We acknowledge the funding support from the Science, Technology, and Innovation Commission of Shenzhen Municipality (No. JCYJ20180306174930894), Zhongshan Municipal Bureau of Science and Technology (2020AG003), and Research Grant Council of Hong Kong (Project 26303018). We also acknowledge Prof. Chi-Ming Che and his funding support from &#34;Laboratory for Synthetic Chemistry and Chemical Biology&#34; under the Health@InnoHK Program launched by Innovation and Technology Commission, The Government of Hong Kong Special Administrative Region of the People&#39;s Republic of China.

1. Optical setup
<ol>
	<li>Direct a 532 nm laser beam (Raman excitation source) into the flex port of the optical tweezer microscope (see Table of Materials).</li>
	<li>Align the 532 nm laser beam into the stereo double-layer pathways of the optical tweezer microscope with a 750 nm long-pass dichroic mirror to combine with the original trapping laser beams to focus on the sample chamber.</li>
	<li>Collect the backscattered light from the sample chamber using a 750 nm long-pass dichroic mirror and redirect it into a spectrometer containing a liquid-nitrogen-cooled charge-coupled device (CCD) camera (see Table of Materials). Place a 532 nm notch filter in front of the entrance slit of the spectrometer before spectral acquisition. 
	&#8203;NOTE: Eye protection must be used when the laser is turned on, and the laser beam must be contained within a safe area.</li>
</ol>
2. Fabrication of AgNPs
<ol>
	<li>Heat 50 mL of 1 mM AgNO3 aqueous solution in a round-bottomed flask while boiling.</li>
	<li>Add 1.0 mL of 0.1 M trisodium citrate solution dropwise into the boiled AgNO3 aqueous solution.</li>
	<li>Keep the mixture boiling for 16 min under constant stirring.</li>
	<li>Cool the mixture to room temperature. Yellowish color is observed.</li>
	<li>Centrifuge the AgNP colloids at 2000 &#215; g for 5 min at room temperature and then remove the supernatant using a pipette.</li>
	<li>Resuspend the AgNP colloids with 1 mL of de-ionized water (resistivity of 18.2 M&#937; cm).</li>
	<li>Repeat steps 2.5 and 2.6 thrice to remove the residual reducing agent.</li>
	<li>Characterize the size distribution of the AgNPs using scanning electron microscopy (SEM) and dynamic light scattering (DLS)33 to confirm the uniformity of the AgNPs (Figure 2). The AgNP concentration was estimated as 0.1 nM by UV absorbance34. 
	NOTES: Due to the low concentration, the AgNP stock solution can be maintained without clustering for 2-3 weeks. No stabilizing agents are required. If a precipitate was observed in the AgNP stock solution, a new AgNP solution was prepared following the above protocol.</li>
</ol>
3. Interaction of the DSNB analyte molecule and AgNP
<ol>
	<li>Add 200 &#181;L of 2 mM DSNB (see Table of Materials) to 1 mL of AgNP colloid and incubate at room temperature for 3 h to coat a layer of DSNB on the surface of AgNP by the formation of the Ag-S bond between AgNP and DSNB35. A schematic representation of this interaction is shown in Figure 3.</li>
	<li>Centrifuge the AgNP at 2,000 &#215; g for 5 min at room temperature and remove the supernatant.</li>
	<li>Resuspend the AgNP-DSNB with 1 mL of de-ionized water.</li>
	<li>Repeat steps 3.2 and 3.3 thrice to remove excess DSNB.</li>
	<li>Record the UV-visible spectra of the AgNP colloid and AgNP-DSNB solution. 
	&#8203;NOTE: This spectra show an absorption peak shift from approximately 420 nm to 450 nm, indicating the successful coating of DSNB on the surface of AgNP (Figure 3).</li>
</ol>
4. Preparation of sample chamber and generation of AgNP assembly for SERS measurement
<ol>
	<li>Clean the glass slide and coverslip with water and ethanol.</li>
	<li>Attach the frame tape (0.25 mm thickness, see Table of Materials) to the glass slide to create a chamber (1.0 cm length &#215; 1.0 cm width &#215; 0.25 mm height).</li>
	<li>Add a few drops of the AgNP-DSNB solution (around 25 &#181;L) into the frame.</li>
	<li>Put the coverslip on the frame tape and seal it (Figure 4).</li>
	<li>Add liquid nitrogen to the container of the liquid nitrogen-cooled CCD camera until the temperature reaches -120 &#176;C.</li>
	<li>Block the Raman probe beam path using a magnetic laser safety screen (see Table of Materials), then turn on the 532 nm Raman excitation source laser.</li>
	<li>Fix the sample chamber with the AgNP-DSNB solution on the chamber holder. Add water to the water-immersed objective (60x magnification with a 1.2 numerical aperture A of 1.2) as shown in Figure 1. Then place the chamber holder immediately onto the microstage above the objective.</li>
	<li>Drop immersion oil on top of the coverslip and position the oil-immersed condenser to visualize particles on the microscope camera.</li>
	<li>Adjust the Z position of the objective by turning the knob of the microscope until the 532 nm Raman probe beam is focused on the bottom glass surface of the chamber, showing a white spot on the microscope camera (Figure 5).
	<ol>
		<li>Adjust the X- and Y-positions of the microstage to move the chamber to place the central region of the chamber at the white spot. Open the optical tweezer control software (see Table of Materials) and use the equipped joystick control to move the 1064 nm trapping laser (indicated by a red circle in the optical tweezer system) to overlap with the white spot (Figure 5).</li>
		<li>Next, tune the knob of the microscope to move the Z position of the objective up. 
		NOTE: The disappearance of the white spot in the microscope camera image indicates that the 532 nm Raman probe beam is focused inside the chamber.</li>
	</ol>
	</li>
	<li>Turn on the 1064 nm trapping laser to attract AgNPs in the sample chamber and create a plasmonic AgNP assembly. 
	NOTE: The gathering of AgNPs results in a dark spot in the sample chamber (Figure 6B).
	<ol>
		<li>Turn down the trapping laser beam to avoid overheating or bubble formation when required. 
		NOTE: Increase the trapping laser power and irradiation time if there is no apparent formation of the AgNP assembly.</li>
	</ol>
	</li>
	<li>Adjust the position of the sample microstage to place the dark spot of the plasmonic AgNP assembly under the focus of the 532 nm Raman probe beam for spectroscopic measurements.</li>
	<li>Place the neutral-density (ND) filters in front of the 532 nm Raman laser outlet to adjust the power to 10 mW. Input the acquisition time (10 s for the present study, Figure 6) in the setting panel in the spectrum software (see Table of Materials) and click on the Acquire button to start the spectral acquisition. 
	NOTE: This generates the SERS spectrum of the analyte molecules (DSNB in the representative result and Figure 6).</li>
</ol>

