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.
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.
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'-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).
1. Optical setup
2. Fabrication of AgNPs
3. Interaction of the DSNB analyte molecule and AgNP
4. Preparation of sample chamber and generation of AgNP assembly for SERS measurement
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−1, 1444 cm−1, and 1579 cm−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.
Figure 1: Schematic representation of the optical tweezer-coupled Raman spectroscopic platform. Please click here to view a larger version of this figure.
Figure 2: Preparation of AgNP for SERS measurement. (A) SEM image of AgNP. (B) Size distribution of AgNP by DLS. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
Figure 4: Schematic of sample chamber preparation. (A) Sample chamber preparation process. (B) Prepared sample chamber. Scale bar = 1 cm. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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%). Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
Supplementary Figure 1: The microscope camera images of AgNP assembly in time series when the trapping laser was turned off. Please click here to download this File.
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.
The authors have nothing to disclose.
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 "Laboratory for Synthetic Chemistry and Chemical Biology" under the Health@InnoHK Program launched by Innovation and Technology Commission, The Government of Hong Kong Special Administrative Region of the People's Republic of China.
1064 nm trapping laser | IPG Photonics, United States | 1064 nm CW Yb fiber laser, 10W | ||
3,3'-Dithiobis[6-nitrobenzoic acid] bis(succinimide) ester | Biosynth Carbosynth | FD15467 | ||
532 nm Raman excitation source | CNI, China | MLL-III-532 | ||
Bluelake software | LUMICKS, Netherlands | version 1.6.12 | optical tweezer control software | |
Frame tape | Thermo Fisher Scientific, Inc | AB-0576 | ||
Immersion oil | Cargille Laboratories, Inc | 16482 | ||
Liquid nitrogen-cooled charge-coupled device (CCD) camera | Teledyn Princeton Instrument, United States | 400B eXcelon | ||
Long-pass dichroic mirror | AHF, Germany | F48-801 | ||
Magnetic laser safety screen | ThorLabs | TPSM2 | ||
Optical tweezer microscope | LUMICKS, Netherlands | m-trap | ||
Silver nitrate | Sigma-Aldrich China, Inc. | S8157 | ||
Spectrometer | Teledyn Princeton Instrument, United States | IsoPlane SCT-320 | ||
Trisodium citrate | Sigma-Aldrich China, Inc. | S4641 | ||
WinSpec software | Teledyn Princeton Instrument, United States | version 2.6.24.0 | spectrum software |