteins based on the presence of associated antibodies. Because of their extensive use in research and clinical settings, ...

Biochemistry

Spatial Separation of Molecular Conformers and Clusters

Gas-phase molecular physics and physical chemistry experiments commonly use supersonic expansions through pulsed valves for the production of cold molecular beams. However, these beams often contain multiple conformers and clusters, even at low rotational temperatures. We present an experimental methodology that allows the spatial separation of these constituent parts of a molecular beam expansion. Using an electric deflector the beam is separated by its mass-to-dipole moment ratio, analogous to a bender or an electric sector mass spectrometer spatially dispersing charged molecules on the basis of their mass-to-charge ratio. This deflector exploits the Stark effect in an inhomogeneous electric field and allows the separation of individual species of polar neutral molecules and clusters. It furthermore allows the selection of the coldest part of a molecular beam, as low-energy rotational quantum states generally experience the largest deflection. Different structural isomers (conformers) of a species can be separated due to the different arrangement of functional groups, which leads to distinct dipole moments. These are exploited by the electrostatic deflector for the production of a conformationally pure sample from a molecular beam. Similarly, specific cluster stoichiometries can be selected, as the mass and dipole moment of a given cluster depends on the degree of solvation around the parent molecule. This allows experiments on specific cluster sizes and structures, enabling the systematic study of solvation of neutral molecules.

We present a technique that allows the spatial separation of different conformers or clusters present in a molecular beam. An electrostatic deflector is used to separate species by their mass-to-dipole moment ratio, leading to the production of gas-phase ensembles of a single conformer or cluster stoichiometry.

Gas-phase molecular physics and physical chemistry experiments commonly use supersonic expansions through pulsed valves for the production of cold ...

Writing and Low-Temperature Characterization of Oxide Nanostructures

Oxide nanoelectronics is a rapidly growing field which seeks to develop novel materials with multifunctional behavior at nanoscale dimensions. Oxide interfaces exhibit a wide range of properties that can be controlled include conduction, piezoelectric behavior, ferromagnetism, superconductivity and nonlinear optical properties. Recently, methods for controlling these properties at extreme nanoscale dimensions have been discovered and developed. Here are described explicit step-by-step procedures for creating LaAlO3/SrTiO3 nanostructures using a reversible conductive atomic force microscopy technique. The processing steps for creating electrical contacts to the LaAlO3/SrTiO3 interface are first described. Conductive nanostructures are created by applying voltages to a conductive atomic force microscope tip and locally switching the LaAlO3/SrTiO3 interface to a conductive state. A versatile nanolithography toolkit has been developed expressly for the purpose of controlling the atomic force microscope (AFM) tip path and voltage. Then, these nanostructures are placed in a cryostat and transport measurements are performed. The procedures described here should be useful to others wishing to conduct research in oxide nanoelectronics.

Oxide nanostructures provide new opportunities for science and technology. The interfacial conductivity between LaAlO3 and SrTiO3 can be controlled with near-atomic precision using a conductive atomic force microscopy technique. The protocol for creating and measuring conductive nanostructures at LaAlO3/SrTiO3 interfaces is demonstrated.

Oxide nanoelectronics is a rapidly growing field which seeks to develop novel materials with multifunctional behavior at nanoscale dimensions.  Oxide ...

Surface Enhanced Raman Spectroscopy Detection of Biomolecules Using EBL Fabricated Nanostructured Substrates

Fabrication and characterization of conjugate nano-biological systems interfacing metallic nanostructures on solid supports with immobilized biomolecules is reported. The entire sequence of relevant experimental steps is described, involving the fabrication of nanostructured substrates using electron beam lithography, immobilization of biomolecules on the substrates, and their characterization utilizing surface-enhanced Raman spectroscopy (SERS). Three different designs of nano-biological systems are employed, including protein A, glucose binding protein, and a dopamine binding DNA aptamer. In the latter two cases, the binding of respective ligands, D-glucose and dopamine, is also included. The three kinds of biomolecules are immobilized on nanostructured substrates by different methods, and the results of SERS imaging are reported. The capabilities of SERS to detect vibrational modes from surface-immobilized proteins, as well as to capture the protein-ligand and aptamer-ligand binding are demonstrated. The results also illustrate the influence of the surface nanostructure geometry, biomolecules immobilization strategy, Raman activity of the molecules and presence or absence of the ligand binding on the SERS spectra acquired.

