WO2015188182A1

WO2015188182A1 - Heterogeneous optical slot antenna and method for single molecule detection

Info

Publication number: WO2015188182A1
Application number: PCT/US2015/034690
Authority: WO
Inventors: Chenglong ZHAO; Yongmin Liu; Jiasen ZHANG
Original assignee: Peking University; Northeastern University Boston
Current assignee: Peking University; Northeastern University Boston
Priority date: 2014-06-06
Filing date: 2015-06-08
Publication date: 2015-12-10
Anticipated expiration: 2016-12-06

Abstract

An optical nanoslot antenna device is capable of detecting a single analyte molecule at picomolar to micromolar concentrations. The device includes a rectangular nanoslot fabricated in a heterogeneous metallic film formed by sequential deposition of plasmonic and nonplasmonic materials on an optically transparent substrate. With light illumination from the substrate, the device provides large field enhancement at the bottom of the nanoslot where the analyte molecule is positioned. The electromagnetic field inside the upper portion of the nanoslot is suppressed by the nonplasmonic layer. Methods of using the device include single molecule detection and analysis for biomedical applications, including single molecule DNA sequencing.

Description

TITLE OF THE INVENTION:

Heterogeneous Optical Slot Antenna and Method for Single Molecule Detection

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the priority of U.S. Provisional Application No. 62/008,571 filed 06 June 2014 and entitled "Single-molecule Detection and Radiation Control in Solutions at High Concentrations via a Heterogeneous Optical Slot Antenna", which is hereby incorporated by reference.

BACKGROUND

The detection of single-molecule fluorescence is a key technique for numerous applications in bioscience, including DNA sequencing, diagnostics, and molecular biology. Unfortunately, the detection volume is limited to femtoliters in conventional diffraction-limited optics. In addition, the concentration of molecules has to be limited to the picomolar or nanomolar level, so that on average only one molecule is excited inside the diffraction- limited optical spot. This concentration is far below the micromolar range where many biologically relevant processes occur. Such a limitation can be overcome by using a so- called zero-mode waveguide (1-3) (ZMW), which consists of nanoscale circular holes milled in an aluminum film. The key design principle of the ZMW is that the light field is mainly confined at the bottom of the nanoholes, which act as small reaction chambers with single molecules inside. The metal film blocks the illuminating light so that only the molecule located at the bottom of the nanoholes can be excited and detected while leaving other molecules unaffected. The ZMW allows reduction of the observation volume by 3 to 6 orders of magnitude, from 10^"15 liters (using a standard confocal microscope) to 10^"18 to 10^"21 liters, allowing for single-molecule detection. ZMWs with different shapes, such as circles, rectangles, bowties, and C-shapes have been designed for single-molecule studies. These ZMW structures are commonly fabricated on an Al film with the light field well confined at the bottom but with less field enhancement in the visible spectrum compared with using silver or gold. The poor field enhancement of ZMWs further limits the fluorescence emission of a molecule inside these structures according to optical reciprocity.

Optical nanoantennas, which enable efficient conversion between free space optical radiation and highly localized energy (4), have attracted extensive attention in recent years l for use in fluorescence enhancement (5-12). Optical antennas consisting of nanoapertures fabricated on Ag or Au films (13-21 ) are useful for applications in single-molecule detection at high concentrations. Both the fluorescence emission rate and its radiation pattern can be controlled using optical antennas due to their strong plasmonic resonances (22-24). Au is preferable in these applications because of its unique properties, such as high resistance to oxidation and a wide range of available self-assembly molecules (25-27). In these nanostructures, the light fields at the top surface (water-gold) and at the bottom surface (substrate-gold) tend to be both enhanced because of the strong plasmonic coupling. However, the field enhancement on the top is detrimental for detection of single molecules in solutions at high concentrations, because molecules on the top surface will also be excited and detected, resulting in increased background noise.

Thus, there is a need to develop new devices and techniques for performing single molecule fluorescence at physiological concentrations of biopolymers.

