A Comparison of Single-Molecule Emission in Aluminum and Gold Zero-Mode Waveguides

Abstract

The effect of gold and aluminum zero-mode waveguides (ZMWs) on the brightness of immobilized single emitters was characterized by probing fluorophores that absorb in the green and red regions of the visible spectrum. Aluminum ZMWs enhance the emission of Atto565 fluorophores upon green excitation, but they do not enhance the emission of Atto647N fluorophores upon red excitation. Gold ZMWs increase emission of both fluorophores with Atto647N showing enhancement that is threefold higher than that observed for Atto565. This work indicates that 200 nm gold ZMWs are better suited for single-molecule fluorescence studies in the red region of the visible spectrum, while aluminum appears more suited for the green region of the visible spectrum.

Zero-mode waveguides can be made better: fluorescence enhancement with rectangular aluminum nanoapertures from the visible to the deep ultraviolet

“…8 It also modies the uorescence photokinetics decay rates, 9 improving the net detected photon count rate per molecule. [10][11][12][13][14][15][16][17][18][19][20] Moreover, as the aperture diameter is largely below half the optical wavelength, the light exponentially decays inside the nanoaperture, 8 leading to an effective detection volume in the attoliter (10 À18 L) range, one thousand fold smaller than with a diffraction-limited confocal microscope. 1,6 These improvements have enabled a wide range of applications, including DNA sequencing, [21][22][23] enzymatic reactions, 13,24,25 protein-protein interactions, [26][27][28][29][30] nanopore detection, [31][32][33][34][35][36] and biomembrane studies.…”

Nanoaperture fabrication via colloidal lithography for single molecule fluorescence analysis

“…However, biological processes often occur at concentrations up to a million-fold greater (micro-/milli-molar). A potential solution is nanofabricated zero-mode waveguides (ZMWs) [5–9], arrays of holes (30–300 nm diameter) in a thin, opaque metal layer (typically aluminum, chromium, or gold) on a glass substrate [7,8,10,11]. These sub-wavelength apertures do not propagate optical modes in the visual spectrum, but illumination will create an evanescent wave, like in TIRF, that decays inside the well [12,13].…”

Real-Time Sensing of Single-Ligand Delivery with Nanoaperture-Integrated Microfluidic Devices

“…A647 was used for all single-molecule studies because it is a better single-molecule fluorophore and the plasmon enhancement in gold ZMWs is larger in the red region of the visible spectrum. 45 While imaging through the microfluidic channel during ligand delivery, diffuse fluorescence was observed and corresponded to EGF-A647 in solution. The presence of this fluorescence signal verified the delivery of EGF to the ZMW, and at this point, we initiated data collection, capturing images at a rate of 5 frames per second.…”

“…Molecules on a glass substrate had a survival time of 2.3 s ( Figure 5 D) and those in mf-ZMWs had a survival time of 1.3 s. The increase in fluorescence intensity and the decrease in photostability are similar to what has been observed for molecules isolated in standard gold ZMWs. 35 Representative time traces of molecules on glass versus that in an mf-ZMW are shown in Figure 5 C. We then calculated the relative number of detected photons for single EGF-A647 ligands using the emission intensity and photostability of individual molecules. Molecules in mf-ZMWs yielded 8580 ± 464 counts versus an average of only 3691 ± 163 counts when isolated on the glass surface ( Figure 5 F).…”

“…Gold ZMWs in particular have recently been observed to contribute to a similar fluorescence enhancement of fluorophores, which excite at 640 nm excitation. 35 …”