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The authors have nothing to disclose.

The present study reports an analytical platform that combines optical trapping and SERS detection for in situ molecular characterizations. A 532 nm Raman probe beam was combined with a 1064 nm trapping laser beam through stereo double-layer pathways to combine focus and collect for additional spectroscopic measurements in backscattering geometry. The trapping laser beam assembled AgNPs to form plasmonic hotspots, followed by excitation of the Raman probe laser beam to generate the SERS signal of the analyte molecules in solution. As a proof of concept, the detection of DSNB was demonstrated, which was coated on the surface of AgNPs. In the AgNP assembly region controlled by the trapping laser beam, an approximately 50-fold enhancement in the signal of DSNB compared to the surrounding dispersed AgNPs, was achieved. A similar high-signal amplification of analyte molecules in the solution-phase SERS measurements on the presented platform was reproducibly obtained.
The critical step affecting SERS signal amplification is forming an optical trapping-induced AgNP assembly. The SERS signal of the analyte molecules can be optimized by fine-tuning experimental parameters such as the trapping laser power, irradiation time, and AgNP concentration. As shown in Figure 8, using a higher trapping laser power can increase the efficiency of AgNP assembly formation. Reproducible AgNP assemblies were obtained by increasing the power of the trapping laser from 450 mW to 700 mW. However, a trapping laser power higher than 950 mW may induce overheating and bubble generation38. Thus, moderate trapping laser power is recommended to create a dynamic AgNP assembly. Analogously, a longer irradiation time is useful for promoting the formation of AgNP assemblies. Figure 8B shows that a clear spherical AgNP assembly was formed when the irradiation time increased from 5-20 s. However, the AgNP assembly was distorted after 60 s irradiation. In addition, the formation of the AgNP assembly was accelerated at a higher AgNP concentration, from 0.01 nM to 0.05 nM, while it was quickly overheated at 0.25 nM, as shown in Figure 8C. If there is no apparent AgNP assembly formation, increasing the trapping laser power and the irradiation time is recommended. Upon generation of a stable AgNP assembly, the trapping laser must be turned down to avoid potential thermal damage.
The SERS activity of the optical trapping-induced AgNP assembly was attributed to an increase in the local AgNP concentration in the trapping laser irradiation region, which is the dark spot in Figure 6B. In the fluidic AgNP solution, the optical trap can continuously attract AgNPs to accumulate and form plasmonic hotspots in a confined space in the interparticle junctions. This yields an enhanced electric field which enhances the SERS effect. It was further verified by the stronger SERS signal obtained at a higher AgNP concentration (1.00 nM) compared to the weaker SERS signal acquired at a lower AgNP concentration (0.05 nM) without the trapping laser, as shown in Figure 6E.
Furthermore, position control of the plasmonic AgNP assembly in solution, against Brownian motion, by optical trapping has significantly improved the efficiency and stability of SERS measurements. High-throughput sensing can be conducted when connected to the microfluidic system. Compared to the traditional salt-induced aggregation of nanoparticles to generate SERS-active substrates, our platform allows the dynamic formation of plasmonic AgNP assemblies, at the designed location and moment, with high flexibility26,28. Moreover, it works efficiently at nanomolar AgNP concentrations and enables the spatial-temporal manipulation of SERS-active hotspots for in situ spectroscopic measurements in solutions. This dynamic AgNP assembly gradually disassembled in a few minutes when the trapping laser was turned off. Without the trapping laser, the AgNP assembly almost disappeared in 20 min, as shown in Supplementary Figure 1. This can minimize the influence on the detection system and exhibits great potential for various bio-applications, especially the detection of biomolecules (DNA, RNA, and protein) under physiological and in vivo conditions. However, this dynamic AgNP assembly provides a smaller enhancement factor than salt-induced AgNP aggregates2, and hence, further modification and development are required.
In conclusion, the integration of optical trapping and SERS detection provides a convenient method to control plasmonic nanoparticles and achieve reproducible SERS signal enhancement to detect analyte molecules in solutions with high efficiency, stability, and biocompatibility.