SUMMARY OF THE INVENTION

The present invention provides an apparatus that is capable of detecting a single analyte molecule in solutions at either low or high concentrations. The apparatus is a heterogeneous optical slot antenna, and includes a rectangular nanoslot fabricated on a heterogeneous metallic film formed by sequential deposition of gold and aluminum on a glass substrate. With light illumination from the glass substrate, the apparatus precisely gives rise to large field enhancement at the bottom of the nanoslot where the analyte molecule is positioned. The electromagnetic field inside the upper portion of the nanoslot is purposely suppressed with a layer of aluminum film. The electromagnetic field inside this apparatus in the lower portion of the nanoslot is 170 times larger than that inside an aluminum zero-mode waveguide. The apparatus also gives rise to large fluorescence enhancement. The fluorescence emission rate of an analyte inside this apparatus is 70 times higher than that of the analyte in free space.

Further features of the invention include: the ability to perform single-molecule analysis at high concentrations; excellent field confinement at the detecting position of the ananlyte molecule; large field and fluorescence enhancement at the detecting position of the ananlyte molecule; less field enhancement away from the detection position; and tunable wavelength response. Compared to previous devices and methods, the invention provides the following advantages: greatly increased field enhancement at the detecting position; greatly increased fluorescence emission rate; excellent balance between performance and cost; tunable resonant wavelength; and the use of heterogeneous metallic layers. The invention provides the following commercial applications: single-molecule analysis at high analyte concentration for biomedical applications; single-molecule DNA sequencing; single- molecule fluorescence detection and enhancement and near-field enhancement.

One aspect of the invention is an optical slot antenna device including an optically transparent substrate, a first layer deposited onto the substrate, and a second layer deposited onto the first layer. The first layer contains a plasmonic material, and the second layer contains a nonplasmonic material. Each of the first and second layers includes an essentially rectangular void. The voids in the first and second layers are aligned and overlap so as to create a nanoslot having nanoscale width, length, and height and closed off on the bottom by the substrate. As used herein, "nanoscale" refers to a structure having at least one dimension, and preferably all dimensions, in the range from 1 nm to 999 nm. Light entering the nanoslot through the substrate is substantially confined to a lower portion of the nanoslot.

Another aspect of the invention is a method of detecting a single analyte molecule in a sample. The method includes the steps of: providing the optical slot antenna device described above, the device containing a sample containing or suspected of containing the analyte disposed in or in and above the nanoslot; irradiating the nanoslot through the substrate of the device with light; and detecting an optical signal from a single molecule of said analyte in the nanoslot from below.

Still another aspect of the invention is a method of making an optical slot antenna device. The method includes the steps of: depositing a first layer comprising a plasmonic material on an optically transparent substrate; depositing a second layer comprising a nonplasmonic material on the first layer; and performing lithography to pattern the first and second layers. The, lithography creates one or more essentially rectangular voids in the first and second layers. The voids in the first and second layers are aligned so as to create one or more nanoslots. Each nanoslot has nanoscale width, length, and height and is closed off on the bottom by the substrate.

Yet another aspect of the invention is a kit for using the nanoslot antenna device described above for single molecule detection or analysis. The kit includes the nanoslot antenna device and one or more reagents or biomolecules, and/or includes instructions for detecting a single analyte molecule using the device.

Further features of the invention are summarized in the items described below.

1. An optical slot antenna device comprising:

an optically transparent substrate;

a first layer deposited onto the substrate, the first layer comprising a plasmonic material; and

a second layer deposited onto the first layer, the second layer comprising a nonplasmonic material; wherein the first and second layers each comprise an essentially rectangular void, wherein the voids in the first and second layers are aligned so as to create a nanoslot having nanoscale width, length, and height and having said substrate as floor, and wherein light entering the nanoslot through the substrate is substantially confined to a lower portion of the nanoslot.

2. The optical slot antenna device of item 1 , wherein the substrate comprises an optically transparent material having a refractive index in the range from about 1.5 to about 2.0.

3. The optical slot antenna device of item 1 or 2, wherein the optically transparent material is selected from the group consisting of glass, quartz, silicon dioxide, silicon nitride, and optically transparent polymer materials.

