This protocol provides spatial and temporal control for the SERS-active nanoparticle assembly in the absence of aggregation agents to achieve sensitive detection of target analytes. The main advantage of our method is no aggregation agents are used to generate the SERS-active nanoparticle assembly so it is used suitable for analyzing sensitive biomolecules under physiological conditions. It is a promising platform for detecting analyte molecules such as disease biomarker in solutions and under physiological conditions in a microfluidic system.
When using this method for the first time, a researcher may need to fine tune the trapping laser power, irradiation time, and silver nanoparticle concentration to achieve the best performance. To begin, direct a 532-nanometer laser beam into the flex port of the optical tweezer microscope. Align the 532-nanometer laser beam into the stereo double-layer pathways of the optical tweezer microscope with a 750-nanometer long-pass dichroic mirror to combine them with the original trapping laser beams to focus on the sample chamber.
Collect the backscattered light from the sample chamber using a 750-nanometer long-pass dichroic mirror and redirect it into a spectrometer containing a liquid-nitrogen-cooled charge-coupled device camera. Place a 532-nanometer notch filter in front of the entrance slit of the spectrometer before spectral acquisition. Clean the glass slide and cover slip with water and ethanol.
Attach the frame tape to the glass slide to create a chamber. Add a few drops of the silver nanoparticle DSNB solution into the frame. Put the cover slip on the frame tape and seal it.
Add liquid nitrogen to the container of the liquid-nitrogen-cooled charge-coupled device camera until the temperature reaches minus 120 degrees Celsius. Block the Raman probe beam path using a magnetic laser safety screen, then turn on the 532-nanometers Raman excitation source laser. Fix the sample chamber with the silver nanoparticle DSNB solution on the chamber holder.
Add water to the water-immersed objective, then place the chamber holder immediately onto the microstage above the objective. Drop immersion oil on top of the cover slip and position the oil-immersed condenser to visualize particles on the microscope camera. Adjust the Z position of the objective by turning the knob of the microscope until the 532-nanometer Raman probe beam is focused on the bottom glass surface of the chamber, showing a white spot on the microscope camera.
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 and use the equipped joystick control to move the 1, 064-nanometer trapping laser to overlap with the white spot. Next, tune the knob of the microscope to move the Z position of the objective up.
Turn on the 1, 064-nanometer trapping laser to attract silver nanoparticles in the sample chamber and create a plasmonic silver nanoparticle assembly. Turn down the trapping laser beam to avoid overheating or bubble formation when required. Adjust the position of the sample microstage to place the dark spot of the plasmonic silver nanoparticle assembly under the focus of the 532-nanometer Raman probe beam for spectroscopic measurements.
Place the neutral density filters in front of the 532-nanometer Raman laser outlet to adjust the power to 10 megawatts. Input the acquisition time in the setting panel in the spectrum software and click on the Acquire button to start the spectral acquisition. Without the trapping laser, the dispersed silver nanoparticles in the sample chamber generated a black spectrum.
Increasing the power and extending the irradiation time of the trapping laser could attract more silver nanoparticles and generate a dark spot. Since the dispersed silver nanoparticles were under Brownian motion, the interparticle junctions were large and unstable. The overall intensities of DSNB in the plasmonic silver nanoparticle assembly were higher than those of the dispersed silver nanoparticle.
Considering the intensity of the characteristic peak at 1, 444-centimeter inverse, the plasmonic silver nanoparticle assembly can provide approximately a 50-fold enhancement of the surface-enhanced Raman spectroscopy signal of DSNB compared to that of the dispersed silver nanoparticles. The intensities of the characteristic peaks of DSNB at 1, 152, 1, 444, and 1, 579-centimeter inverse across these 20 surface-enhanced Raman spectra were plotted as histograms with relative standard deviations of 6.88, 6.59, and 5.48%respectively. The most important thing in this procedure is locating the position of the 532-nanometer Raman probe laser and overlapping it with the 1, 064-nanometer trapping laser.
This technique paves the way for researchers to detect analyte molecules with spatial and temporal control under physiological conditions for future in vivo analysis.