Surface-enhanced Raman spectroscopy (SERS) is a sensitive analytical technique for directly detecting the chemical structure of target molecules at ultralow concentrations or even at the single-molecule level1,2,3,4. Laser irradiation induces localized surface plasmon resonance in metallic nanostructures, used as SERS substrates to amplify the Raman signals of target molecules. Salt-induced nanoparticle aggregates are the widely used SERS substrates, which spontaneously undergo Brownian motion in colloidal suspension liquids5,6. Further drying allows stable SERS measurements; however, impurity concentration may occur, which introduces background noise and causes irreversible damage to biological samples7. Hence, it is pertinent to develop salt-free nanoparticle aggregations, control their movement in solution, and improve biocompatibility while maintaining measurement efficiency.
Optical trapping has been adopted to control various metallic substrates and facilitate SERS detections8,9,10,11,12,13,14. An optical trap is generated by tightly focusing a laser beam to generate an optical force field, which attracts small objects to the highest-intensity region around the focus15,16. Recently, optical traps have been used to develop reproducible and sensitive plasmonic sensing platforms for various applications, displaying their unique advantages in locating and controlling the position of SERS-active metallic nanostructures in solutions17,18,19,20,21,22,23,24. The present protocol introduces an approach to combine optical tweezers and Raman spectro-microscopy to dynamically assemble silver nanoparticles (AgNPs) and stabilize them against Brownian motion in solution for efficient SERS measurements. In the AgNP assembly region, the signal of the 3,3&#39;-dithiobis[6-nitrobenzoic acid] bis(succinimide) ester (DSNB), analyte molecules coated on the surface of AgNPs, can be enhanced by approximately 50 folds. This approach is suitable for analyzing sensitive biomolecules incompatible with chemical capping agents25,26,27. Moreover, it provides spatial and temporal control to generate the SERS-active AgNP assembly. This enables in situ detection in aqueous environments, which could lower the usage of AgNPs and minimize perturbation for in vivo analysis28,29,30. In addition, the optical trapping-induced AgNP assembly is stable, reproducible, and reversible31,32. Hence, it is a promising platform for detecting analyte molecules in solutions and under physiological conditions where salt-induced aggregation is not applicable.
In the present study, a 1064 nm trapping laser, force detection module, and brightfield illumination source are integrated into the optical tweezer microscopy system for optical manipulation and visualization of particles. A 532 nm Raman probe laser was also incorporated into the microscope and aligned with the trapping laser in the sample chamber. For spectral acquisition, backscattered light was collected and redirected into a spectrometer (Figure 1).

<table><tbody><tr><td>1064 nm trapping laser</td><td>IPG Photonics, United States</td><td>1064 nm CW Yb fiber laser, 10W</td><td></td></tr><tr><td>3,3&#039;-Dithiobis[6-nitrobenzoic acid] bis(succinimide) ester&amp;nbsp;</td><td>Biosynth Carbosynth</td><td>FD15467</td><td></td></tr><tr><td>532 nm Raman excitation source</td><td>CNI, China</td><td>MLL-III-532</td><td></td></tr><tr><td>Bluelake software</td><td>LUMICKS, Netherlands</td><td>version 1.6.12</td><td>optical tweezer control software</td></tr><tr><td>Frame tape</td><td>Thermo Fisher Scientific, Inc</td><td>AB-0576</td><td></td></tr><tr><td>Immersion oil</td><td>Cargille Laboratories, Inc</td><td>16482</td><td></td></tr><tr><td>Liquid nitrogen-cooled charge-coupled device (CCD) camera</td><td>Teledyn Princeton Instrument, United States</td><td>400B eXcelon</td><td></td></tr><tr><td>Long-pass dichroic mirror</td><td>AHF, Germany</td><td>F48-801</td><td></td></tr><tr><td>Magnetic laser safety screen</td><td>ThorLabs</td><td>TPSM2</td><td></td></tr><tr><td>Optical tweezer microscope</td><td>LUMICKS, Netherlands</td><td>m-trap</td><td></td></tr><tr><td>Silver nitrate</td><td>Sigma-Aldrich China, Inc.</td><td>S8157</td><td></td></tr><tr><td>Spectrometer</td><td>Teledyn Princeton Instrument, United States</td><td>IsoPlane SCT-320</td><td></td></tr><tr><td>Trisodium citrate</td><td>Sigma-Aldrich China, Inc.</td><td>S4641</td><td></td></tr><tr><td>WinSpec software</td><td>Teledyn Princeton Instrument, United States</td><td>version 2.6.24.0</td><td>spectrum software</td></tr></tbody></table>

