Enhancement of fluorescence by metal nanostructures has recently become one of the most exciting interdisciplinary research areas in nanobiotechnology because of potential applications in surface-enhanced spectroscopies, [1] biosensors, [2][3][4] and optical devices. [5] Metal-enhanced fluorescence (MEF) methods [6][7][8][9][10][11][12][13][14] use effective coupling between excited fluorophores and the localized surface plasmon resonance (LSPR) [15][16][17][18] in metal nanostructures to enhance fluorescence emission. Such interactions can be modulated in several ways, [6,12] including local field enhancement of incident optical density, increase in radiative decay rate of the fluorophore, and modulation of the far-field radiative coupling of fluorescence emission through nanoparticle scattering. In contrast to the explosive use of MEF, biological uses of MEF have not focused on the limits of detection for biomolecules or how optically-tunable, metal nanopatterns can be used for highly sensitive fluorescence sensing. Thus far the MEF effect has been limited to relatively high concentrations of target molecules which produce low signal enhancement and thus low detection. [7][8][9] This is due to relatively poor ordering of the metal nanostructures over the sensor area and imprecise spectral coupling between the flourophores and nanostructures, constituting major drawbacks to practical applications of MEF.Here, we report a significantly increased sensitivity of MEF-based biological detection by using a combination of a large-area metal nanopattern (2.7 mm  2.7 mm) and layerby-layer assembly (LbL) of polyelectrolyte films. We use the nonlinear optical process of two-photon excitation (TPE) [19][20][21] which has been finding increasing use in biology because of its smaller focal spot size, intrinsic three dimensionality, and reduced damage to living organisms when compared to one-photon systems. We demonstrate TPE coupled with our structures can lead to highly enhanced fluorescence with two orders of magnitude increased emission. Detection of extremely low concentrations of target Cy5-DNA molecules ($10 À11 M) was achieved by controlling feature size and polyelectrolyte film thickness, allowing precise spectral tailoring. Figure 1 is a schematic representation of enhanced two-photon excited fluorescence (TPEF) DNA detection using large-area metal nanopatterns. To prepare a desired substrate, capillary force lithography (CFL) [22,23] was employed to fabricate metal nanopatterns with various feature sizes over a large, patterned area (2.7 mm  2.7 mm). We used the versatility of CFL to generate patterns of gold metal dots of diameter (D) 110 or 150 nm, with a period (S) of 250 nm. A dot height of 35 nm was produced consistently across all samples to give the most sensitive LSPR. [11,15] It is noteworthy that the feature diameter of the gold dots can be further controlled by varying the reactive ion etching (RIE) time.[22] Various feature diameters (110-150 nm) of gold patterns can be prepared from a single dot (D ¼ ...