4. The optical slot antenna device of item 3, wherein the substrate is glass.

5. The optical slot antenna device of any of the preceding items, wherein the first layer comprises Au, Ag, or Al.

6. The optical slot antenna device of any of the preceding items, wherein the first layer consists essentially of Au, Ag, or Al.

7. The optical slot antenna device of any of the preceding items, wherein the first layer consists essentially of Au.

8. The optical slot antenna device of any of the preceding items, wherein the thickness of the first layer is in the range from about 50 nm to about 300 nm.

9. The optical slot antenna device of any of the preceding items, wherein the second layer comprises Al, Cr, or Ti, with the proviso that if the first layer comprises Al, the second layer does not comprise Al.

10. The optical slot antenna device of any of the preceding items, wherein the second layer consists essentially of Al, Cr, or Ti, with the proviso that if the first layer comprises Al, the second layer consists essentially of Cr or Ti.

1 1 . The optical slot antenna device of any of the preceding items, wherein the thickness of the second layer is about 20 nm or greater.

12. The optical slot antenna device of any of the preceding items, wherein the width of the nanoslot is in the range from about 10 nm to about 50 nm.

13. The optical slot antenna device of any of the preceding items, wherein the length of the nanoslot is in the range from about 50 nm to about 200 nm.

14. The optical slot antenna device of any of the preceding items, wherein the height of the nanoslot is in the range from about 70 nm to about 500 nm.

15. The optical slot antenna device of any of the preceding items, wherein the first layer consists essentially of Au, the second layer consists essentially of Al, the thickness of the first layer is about 100 nm, the thickness of the second layer is about 50 nm, the width of the nanoslot is about 40 nm, the length of the nanoslot is about 1 10 nm, and the height of the nanoslot is about 150 nm.

16. The optical slot antenna device of any of the preceding items, further comprising one or more additional nanoslots formed by one or more additional overlapping voids in the first and second layers.

17. The optical slot antenna device of any of the preceding items, wherein said nanoslots form a two-dimensional array of nanoslots.

18. The optical slot antenna device of any of the preceding items, further comprising a light source for illuminating the nanoslot through the substrate.

19. The optical slot antenna device of any of the preceding items, further comprising a detector for light emitted from the nanoslot.

20. The optical slot antenna device of any of the preceding items, which is capable of detecting a single analyte molecule present in a solution disposed in the nanoslot by means of fluorescence, surface plasmon resonance, Raman spectroscopy, or a nonlinear optical property.

21 . The optical slot antenna device of any of the preceding items, wherein fluorescence of an analyte molecule disposed in the lower portion of the optical slot is enhanced about 70-fold compared to fluorescence of said analyte molecule in free space.

22. The optical slot antenna device of any of the preceding items, capable of detecting an analyte molecule present in a solution disposed in the nanoslot at a concentration in the range from about 10^"12 molar (picomolar) to 10^"6 molar (micromolar).

23. The optical slot antenna device of any of the preceding items, which does not act as a zero mode waveguide.

24. The optical slot antenna device of any of the preceding items, configured for use with a fluorescence microscope.

25. The optical slot antenna device of any of the preceding items which is capable of confining light ranging from about 300 nm to about 2000 nm in wavelength in said lower portion of said nanoslot.

26. The optical slot antenna device of any of the preceding items, further comprising a biomolecule immobilized in said lower portion of the nanoslot.

27. The optical slot antenna device of item 26, wherein the biomolecule is a DNA polymerase or a fluorescent biomolecule.

28. A method of detecting a single analyte molecule in a sample, the method comprising the steps of: (a) providing the optical slot antenna device of item 1 , the device comprising a sample suspected of containing said analyte disposed in and above said nanoslot;

(b) irradiating the nanoslot through the substrate of the device with light; and

(c) detecting an optical signal from a single molecule of said analyte in the nanoslot from below.

29. The method of item 28, wherein the optical signal is single molecule fluorescence, a Raman signal, or a nonlinear optical signal such as second or higher order harmonic generation.