optical trapping of plasmonic nanoparticles for in situ surface-enhanced raman spectroscopy characterizations

Surface-enhanced Raman spectroscopy (SERS) enables the ultrasensitive detection of analyte molecules in various applications due to the enhanced electric field of metallic nanostructures. Salt-induced silver nanoparticle aggregation is the most popular method for generating SERS-active substrates; however, it is limited by poor reproducibility, stability, and biocompatibility. The present protocol integrates optical manipulation and SERS detection to develop an efficient analytical platform to address this. A 1064 nm trapping laser and a 532 nm Raman probe laser are combined in a microscope to assemble silver nanoparticles, which generate plasmonic hotspots for in situ SERS measurements in aqueous environments. Without aggregating agents, this dynamic plasmonic silver nanoparticle assembly enables an approximately 50-fold enhancement of the analyte molecule signal. Moreover, it provides spatial and temporal control to form the SERS-active assembly in as low as 0.05 nM analyte-coated silver nanoparticle solution, which minimizes the potential perturbation for in vivo analysis. Hence, this optical trapping-integrated SERS platform holds great potential for efficient, reproducible, and stable molecular analyses in liquids, especially in aqueous physiological environments.

As proof of concept, DSNB was chosen as the analyte molecule and coated onto the surface of AgNPs. The typical SERS spectra of DSNB enhanced by the plasmonic AgNP assembly and dispersed AgNP are shown in Figure 6. Without the trapping laser, the dispersed AgNPs in the sample chamber generated a black spectrum (Figure 6A) upon excitation by the Raman probe laser. A weak and broad SERS signal was observed at approximately 1380-1450 cm-1, the characteristic peak of DSNB from its symmetric NO2 stretch, which is consistent with literature reports35,36. Since the dispersed AgNPs were under Brownian motion, the interparticle junctions were large and unstable, as illustrated in Figure 6C. Thus, the SERS signal amplification of DSNB was low for the dispersed AgNPs.
AgNPs are gathered to form a plasmonic AgNP assembly when the trapping laser is on. Increasing the power and extending the irradiation time of the trapping laser could attract more AgNPs and generate a dark spot, as shown in Figure 6B. Here, we applied a trapping laser power of 700 mM and a 20 s irradiation time to create a plasmonic AgNP assembly in a 0.05 nM DSNB-coated AgNP solution at a designated location and moment. The SERS spectrum of DSNB was obtained in the region of the plasmonic AgNP assembly (Figure 6A, red). The strong Raman band at 930 cm-1 is assigned to the nitro scissoring vibration, and the large bands at 1078 cm-1, 1152 cm-1, and 1191 cm-1 likely correspond to the succinimidyl N-C-O stretch overlapping with the aromatic ring modes of DSNB35,37. The feature bands at 1385 cm-1 and 1444 cm-1 arise from the symmetric nitro stretch of DSNB and are significantly enhanced and slightly shifted due to the reaction with the surface of AgNP35,37. Based on the previously reported SERS fingerprints of DSNB35,36,37, the band at 1579 cm-1 was assigned to the aromatic ring mode of DSNB. The overall intensities of DSNB in the plasmonic AgNP assembly were higher than those of the dispersed AgNP. Considering the intensity of the characteristic peak at 1444 cm-1, the plasmonic AgNP assembly can provide approximately a 50-fold enhancement of the SERS signal of DSNB compared to that of the dispersed AgNP. As shown in Figure 7, SERS spectra of DSNB were recorded repeatedly (20 times) for the AgNP assembly in the experiment, demonstrating identical vibrational features. The intensities of the characteristic peaks of DSNB at 1152 cm&#8722;1, 1444 cm&#8722;1, and 1579 cm&#8722;1 across these 20 SERS spectra were plotted as histograms with relative standard deviations (RSD) of 6.88%, 6.59%, and 5.48%, respectively. This further verified the reproducibility and stability. Hence, this approach is reliable for manipulating plasmonic nanoparticles and SERS detection of analyte molecules in solution.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63862/63862fig01.jpg" /> 
Figure 1: Schematic representation of the optical tweezer-coupled Raman spectroscopic platform. <a href="https://www.jove.com/files/ftp_upload/63862/63862fig01large.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/63862/63862fig02.jpg" /> 