We describe the fabrication and characterization of nano-biological systems interfacing nanostructured substrates with immobilized proteins and aptamers. The relevant experimental steps involving lithographic fabrication of nanostructured substrates, bio-functionalization, and surface-enhanced Raman spectroscopy (SERS) characterization, are reported. SERS detection of surface-immobilized proteins, and probing of protein-ligand and aptamer-ligand binding is demonstrated.

Fabrication and characterization of conjugate nano-biological systems interfacing metallic nanostructures on solid supports with immobilized biomolecules ...

Ohmic Contact Fabrication Using a Focused-ion Beam Technique and Electrical Characterization for Layer Semiconductor Nanostructures

Layer semiconductors with easily processed two-dimensional (2D) structures exhibit indirect-to-direct bandgap transitions and superior transistor performance, which suggest a new direction for the development of next-generation ultrathin and flexible photonic and electronic devices. Enhanced luminescence quantum efficiency has been widely observed in these atomically thin 2D crystals. However, dimension effects beyond quantum confinement thicknesses or even at the micrometer scale are not expected and have rarely been observed. In this study, molybdenum diselenide (MoSe2) layer crystals with a thickness range of 6-2,700 nm were fabricated as two- or four-terminal devices. Ohmic contact formation was successfully achieved by the focused-ion beam (FIB) deposition method using platinum (Pt) as a contact metal. Layer crystals with various thicknesses were prepared through simple mechanical exfoliation by using dicing tape. Current-voltage curve measurements were performed to determine the conductivity value of the layer nanocrystals. In addition, high-resolution transmission electron microscopy, selected-area electron diffractometry, and energy-dispersive X-ray spectroscopy were used to characterize the interface of the metal&#8211;semiconductor contact of the FIB-fabricated MoSe2 devices. After applying the approaches, the substantial thickness-dependent electrical conductivity in a wide thickness range for the MoSe2-layer semiconductor was observed. The conductivity increased by over two orders of magnitude from 4.6 to 1,500 &#937;&#8722;1 cm&#8722;1, with a decrease in the thickness from 2,700 to 6 nm. In addition, the temperature-dependent conductivity indicated that the thin MoSe2 multilayers exhibited considerably weak semiconducting behavior with activation energies of 3.5-8.5 meV, which are considerably smaller than those (36-38 meV) of the bulk. Probable surface-dominant transport properties and the presence of a high surface electron concentration in MoSe2 are proposed. Similar results can be obtained for other layer semiconductor materials such as MoS2 and WS2.

We describe the approaches for the device fabrication and electrical characterization of molybdenum diselenide (MoSe2) layer semiconductor nanostructures with different thicknesses. In addition, the fabrication of ohmic contacts for MoSe2-layer nanocrystals by the focused-ion beam deposition method using platinum (Pt) as a contact metal is described.

Layer semiconductors with easily processed two-dimensional (2D) structures exhibit indirect-to-direct bandgap transitions and superior transistor ...

Single-Molecule SERS Measurements Enabled by Plasmonic DNA Origami Nanoantennas

Single-Molecule Surface-Enhanced Raman Scattering Measurements Enabled by Plasmonic DNA Origami Nanoantennas

Surface-enhanced Raman scattering (SERS) has the capability to detect single molecules for which high field enhancement is required. Single-molecule (SM) SERS is able to provide molecule-specific spectroscopic information about individual molecules and therefore yields more detailed chemical information than other SM detection techniques. At the same time, there is the potential to unravel information from SM measurements that remain hidden in Raman measurements of bulk material. This protocol outlines the SM SERS measurements using a DNA origami nanoantenna (DONA) in combination with atomic force microscopy (AFM) and Raman spectroscopy. A DNA origami fork structure and two gold nanoparticles are combined to form the DONAs, with a 1.2-2.0 nm gap between them. This allows an up to 1011-fold SERS signal enhancement, enabling measurements of single molecules. The protocol further demonstrates the placement of a single analyte molecule in a SERS hot spot, the process of AFM imaging, and the subsequent overlaying of Raman imaging to measure an analyte in a single DONA.

