Single-Molecule Studies on Fluorescently Labeled Silver Particles: Effects of Particle Size

Abstract

We studied the dependence of single-molecule fluorescence on the size of nearby metal particles. The silver particles were synthesized with average diameters of metal cores being 5, 20, 50, 70, and 100 nm, respectively. A single-stranded oligonucleotide was chemically bound to a single silver particle and a Cy5-labeled complementary single-stranded oligonucleotide was hybridized with the particle-bound oligonucleotide. The space between the fluorophore and metal core was separated by a rigid hybridized DNA duplex of 8 nm length. The single fluorescence images and intensity traces were recorded by scanning confocal microscopy. The single fluorophore-labeled 50 nm silver particles displayed the most enhanced intensity, a factor of 17-fold increase relative to the free fluorophores in the absence of metal. Numerical simulations by the finite-difference time-domain (FDTD) method and results from Mie theory were used to compare with the experimental results. The 50 nm silver particles were also labeled by multiple fluorophores. The fluorescence intensity of multiple fluorophore-labeled metal particles increases dramatically with the loading number and reached 400-fold relative to the free single fluorophore when the loading number of fluorophore per metal particle was 50. The fluorophore also displayed better photostability when binding on the metal particle. These results can aid us to develop novel nanoscale fluorophores for clinical diagnostics and bioassay.

Figure 1

Absorbance spectra of tiopronin-coated silver particles with the different metal core sizes.

Figure 2

Transmission electron micrograph (TEM) images of silver particles with the different metal core sizes of (a) 5, (b) 20, (c) 50, (d) 70, and (e) 100 nm. Histograms of the size distributions of the metal particles were inserted correspondingly.

Figure 3

Ensemble emission spectra of Cy5-labeled oligonucleotide in the absence of metal and bound on the 20 nm silver particles.

Figure 4

Respective fluorescence images of single-labeled (a) free Cy5; a single fluorophore on (b) 5, (c) 20, (d) 50, (e) 70, and (f) 100 nm silver particle; and (g) 50 fluorophores on 50 nm silver particle. The intensity bar for a-f is 0-200 and that for g is 0-1000. Sample g was excited at 20% laser intensity of the other samples. The 5 × 5 μm images are 100 × 100 pixels within integration time of 0.6 ms/pixel.

Figure 5

Histograms of emission intensity from single Cy5 fluorophores loaded on different-size silver particles.

Figure 6

Respective time traces of free Cy5-labeled oligonucleotide, single Cy5 on the metal particles with the different metal core sizes of 5, 20, 50, 70, and 100 nm, and 50 fluorophores loaded on a single 50 nm silver particle.

Figure 7

Dependence of enhancement efficiency of single-fluorophore-labeled metal particle and the electric field intensity at 8 nm away from the metal core on the size of the metal core.

Figure 8

Intensity distribution of the electric field near the silver particles with different core sizes of 5, 20, 50, 70, and 100 nm. The field images were achieved by FDTD calculations. The circles represent the distance of 8 nm away from the metal cores.

Figure 9

Dependence of enhancement efficiency on the loading number per metal particle for the multiple-fluorophore-labeled silver particle with 50 nm diameter.

SCHEME 1. Succinimidylated Silver Particle Covalently Bound with Aminated Single-Stranded Oligonucleotide and Fluorescently Labeled by Complementary Single-Stranded Cy5-Labeled Oligonucleotide^a

^aOligonucleotide sequences used in the experiments are given.