30. The method of item 28 or 29, wherein the analyte is fluorescent or is a material or particle with a nonlinear optical property.

31 . The method of any of items 28-30, wherein the sample is an aqueous solution containing said analyte.

32. The method of item 31 , wherein the aqueous solution further comprises an enzyme.

33. The method of item 32, wherein the enzyme is a DNA polymerase, and the method is used for nucleic acid sequencing.

34. A method of making an optical slot antenna device, the method comprising the steps of:

(a) depositing a first layer comprising a plasmonic material on an optically transparent substrate;

(b) depositing a second layer comprising a nonplasmonic material on the first layer; and

(c) performing lithography to pattern the first and second layers, whereby one or more essentially rectangular voids are created in the first and second layers, the voids in the first and second layers being aligned so as to create one or more nanoslots, each nanoslot having nanoscale width, length, and height and having said substrate as floor.

35. The method of item 34, wherein the first layer comprises Au, Ag or Al.

36. The method of item 34, wherein the first layer consists essentially of Au, Ag, or Al.

37. The method of item 36, wherein the first layer consists essentially of Au.

38. The method of item any of items 34-37, wherein the second layer comprises Al, Cr, or Ti, with the proviso that if the first layer comprises Al, the second layer comprises Cr or Ti.

39. The method of item any of items 34-37, wherein the second layer consists essentially of Al, Cr, or Ti.

40. The method of item 39, wherein the second layer consists essentially of Al. 41 . The method of any of items 34-40, wherein the width of the nanoslot is in the range from about 10 nm to about 50 nm, the length of the nanoslot is in the range from about 50 nm to about 200 nm, and the height of the nanoslot is in the range from about 70 nm to about 500 nm, preferably in the range from about 100 to 150 nm.

42. The method of any of items 34-41 , wherein a plurality of said nanoslots are formed, and said nanoslots form a two-dimensional array of nanoslots.

43. The method of any of items 34-42, further comprising immobilizing a biomolecule in a lower portion of the nanoslot or nanoslots.

44. A kit comprising the nanoslot antenna device of any of items 1-27 and one or more reagents or biomolecules, or comprising instructions for detecting a single analyte molecule using the device.

45. A microfluidic device comprising the nanoslot antenna device of item 1 , wherein said microfluidic device comprises one or more channels for delivery of a fluid sample or other liquids to the nanoslot of the nanoslot antenna device.

BRIEF DESCRIPTION OF THE DRAWINGS

Figs. 1A-1 D show schematic illustrations of different nanostructures for single- molecule detection. Fig. 1A shows a zero-mode waveguide (ZMW). Fig. 1 B shows an optical slot antenna (OSA) made using an aluminum film on a glass substrate. Fig. 1 C shows an optical slot antenna on a gold film. Fig. 1 D shows a heterogeneous-OSA of the present invention made using gold and aluminum layers on a glass substrate. For Figs. 1A- 1 D, the upper row shows the top view of the respective nanostructures in the xy plane, and the lower row shows the side view of the respective nanostructures in the xz plane.

Fig. 2A shows the wavelength dependence of the near-field intensity for a ZMW (lower curve at right side, scale on right) and an AI-OSA (upper curve with peak, scale at left). Near field intensity was recorded at the origin (x = 0, y = 0, and z = 0) of the coordinates as indicated in Fig. 1. Near-field distribution in the xz plane for the ZMW is shown in Fig. 2B, and AI-OSA is shown in Fig. 2C, both at wavelength λ = 680 nm.