Author Spotlight: Single-Molecule Surface-Enhanced Raman Scattering Measurements Enabled by Plasmonic DNA Origami Nanoantennas  

This protocol demonstrates single-molecule surface-enhanced Raman scattering (SERS) measurements using a DNA origami nanoantenna (DONA) combined with colocalized atomic force microscopy (AFM) and Raman measurements.

Surface-enhanced Raman scattering (SERS) has the capability to detect single molecules for which high field enhancement is required. Single-molecule (SM) ...

Chemistry

Biomolecular Imaging of Cellular Uptake of Nanoparticles using Multimodal Nonlinear Optical Microscopy

Probing gold nanoparticles (AuNPs) in living systems is essential to reveal the interaction between AuNPs and biological tissues. Moreover, by integrating nonlinear optical signals such as stimulated Raman scattering (SRS), two-photon excited fluorescence (TPEF), and transient absorption (TA) into an imaging platform, it can be used to reveal biomolecular contrast of cellular structures and AuNPs in a multimodal manner. This article presents a multimodal nonlinear optical microscopy and applies it to perform chemically specific imaging of AuNPs in cancer cells. This imaging platform provides a novel approach for developing more efficient functionalized AuNPs and determining whether they are within vasculatures surrounding the tumor, pericellular, or cellular spaces.

This article presents the integration of a spectral-focusing module and a dual-output pulse laser, enabling rapid hyperspectral imaging of gold nanoparticles and cancer cells. This work aims to demonstrate the details of multimodal nonlinear optical techniques on a standard laser scanning microscope.

Probing gold nanoparticles (AuNPs) in living systems is essential to reveal the interaction between AuNPs and biological tissues. Moreover, by integrating ...

Bioengineering

Label-Free SERS Analysis: Au@Carbon Dot Nanoprobes for Molecular Fingerprinting

Label-Free Surface-Enhanced Raman Scattering Bioanalysis Based on Au@Carbon Dot Nanoprobes

Surface-enhanced Raman scattering (SERS) technology has attracted more and more attention in the biomedical field due to its ability to provide molecular fingerprint information of biological samples, as well as its potential in single-cell analysis. This work aims to establish a simple strategy for label-free SERS bioanalysis based on Au@carbon dot nanoprobes (Au@CDs). Here, polyphenol-derived CDs are utilized as the reductant to rapidly synthesize core-shell Au@CD nanostructures, which allows powerful SERS performance even when the concentration of methylene blue (MB) is as low as 10-9 M, due to the cooperative Raman enhancement mechanism. For bioanalysis, Au@CDs can serve as a unique SERS nanosensor to identify the cellular components of biosamples (e.g., cancer cells and bacteria). The molecular fingerprints from different species can be further distinguished after combination with the principal component analysis. In addition, Au@CDs also enable label-free SERS imaging to analyze intracellular composition profiles. This strategy offers a feasible, label-free SERS bioanalysis, opening up a new prospect for nanodiagnosis.

Author Spotlight: Advancing SERS Technology: Au@Carbon Dot Nanoprobes for Label-Free Analysis and Imaging 

In this study, we developed a low-cost surface-enhanced Raman scattering (SERS)-based fingerprint nanoprobe with favorable biocompatibility to show label-free live cell bioimaging and detect two bacterial strains, showing in detail how to get SERS spectra of living cells in a non-destructive method.

Surface-enhanced Raman scattering (SERS) technology has attracted more and more attention in the biomedical field due to its ability to provide molecular ...