Observation and Analysis of Blinking Surface-enhanced Raman Scattering

Summary

This protocol describes the analysis of blinking surface-enhanced Raman scattering due to the random walk of a single molecule on a silver surface using power laws.

Abstract

From a single molecule at a silver nanoaggregate junction, blinking surface-enhanced Raman scattering (SERS) is observed. Here, a protocol is presented on how to prepare the SERS-active silver nanoaggregate, record a video of certain blinking spots in the microscopic image, and analyze the blinking statistics. In this analysis, a power law reproduces the probability distributions for bright events relative to their duration. The probability distributions for dark events are fitted by a power law with an exponential function. The parameters of the power law represent molecular behavior in both bright and dark states. The random walk model and the speed of the molecule across the entire silver surface can be estimated. It is difficult to estimate even when using averages, autocorrelation functions, and super-resolution SERS imaging. In the future, power law analyses should be combined with spectral imaging, because the origins of blinking cannot be confirmed by this analysis method alone.

Introduction

Surface-enhanced Raman scattering (SERS) is highly sensitive Raman spectroscopy from a noble metal surface. Since the Raman spectrum provides detailed information about molecular structure based on the sharp peak positions, through the vibrational modes of functional groups in the molecules, the information of a single molecule on a metal surface can be investigated using SERS^1,2,3. From a silver nanoaggregate with an adsorbate at the single-molecule level, a blinking signal is observed^1,2,

Protocol

1. Sample Preparation

1. Preparation of silver colloidal nanoparticles²⁰
   1. To fabricate silver colloidal nanoparticles, dissolve 0.030 g of silver nitrate and 0.030 g of trisodium citrate dihydrate in 150 mL of water in a 200-mL round bottom flask.
   2. Combine the flask with a reflux (Dimroth) condenser.
   3. Stir the solution in the flask with a magnetic stirrer and stir bar. Then, heat the stirring solution in the flask in an oil bath at 150 °C for 60 min.
      NOTE: The solution will turn yellow, then milky grey.
   4. Cool the suspension at room temperature, and keep the suspension in t....

Results

From the silver nanoaggregates with poly-L-lysine prepared by protocol 1.2, multicolored blinking spots from SERS and surface-enhanced fluorescence are observed, as shown in Figure 1¹¹. In contrast, monotonous colored blinking spots from SERS were observed for the silver nanoaggregates with the dye molecules prepared by protocol 1.3^{7,8,9,.......}

Discussion

From the silver nanoaggregate junction, SERS is emitted. Thus, we need to prepare nanoaggregates rather than colloidal nanoparticles, which are covered with citrate anions. Silver aggregates are formed from the salting out effect created by the addition of poly-L-lysine, which has -NH₃⁺ and is the origin of the SERS, or Na⁺ cations from NaCl, as shown in Figure S2 of the supplementary material. Moreover, to illuminate the many spots in the wide area, the unfocused laser b.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Silver nitrate, 99.8%	Wako	194-00832
Trisodium citrate dihydrate, 99. %	Wako	191-01785
Poly-L-lysine aqueous solution, 0.1%	Sigma-Aldrich	P8920
3,3'-disulfopropylthiacyanine triethylamine	Hayashibara Biochemical Laboratories	NK-2703	a kind of thiacyanine dyes
3,3'-diethyl-5,5'-dichloro-9-methylthiacarbocyanine iodine salt	Hayashibara Biochemical Laboratories	SMP-9	a kind of thiacarobocyanine dyes
Sodium chloride, 99.5%	Wako	191-01665
Dimroth condenser	Iwaki	61-9722-22	perchased from AS ONE
Magnetic stirrer	Corning	DC-420D
Oil bath	Advantech	OS-220
Glass plate	Matsunami	S-1112	Microscope slide
Blower	Hozan	Z-288	Air duster
Liquid blocker pen	Daido Sangyo		LIQUID BLOCKER (Super Pap Pen). Ready-to-use hydrophobic barrier pen designed for immunohistochemistry applications
Inverted microscope	Olympus	IX-70
Objective lens	Olympus	LCPlanFl 60×	NA 0.7
Dark field condenser	Olympus	U-DCD	NA 0.8–0.92
Cooled digital CCD camera	Hamamatsu	ORCA-AG	controlled by software Aqua Cosmos
Software for the cooled digital CCD camera	Hamamatsu	AquaCosmos	used for also derivation of the time-profiles from the blinking spots in the video
Color CCD camera	ELMO	TNC-C920	not used for analysis
DPSS laser	RGB laser system	NovaPro532-75	λ = 532 nm; 60 mW (corresponds to a power density of 600 W/cm2)
Interference filter	Semrock	LL01-532-12.5
Long pass filter	Semrock	BLP01-532R-25
Software for the distinguishment and counting of the bright/dark events	home-maid		programmed by C++
Software for the fitting by a power law	LightStone	Origin6.1