Single molecule detection from a large-scale SERS-active Au₇₉Ag₂₁ substrate

Abstract

Detecting and identifying single molecules are the ultimate goal of analytic sensitivity. Single molecule detection by surface-enhanced Raman scattering (SM-SERS) depends predominantly on SERS-active metal substrates that are usually colloidal silver fractal clusters. However, the high chemical reactivity of silver and the low reproducibility of its complicated synthesis with fractal clusters have been serious obstacles to practical applications of SERS, particularly for probing single biomolecules in extensive physiological environments. Here we report a large-scale, free standing and chemically stable SERS substrate for both resonant and nonresonant single molecule detection. Our robust substrate is made from wrinkled nanoporous Au₇₉Ag₂₁ films that contain a high number of electromagnetic "hot spots" with a local SERS enhancement larger than 10⁹. This biocompatible gold-based SERS substrate with superior reproducibility, excellent chemical stability and facile synthesis promises to be an ideal candidate for a wide range of applications in life science and environment protection.

Figure 1. Wrinkled nanoporous substrate.

(a) Schematic diagram of the preparation of wrinkled nanoporous films. (b) Photograph of a plasmonic substrate consisting in a wrinkled nanoporous film with dimensions 30 mm × 15 mm supported on a polymer substrate. (c) Photograph of the substrate used in this work with dimensions of 8 mm × 8 mm.

Figure 2. Microstructure characterization of wrinkled nanoporous films.

(a) SEM micrographs of the flat nanoporous film with a characteristic length of 20 ∼ 25 nm. (b) Microstructure of a wrinkled nanoporous film with a quasi-periodic wavelength of 10 ∼ 15 µm. (c) Chemical composition of a nanoporous film measured by energy dispersive X-ray spectroscopy. (d) Microstructure of wrinkle ridges showing nanogaps, interleaving broken ligaments and linear chains of self-similar nanocavities.

Figure 3. Single molecule detection of R6G molecules.

(a) Raman spectra of R6G from a flat nanoporous Au₇₉Ag₂₁ film and wrinkled one. (b) Typical Raman map of R6G (10⁻¹² M) on the wrinkled Au₇₉Ag₂₁ film. The pixel size in the Raman map is 250 × 250 nm². (c) Overlay of the image in b and the corresponding optical microscopic image. The hot spots can be identified along the broken gaps of the wrinkled nanoporous film. (d) Typical SERS spectrum (blue) taken from a hot spot for 10⁻¹⁰ M R6G. Single molecule SERS spectra (A) (B) (C) for 10⁻¹²M R6G taken from the three hot spots in the same colour squares in b. The inset in d gives the statistical distribution of Raman intensity of 28 hot spots in a Raman map of 10⁻¹² M R6G. The laser excitation is 532 nm.

Figure 4. Single molecule detection of DNA adenine molecules.

(a) Typical Raman map of adenine (10⁻⁹ M) (left), corresponding optical microscopic image (middle), and their overlay image (right). The hot spots can be directly observed at the narrower gaps of the wrinkled nanoporous film. (b) Fluctuation field intensity with 3 chained hot spots (adenine 10⁻⁹ M). The configuration is analogous to the “superlenses” with a linear chain of nanocavities suggested by computer simulations. Raman map size: 4.0 × 5.5 μm². The spectral interval is 250 nm. (c) SERS spectrum of 10⁻⁹ M adenine taken from a hot spot. (d) Single molecule SERS spectrum of 10⁻¹²M adenine with selective Raman bands. The laser excitation is 785 nm.