Figure 2: Preparation of AgNP for SERS measurement. (A) SEM image of AgNP. (B) Size distribution of AgNP by DLS. <a href="https://www.jove.com/files/ftp_upload/63862/63862fig02large.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/63862/63862fig03.jpg" /> 
Figure 3: Interaction of AgNP and DSNB. (A) Schematic of the coating of DSNB on the surface of AgNP. (B) UV-visible spectra of AgNP and AgNP-DSNB. <a href="https://www.jove.com/files/ftp_upload/63862/63862fig03large.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/63862/63862fig04.jpg" /> 
Figure 4: Schematic of sample chamber preparation. (A) Sample chamber preparation process. (B) Prepared sample chamber. Scale bar = 1 cm. <a href="https://www.jove.com/files/ftp_upload/63862/63862fig04large.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/63862/63862fig05.jpg" /> 
Figure 5: Position overlapping of 532 nm Raman laser and 1064 nm trapping laser. (A) Position of 532 nm Raman laser indicated by white spot. (B) Position of 1064 nm trapping laser indicated by red circle. <a href="https://www.jove.com/files/ftp_upload/63862/63862fig05large.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/63862/63862fig06.jpg" /> 
Figure 6: Typical SERS spectra of the analyte molecules enhanced by the plasmonic AgNP assembly. (A) SERS spectra of DSNB at the plasmonic AgNP assembly (red) and the dispersed AgNP (black). (B) The plasmonic AgNP assembly when the trapping laser is on shows a dark spot under microscopic visualization. (C) The dispersed AgNP when the trapping laser is off. (D) Illustration of the mechanism of AgNP assembly formation. (E) Concentration-dependent SERS intensity in the absence of the trapping laser. <a href="https://www.jove.com/files/ftp_upload/63862/63862fig06large.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/63862/63862fig07.jpg" /> 
Figure 7: Reproducibility of SERS signal of DSNB. (A) 20 SERS spectra of DSNB at the plasmonic AgNP assembly recorded repeatly in the experiment. (B) Histograms of the intensities of the characteristic DSNB peaks at 1152 cm-1 (RSD = 6.88%), 1444 cm-1 (RSD = 6.59%), and 1579 cm-1 (RSD = 5.48%). <a href="https://www.jove.com/files/ftp_upload/63862/63862fig07large.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/63862/63862fig08.jpg" /> 
Figure 8: AgNP assembly generated under different experimental parameters. (A) Different trapping laser power; irradiation time 20 s and AgNP concentration 0.05 nM. (B) Different irradiation time; trapping laser power 700 mW and AgNP concentration 0.05 nM. (C) Different AgNP concentration; irradiation time 20 s and trapping laser power 700 mW. <a href="https://www.jove.com/files/ftp_upload/63862/63862fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1: The microscope camera images of AgNP assembly in time series when the trapping laser was turned off. <a href="https://www.jove.com/files/ftp_upload/63862/Figure S1.zip" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Optical Trapping of Plasmonic Nanoparticles for In Situ Surface-Enhanced Raman Spectroscopy Characterizations at JoVE.com

Optical Trapping of Plasmonic Nanoparticles for In Situ Surface-Enhanced Raman Spectroscopy Characterizations

The present protocol describes a convenient approach to integrating optical trapping and surface-enhanced Raman spectroscopy (SERS) to manipulate plasmonic nanoparticles for sensitive molecular detection. Without aggregating agents, the trapping laser assembles plasmonic nanoparticles to enhance the SERS signals of target analytes for in situ spectroscopic measurements.

Optical Trapping of Plasmonic Nanoparticles for In Situ Surface-Enhanced Raman Spectroscopy Characterizations

Surface-enhanced Raman spectroscopy (SERS) enables the ultrasensitive detection of analyte molecules in various applications due to the enhanced electric ...

optical-trapping-plasmonic-nanoparticles-for-situ-surface-enhanced

Research

JoVE Journal

Bioengineering

2.5K Views.  HKUST-Shenzhen Research Institute. The present protocol describes a convenient approach to integrating optical trapping and surface-enhanced Raman spectroscopy (SERS) to manipulate plasmonic nanoparticles for sensitive molecular detection. Without aggregating agents, the trapping laser assembles plasmonic nanoparticles to enhance the SERS signals of target analytes for in situ spectroscopic measurements.