Fig. 3A shows near-field intensities of an Au-OSA (having a peak at 750 nm) and a heterogeneous OSA (peak at 680 nm) versus wavelength. The inset shows the near-field distribution of a heterogeneous OSA on the xy plane at 20 nm below the lower Au surface (z = -20 nm). Fig. 3B shows the near-field distribution for an Au-OSA in the xz plane at its plasmonic resonant wavelength (λ = 750 nm). Fig. 3C shows the near-field distribution for a heterogeneous OSA of the invention in the xz plane at its plasmonic resonant wavelength (λ = 680 nm). In Figs. 3B and 3C, each molecule is marked as a blue arrow along with a dot. Fig. 4A shows the wavelength dependence of the near-field intensity for a heterogeneous OSA of the invention with different Au film thicknesses (hi = 40 (bottom curve), 60 (2^nd from bottom), 80 (3^rd from bottom), and 100 nm (top curve)). The thickness of the Al film is kept constant at h2 = 50 nm. The inset schematically shows the structure of the heterogeneous OSA. Near-field intensity was calculated at origin (x = 0, y = 0, z = 0 nm). Fig. 4B shows the wavelength dependence of the near-field intensity for a heterogeneous OSA of the invention with different metal compositions: Al-Au (top curve), Ti-Au (bottom curve), and Cr-Au (middle curve). The thickness of the bottom layer (Au) is hi = 100 nm, and the thickness of the top layer (Al, Ti, or Cr) is h2 = 50 nm.

Fig. 5A shows the wavelength dependence of the normalized excitation rate and the quantum yield of a molecule located at x = 0, y = 0, and z = 10 nm as shown in Fig. 1 C. Fig. 5B shows the wavelength dependence on the fluorescence enhancement. The inset shows a molecule, represented by a dipole with dipole moment along the x axis (marked with blue arrow), inside the heterogeneous OSA.

Fig. 6A shows the wavelength dependence of the near-field intensity of an Au-OSA with water (peaks at 600 and 750 nm) or oil (peaks as 626 and 808 nm) as the surrounding medium. The inset shows the OSA structure within the xz plane. The inset in the black box schematically shows the plasmonic mode hybridization. Fig. 6B shows the field distribution for the symmetry and anti-symmetry modes in the xz plane. The green arrows show the instantaneous direction of the x compenent, Jx, of the current flow, and the corresponding signs of the charges are also marked.

Fig. 7 shows the wavelength dependence of the near-field intensity for an Au-OSA with three different gold film thickness (h = 100, 150, and 300 nm). The inset schematically shows the structure of the Au-OSA. Near-field intensity was recorded at the origin (x=0, y=0, z=0 nm).

Fig. 8 shows the plasmonic resonance variation as a function of the width, length, and depth of the Au-OSA. The black solid curve corresponds to the plasmonic resonant peak of an Au-OSA with parameters w = 40, I = 1 10 and h = 100 nm, the same curve shown in Fig. 3A.

Fig. 9A shows the wavelength dependence of the near-field intensity for a heterogeneous OSA with different Al film thickness (h2 =10 (bottom curve), 30 (middle curve), and 50 nm (top curve)). The thickness of the gold film is kept constant at h1 = 100nm. The inset schematically shows the structure of the heterogeneous OSA. Near-field intensity was recorded at the origin (x=0, y=0, z=0 nm). Fig. 9B shows the field distribution for a heterogeneous OSA (Au thickness hi = 100nm, Al thickness h2 = 30nm) in the xz plane at its plasmonic resonant wavelength λ = 700 nm. Fig. 9C shows the field distribution for a heterogeneous OSA (Au thickness hi = 100nm, Al thickness h2 = 10nm) in the xz plane at its plasmonic resonant wavelength λ = 710 nm.

Fig. 10 shows the wavelength dependence of the normalized radiative (peak = 670 nm) and nonradiative (peak = 665 nm) decay rates for a molecule located near the bottom of a heterogeneous OSA (x=0, y=0, z=10 nm), 10 nm above the glass substrate.

DETAILED DESCRIPTION OF THE INVENTION An optical nanoslot antenna device according to the present invention confines and enhances a light field in the lower portion of a nanoscale slot where a single molecule can be excited and detected. The device design optimized for optical single-molecule detection at high (micromolar) to low (picomolar and below) concentrations. The optical nanoantenna of the invention has excellent field confinement at the bottom and negligible field enhancement on the top of the nanoslot, a large enhancement of the electromagnetic field and fluorescence, and excellent balance between performance and cost.

