Increased intensities of YOYO-1 labeled DNA oligomers near silver particles[para]

Photochemistry and Photobiology, Jun 2003 by Lakowicz, Joseph R, Malicka, Joanna, Gryczynski, Ignacy

ABSTRACT

DNA detection is usually performed using fluorescence probes. Using a DNA oligomer stained with the widely used dye 1,1'-[1,3-propanediylbis[(dimethylimino)-3,1-propanediyl]]-bis[4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]]-quinolinum tetraiodide (YOYO-1), we show that a substrate containing silver particles can lead to a greater than 10-fold increase in the fluorescence intensity. Proximity to silver particles also increases the photostability of YOYO-1-DNA. These results suggest that substrates or gels containing silver particles may be used for increased sensitivity in DNA detection.

INTRODUCTION

The intrinsic fluorescence from DNA is extremely weak. As a result, DNA detection is often performed using fluorescent probes. The TOTO and YOYO series are a valuable class of probes (1-3). These dyes contain multiple positive charges, bind strongly to DNA and display almost no fluorescence in water. As a result, these dyes are useful for the observation of DNA in electrophoretic gels.

During the past 2 years we have investigated the effects of metallic silver particles on fluorescence (4-7). We found that the positioning of fluorophores 50-100 a from a metallic silver surface (8) results in increased emission intensities, decreased lifetimes and modest increases in photostability. These effects appear to be due to interactions of the excited fluorophore with the surface plasmon resonance (SPR) displayed by subwavelength size silver particles (9). The SPR is due to electron oscillations on the metallic surface induced by an incident field. For clarity, we note that our studies are not the usual SPR assays, which depend on a decreased reflectance from a continuous gold surface at specific angles of incidence.

In the present article, we examine the effects of silver particles on DNA oligomers labeled with 1,1'-[1,3-propanediylbis[(dimethylimino)-3,1-propanediyl]]bis[4-[(3-methyl-2(3H)-benzoxazolylidene) methyl]]-quinolinum tetraiodide (YOYO-1) (Scheme 1). Binding to a surface coated with silver particles was found to result in dramatically increased emission intensities of YOYO-1-DNA.

MATERIALS AND METHODS

Biotinylated oligonucleotide 5'-GAA GAT GGC CAG TGG TGT GTG GA-3'-biotin and complementary oligonucleotide 5'-TCC ACA CAC CAC TGG CCA TCT TC-3' were obtained from the Biopolymer Core Facility at the University of Maryland, School of Medicine (Baltimore, MD). YOYO-1 and avidin (egg white) were purchased from Molecular Probes (Eugene, OR). Bovine serum albumin (BSA)-biotin (bovine albumin-biotinamidocaproyl labeled) was from Sigma (St. Louis, MO). Other compounds used for silver island film (SIF) and buffer preparation were from Sigma-Aldrich (St. Louis, MO).

SIF are particles of silver on inert substrates formed by chemical reduction. Such films are widely used in surface-enhanced Raman scattering. Our SIF were deposited on clean quartz slides (Starna Cell Inc., Atascadero, CA) using an ammonia solution of silver nitrate and D-glucose as a reducing agent as described previously (5,10). The size distribution of the SIF based on atomic force microscopy (AFM) image is described by Lakowicz et al. (5).

We used protein-coated surfaces to bind DNA to quartz or SIF (Scheme 1). Each slide (12.5 x 45 mm, half coated with SIF) was covered with 250 [mu]L of 10 [mu]M BSA-biotin aqueous solution and placed in a humid chamber for 20 h (5[degrees]C, cold room). After washing three times with water, slides were placed again in the humid chamber and 250 [mu]L of 5 [mu]M avidin in 0.1x phosphate-buffered saline (PBS) was deposited on each BSA-biotin-coated surface for 40 min at room temperature. The slides were then washed three times with 0.1x PBS buffer, and 250 [mu]L of YOYO-1-DNA-biotin (3:1) solution was deposited for 60 min at room temperature.

Double-stranded (ds)-DNA sample (DNA-biotin) was prepared by mixing complementary oligonucleotides in 5 mM N-(2-hydroxyethyl)piperazine-N-(2-ethanesulfonic acid) (HEPES; pH 7.5), 0.1 M KCl and 0.25 mM ethylenediaminetetraacetic acid (EDTA) solution to a final concentration 1 [mu]M and very slow cooling after incubation at 70[degrees]C for 2 min. To the above solution, 1 mM YOYO-1 solution in dimethyl sulfoxide (DMSO) was added to a final concentration of 3 [mu]M, yielding a YOYO-1/DNA ratio of 3:1. After YOYO-1-DNA-biotin deposition, the slides were washed three times with hybridization buffer (5 mM HEPES, pH 7.5; 0.1 M KCl; 0.25 mM EDTA) and covered with one part of a 0.5 mm demountable cuvette filled with the same buffer. For titration, we used a 1 [mu]M DNA-biotin solution in the hybridization buffer and an appropriate amount of 1 mM YOYO-1 in DMSO.

To check for the presence and possible emission of unbound YOYO-1 on one of the above SIF slides coated with a monolayer of BSA-biotin-avidin instead of YOYO-1-DNA-biotin solution, we deposited a similar sample but without DNA-biotin.

The titration experiment was performed in a 1 cm^sup 2^ cuvette using a Cary Eclipse fluorometer (Varian, Walnut Creek, CA) and an excitation wavelength of 470 nm. Emission spectra of YOYO-1-DNA-biotin on the protein layer were measured in front face geometry on a SLM 8000 spectrofluorometer with 470 nm excitation from a xenon lamp. Lifetimes were measured on a 10 GHz frequency-domain (FD) fluorometer using a mode-locked argon ion laser (514 nm, 76 MHz repetition rate) (11). Excitation and emission polarizers were in the magic angle orientation. Emission was collected with a combination of a 520 nm long pass filter and a 540 nm interference filter.