Video: Optical Trapping of Plasmonic Nanoparticles for In Situ Surface-Enhanced Raman Spectroscopy Characterizations

Optical Trapping of Nanoparticles

Optical trapping is a technique for immobilizing and manipulating small objects in a gentle way using light, and it has been widely applied in trapping and manipulating small biological particles. Ashkin and co-workers first demonstrated optical tweezers using a single focused beam1. The single beam trap can be described accurately using the perturbative gradient force formulation in the case of small Rayleigh regime particles1. In the perturbative regime, the optical power required for trapping a particle scales as the inverse fourth power of the particle size. High optical powers can damage dielectric particles and cause heating. For instance, trapped latex spheres of 109 nm in diameter were destroyed by a 15 mW beam in 25 sec1, which has serious implications for biological matter2,3.
A self-induced back-action (SIBA) optical trapping was proposed to trap 50 nm polystyrene spheres in the non-perturbative regime4. In a non-perturbative regime, even a small particle with little permittivity contrast to the background can influence significantly the ambient electromagnetic field and induce a large optical force. As a particle enters an illuminated aperture, light transmission increases dramatically because of dielectric loading. If the particle attempts to leave the aperture, decreased transmission causes a change in momentum outwards from the hole and, by Newton's Third Law, results in a force on the particle inwards into the hole, trapping the particle. The light transmission can be monitored; hence, the trap can become a sensor. The SIBA trapping technique can be further improved by using a double-nanohole structure.
The double-nanohole structure has been shown to give a strong local field enhancement5,6. Between the two sharp tips of the double-nanohole, a small particle can cause a large change in optical transmission, thereby inducing a large optical force. As a result, smaller nanoparticles can be trapped, such as 12 nm silicate spheres7 and 3.4 nm hydrodynamic radius bovine serum albumin proteins8. In this work, the experimental configuration used for nanoparticle trapping is outlined. First, we detail the assembly of the trapping setup which is based on a Thorlabs Optical Tweezer Kit. Next, we explain the nanofabrication procedure of the double-nanohole in a metal film, the fabrication of the microfluidic chamber and the sample preparation. Finally, we detail the data acquisition procedure and provide typical results for trapping 20 nm polystyrene nanospheres.

The following setup approach details low power optical trapping of dielectric nanoparticles using a double-nanohole in metal film.

Optical trapping is a technique for immobilizing and manipulating small objects in a gentle way using light, and it has been widely applied in trapping ...

Engineering

Utilization of Plasmonic and Photonic Crystal Nanostructures for Enhanced Micro- and Nanoparticle Manipulation

A method to manipulate the position and orientation of submicron particles nondestructively would be an incredibly useful tool for basic biological research. Perhaps the most widely used physical force to achieve noninvasive manipulation of small particles has been dielectrophoresis(DEP).1 However, DEP on its own lacks the versatility and precision that are desired when manipulating cells since it is traditionally done with stationary electrodes. Optical tweezers, which utilize a three dimensional electromagnetic field gradient to exert forces on small particles, achieve this desired versatility and precision.2 However, a major drawback of this approach is the high radiation intensity required to achieve the necessary force to trap a particle which can damage biological samples.3 A solution that allows trapping and sorting with lower optical intensities are optoelectronic tweezers (OET) but OET's have limitations with fine manipulation of small particles; being DEP-based technology also puts constraint on the property of the solution.4,5
This video article will describe two methods that decrease the intensity of the radiation needed for optical manipulation of living cells and also describe a method for orientation control. The first method is plasmonic tweezers which use a random gold nanoparticle (AuNP) array as a substrate for the sample as shown in Figure 1. The AuNP array converts the incident photons into localized surface plasmons (LSP) which consist of resonant dipole moments that radiate and generate a patterned radiation field with a large gradient in the cell solution. Initial work on surface plasmon enhanced trapping by Righini et al and our own modeling have shown the fields generated by the plasmonic substrate reduce the initial intensity required by enhancing the gradient field that traps the particle.6,7,8 The plasmonic approach allows for fine orientation control of ellipsoidal particles and cells with low optical intensities because of more efficient optical energy conversion into mechanical energy and a dipole-dependent radiation field. These fields are shown in figure 2 and the low trapping intensities are detailed in figures 4 and 5. The main problems with plasmonic tweezers are that the LSP's generate a considerable amount of heat and the trapping is only two dimensional. This heat generates convective flows and thermophoresis which can be powerful enough to expel submicron particles from the trap.9,10 The second approach that we will describe is utilizing periodic dielectric nanostructures to scatter incident light very efficiently into diffraction modes, as shown in figure 6.11 Ideally, one would make this structure out of a dielectric material to avoid the same heating problems experienced with the plasmonic tweezers but in our approach an aluminum-coated diffraction grating is used as a one-dimensional periodic dielectric nanostructure. Although it is not a semiconductor, it did not experience significant heating and effectively trapped small particles with low trapping intensities, as shown in figure 7. Alignment of particles with the grating substrate conceptually validates the proposition that a 2-D photonic crystal could allow precise rotation of non-spherical micron sized particles.10 The efficiencies of these optical traps are increased due to the enhanced fields produced by the nanostructures described in this paper.

Plasmonic tweezers and photonic crystal nanostructures are shown to produce useful enhancements in the efficiency and orientation control of optically trapping micro- and nano-particles.