Compared to zero mode waveguides (ZMWs), the heterogeneous optical slot antenna (OSA) of the invention, also referred to herein as a nanoslot antenna device, substantially enhance the fluorescence of single molecules, while yielding excellent field confinement and enhancement at the bottom of the antenna nanoslot. In a preferred embodiment, the nanoslot antenna device includes a rectangular nanoslot on a heterogeneous metallic bilayer film, formed by depositing a plasmonic material film (e.g., Au) and a nonplasmonic material film (e.g., Al) in sequence on an optically transparent substrate. The nonplasmonic film can greatly quench the light field on the top of the plasmonic layer but allows the large field enhancement at the bottom (i.e., in the plasmonic layer). The field enhancement within a heterogeneous OSA is 170 times larger than that inside a ZMW using aluminum alone. This selective enhancement of the optical field at the bottom of the antenna makes it especially suitable for enhancing single molecule detection in solution at high concentrations, allowing a fluorescence enhancement factor of 70 for single molecules inside the heterogeneous OSA compared to that emitted in free space. The performance and cost of the OSA devices is well balanced. This design of the nanoslot antenna device of the present invention enables a new paradigm for developing plasmonic nanostructures for applications in biomolecule and enzyme dynamics at the single-molecule level.

Figs.1A-1 C show schematic structures of prior art waveguide and slot antenna designs. Fig. 1A shows a typical ZMW structure that consists of a circular nano-hole milled into 100 nm thick Al film on a glass substrate. In the simulation which follows, the diameter and depth of the ZMW are chosen to be D = 50 and h = 100 nm, respectively, a typical size parameter that has been used in most experiments in the literature. Fig. 1 B shows an optical slot antenna that was etched into an Al film (AI-OSA). The AI-OSA consists of a rectangular nanoslot formed in a 100 nm thick Al film on a glass substrate. The width, length and depth of the AI-OSA is w = 40 nm, I = 160 nm, and h = 100 nm, respectively. Fig. 1 C shows an optical slot antenna fabricated on a 100 nm Au film (Au-OSA) with parameters w = 40 nm, 1 = 1 10 nm, and h = 100 nm, respectively. Fig. 1 D shows a heterogeneous optical slot antenna of the invention (heterogeneous-OSA) with parameters w = 40 nm, 1 = 1 10 nm, and h = 150 nm. The heterogeneous-OSA is made on a heterogeneous film that consists of one layer of 100 nm thick Au film and another layer of 50 nm thick Al film. The three rectangular optical slot antennas shown in Figs. 1 B, 1 C, and 1 D have better performance than a conventional, circular ZMW, as will be discussed below. The designed geometry of OSA can be routinely fabricated using current nanofabrication technology, such as focused ion beam (FIB) milling. The origin (x=0, y=0, z=0) of the system is defined at the bottom center of the nanostructures so that the xy plane at z=0 overlaps with the metal-glass interface. The wavelength dependence of the near-filed intensity is recorded at the origin and all the structures are with water as the superstrate in the following discussions, unless otherwise stated.

Referring again to Fig 1 D, an embodiment of a nanoslot antenna device 10 is shown as a top view (upper portion of figure) and as a cross-section (lower portion of figure). Substrate 20 is a glass layer, which is covered by a first layer 30 consisting of Au, which in turn is covered by a second layer 40 consisting of Al. The rectangular slot or void 50 extends through both first and second layers, and has nanoscale dimensions of width (w), length (I), and height (z).