A method to manipulate the position and orientation of submicron particles nondestructively would be an incredibly useful tool for basic biological ...

Analyzing the Movement of the Nauplius 'Artemia salina' by Optical Tracking of Plasmonic Nanoparticles

We demonstrate how optical tweezers may provide a sensitive tool to analyze the fluidic vibrations generated by the movement of small aquatic organisms. A single gold nanoparticle held by an optical tweezer is used as a sensor to quantify the rhythmic motion of a Nauplius larva (Artemia salina) in a water sample. This is achieved by monitoring the time dependent displacement of the trapped nanoparticle as a consequence of the Nauplius activity. A Fourier analysis of the nanoparticle&#39;s position then yields a frequency spectrum that is characteristic to the motion of the observed species. This experiment demonstrates the capability of this method to measure and characterize the activity of small aquatic larvae without the requirement to observe them directly and to gain information about the position of the larvae with respect to the trapped particle. Overall, this approach could give an insight on the vitality of certain species found in an aquatic ecosystem and could expand the range of conventional methods for analyzing water samples.

Analyzing the Movement of the Nauplius 'Artemia salina' by Optical Tracking of Plasmonic Nanoparticles

We use optical tracking of plasmonic nanoparticles to probe and characterize the frequency movements of aquatic organisms.

We demonstrate how optical tweezers may provide a sensitive tool to analyze the fluidic vibrations generated by the movement of small aquatic organisms. A ...

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 ...

Plasmonic Trapping and Release of Nanoparticles in a Monitoring Environment

Plasmonic tweezers use surface plasmon polaritons to confine polarizable nanoscale objects. Among the various designs of plasmonic tweezers, only a few can observe immobilized particles. Moreover, a limited number of studies have experimentally measured the exertable forces on the particles. The designs can be classified as the protruding nanodisk type or the suppressed nanohole type. For the latter, microscopic observation is extremely challenging. In this paper, a new plasmonic tweezer system is introduced to monitor particles, both in directions parallel and orthogonal to the symmetric axis of a plasmonic nanohole structure. This feature enables us to observe the movement of each particle near the rim of the nanohole. Furthermore, we can quantitatively estimate the maximal trapping forces using a new fluidic channel.

A microchip fabrication process that incorporates plasmonic tweezers is presented here. The microchip enables the imaging of a trapped particle to measure maximal trapping forces.

Plasmonic tweezers use surface plasmon polaritons to confine polarizable nanoscale objects. Among the various designs of plasmonic tweezers, only a few ...

Construction and Operation of a Light-driven Gold Nanorod Rotary Motor System

The possibility to generate and measure rotation and torque at the nanoscale is of fundamental interest to the study and application of biological and artificial nanomotors and may provide new routes towards single cell analysis, studies of non-equilibrium thermodynamics, and mechanical actuation of nanoscale systems. A facile way to drive rotation is to use focused circularly polarized laser light in optical tweezers. Using this approach, metallic nanoparticles can be operated as highly efficient scattering-driven rotary motors spinning at unprecedented rotation frequencies in water.
In this protocol, we outline the construction and operation of circularly-polarized optical tweezers for nanoparticle rotation and describe the instrumentation needed for recording the Brownian dynamics and Rayleigh scattering of the trapped particle. The rotational motion and the scattering spectra provides independent information on the properties of the nanoparticle and its immediate environment. The experimental platform has proven useful as a nanoscopic gauge of viscosity and local temperature, for tracking morphological changes of nanorods and molecular coatings, and as a transducer and probe of photothermal and thermodynamic processes.

Plasmonic gold nanorods can be trapped in liquids and rotated at kHz frequencies using circularly-polarized optical tweezers. Introducing tools for Brownian dynamics analysis and light scatteringspectroscopy leads to a powerful system for research and application in numerous fields of science.

The possibility to generate and measure rotation and torque at the nanoscale is of fundamental interest to the study and application of biological and ...

﻿Analyzing the Heterogeneous Performance of Single Nanoparticles with Electrochemical Methods

Tracking Electrochemistry on Single Nanoparticles with Surface-Enhanced Raman Scattering Spectroscopy and Microscopy

Studying electrochemical reactions on single nanoparticles is important to understand the heterogeneous performance of individual nanoparticles. This nanoscale heterogeneity remains hidden during the ensemble-averaged characterization of nanoparticles. Electrochemical techniques have been developed to measure currents from single nanoparticles but do not provide information about the structure and identity of the molecules that undergo reactions at the electrode surface. Optical techniques such as surface-enhanced Raman scattering (SERS) microscopy and spectroscopy can detect electrochemical events on individual nanoparticles while simultaneously providing information on the vibrational modes of electrode surface species. In this paper, a protocol to track the electrochemical oxidation-reduction of Nile Blue (NB) on single Ag nanoparticles using SERS microscopy and spectroscopy is demonstrated. First, a detailed protocol for fabricating Ag nanoparticles on a smooth and semi-transparent Ag film is described. A dipolar plasmon mode aligned along the optical axis is formed between a single Ag nanoparticle and Ag film. The SERS emission from NB fixed between the nanoparticle and the film is coupled into the plasmon mode, and the high-angle emission is collected by a microscope objective to form a donut-shaped emission pattern. These donut-shaped SERS emission patterns allow for the unambiguous identification of single nanoparticles on the substrate, from which the SERS spectra can be collected. In this work, a method for employing the SERS substrate as a working electrode in an electrochemical cell compatible with an inverted optical microscope is provided. Finally, tracking the electrochemical oxidation-reduction of NB molecules on an individual Ag nanoparticle is shown. The setup and the protocol described here can be modified to study various electrochemical reactions on individual nanoparticles.

Author Spotlight: Tracking Electrochemistry on Single Nanoparticles with Surface-Enhanced Raman Scattering Spectroscopy and Microscopy  

The protocol describes how to monitor electrochemical events on single nanoparticles using surface-enhanced Raman scattering spectroscopy and imaging.

Studying electrochemical reactions on single nanoparticles is important to understand the heterogeneous performance of individual nanoparticles. This ...

Chemistry

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) ...

Trapping of Micro Particles in Nanoplasmonic Optical Lattice

The plasmonic optical tweezer has been developed to overcome the diffraction limits of the conventional far field optical tweezer. Plasmonic optical lattice consists of an array of nanostructures, which exhibit a variety of trapping and transport behaviors. We report the experimental procedures to trap micro-particles in a simple square nanoplasmonic optical lattice. We also describe the optical setup and the nanofabrication of a nanoplasmonic array. The optical potential is created by illuminating an array of gold nanodiscs with a Gaussian beam of 980 nm wavelength, and exciting plasmon resonance. The motion of particles is monitored by fluorescence imaging. A scheme to suppress photothermal convection is also described to increase usable optical power for optimal trapping. Suppression of convection is achieved by cooling the sample to a low temperature, and utilizing the near-zero thermal expansion coefficient of a water medium. Both single particle transport and multiple particle trapping are reported here.

We describe a procedure to optically trap micro-particles in nanoplasmonic optical lattice.

The plasmonic optical tweezer has been developed to overcome the diffraction limits of the conventional far field optical tweezer. Plasmonic optical ...

Monitoring Conformational Dynamics of Single Unmodified Proteins using Plasmonic Nanotweezers

Current single-molecule techniques to characterize proteins typically require labels, tethers, or the use of non-native solution conditions. Such changes can alter protein biophysics and reduce the usefulness of the data acquired. Plasmonic nanotweezers is a technique that uses localized surface plasmon resonance (LSPR) on gold nanostructures to enhance the electric field within a confined hotspot region. This field enhancement allows for the use of low laser powers to trap single nanoparticles far smaller than conventional optical tweezers, down to only a few nanometers in diameter, such as single proteins. Trapping of single protein molecules within the hotspot region induces a shift in the local refractive index (nprotein &#62; nwater), altering light scattering as a product of the molecule&#39;s polarisability, which is affected by its volume, shape anisotropy, and refractive index. An avalanche photodiode (APD) collects the subsequent changes in light scattering. These alterations can then be analyzed to determine changes in the trapped molecule, including its size, global conformation, and dynamics of conformational change over time. The incorporation of microfluidics within the system allows for controlled environmental changes and real-time monitoring of their subsequent effects on the molecule. In this protocol, we demonstrate the steps to trap single protein molecules, alter their environmental solution conditions, and monitor their corresponding conformational changes using a plasmonic nanotweezers system.

Plasmonic nanotweezers use localized surface plasmon resonance in gold nanostructures to trap single nanoparticles, including proteins, within a nanometer-scale optical field. Changes in the scattered signal reveal protein presence and conformational dynamics, enabling monitoring without fluorophore modifications or surface tethering.

Current single-molecule techniques to characterize proteins typically require labels, tethers, or the use of non-native solution conditions. Such changes ...

Optical Trapping of Plasmonic Nanoparticles for In Situ Surface-Enhanced Raman Spectroscopy Characterizations

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Chapters in this video

Optical Trapping of Nanoparticles

Utilization of Plasmonic and Photonic Crystal Nanostructures for Enhanced Micro- and Nanoparticle Manipulation

Analyzing the Movement of the Nauplius 'Artemia salina' by Optical Tracking of Plasmonic Nanoparticles

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

Plasmonic Trapping and Release of Nanoparticles in a Monitoring Environment

Construction and Operation of a Light-driven Gold Nanorod Rotary Motor System

Tracking Electrochemistry on Single Nanoparticles with Surface-Enhanced Raman Scattering Spectroscopy and Microscopy

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

Trapping of Micro Particles in Nanoplasmonic Optical Lattice

Monitoring Conformational Dynamics of Single Unmodified Proteins using Plasmonic Nanotweezers