The substrate is an optically transparent material whose purpose is both to provide mechanical support for the first and second layers and to provide an optically transparent window into the nanoslot space for entry and exit of light. Substrate geometry is typically planar, though other forms can be used. Thickness of the substrate can vary over a wide range, and can be selected according to the material, the desired interface with other equipment, and a balance between rigidity and light transmission. Suitable materials for the substrate are those having a refractive index of about 1.5 and above, such as about 1 .5 to about 2.0. Examples of suitable materials include glass, quartz, silicon dioxide (silica), silicon nitride (Si₃N₄), and optically transparent polymers such as polycarbonate and polyethylene terephthalate (PET). The thickness of the substrate is preferably smaller than 170 μηη to permit use of a high numerical aperture (NA) objective lens to collect the detection light. Substrate thickness larger than 170 μηη is acceptable if a low NA lens is used for collecting the detection light. The first layer is formed of a plasmonic material, which is a metal, metal-like, or metamaterial having negative permittivity. Examples of suitable materials include Au, Ag, Al, and combinations thereof. The first layer can comprise, consist essentially of, or consist of these materials. The second layer is formed of a nonplasmonic material, which is a metal or metal-like material that does not show strong surface plasmon resonance in the visible or infrared spectrum. Suitable materials include Al, Cr, Ti, and combinations thereof. The second layer can comprise, consist essentially of, or consist of these materials, with the proviso that if the first layer contains Al, the second layer does not contain Al. The thickness of the first and second layers together determines the height of the nanoslot. The thickness may be adjusted, such that if the first layer is thicker, then the second layer should be thinner. The ratios of the first layer thickness to the second layer thickness is preferably in the range from 1 to 2.

Each of the first and second layers possesses a void, or empty space. The voids of the first and second layers overlap and are preferably of the same size and shape. The voids of the first and second layers combine to form a single void of the nanoslot. The voids of the first and second layers, as well as the nanoslot itself, is essentially rectangular in the planes of the layers; rectangular voids with rounded corners (e.g., due to fabrication errors) are also acceptable. Preferably, the angles at all vertices are about 90 degrees, but may vary slightly therefrom within a range of angles from about 90 to about 120 degrees. It is also acceptable if the slot is V-shaped in the vertical direction (e.g., due to fabrication errors). In three dimensions, the combined void of the nanoslot essentially has the shape of a rectangular prism or V-shaped prism, with all vertices having angles of about 90 degrees, or within a range from about 90 to about 120 degrees.

For the device to be used in the ultraviolet or violet regions of the spectrum, the first layer can be fabricated from Al, and the second layer can be Ti or Cr. If the device will be used in the visible portion of the spectrum, either Ag or Au can be used as the first layer, and the second layer can be Al, Ti or Cr. If the device will be used in the infrared portion of the spectrum, Au is preferred for the first layer, while the second layer can be Al, Ti or Cr. In all cases, the length of the slot is typically much smaller than the light wavelength (for example, the length of the slot is 0.16 times the light wavelength in the case of the parameter described in item 15), and the length of the slot can be readily adjusted to have a surface plasmon resonance (SPR) at or around the illuminating light wavelength.

To form the first and second layers, any know method of chemical or physical deposition can be used, such as chemical vapor deposition or sputtering. The pattern of one or more voids can be established by performing a lithographic method, such as electron beam lithography, focused ion beam milling, or reactive ion etching. The nanoslot antenna device can be further outfitted with a light source, such as a lamp, laser, or laser diode, for illuminating analyte molecules through the substrate. The device also can be outfitted with a detector, such as a photomultiplier tube (PMT), diode array, avalanche photodiode (APD), or imaging device for detecting and/or tracking analyte molecules. In certain embodiments, the device can be configured for use with a standard fluorescence microscope, or a customized analytical instrument. Two or more nanoslots can be included on a single device or chip. For example, the device can be configured as an array containing 100 or more, 1000 or more, 10000 or more, 100000 or more, or 1 million or more nanoslots. Such arrays can be used for the simultaneous analysis of many samples in parallel. Samples for analysis can be, for example, biological fluid samples, tissue or cell homogenates, samples containing or suspected of containing certain chemicals such as toxins, or environmental testing samples. The samples for analysis can be delivered to the nanoslots from above by one or more microfluidic devices. Thus, the nanoslot device of the present invention can be fully integrated into a microfludic device.

Analytes can be detected, analyzed or manipulated at the single molecule level using the nanoslot antenna device. For example, an analyte molecule can have its own intrinsic fluorescence, or it can be detected through binding to an analyte detecting moiety, such as an antibody, nucleic acid, aptamer, enzyme, receptor, or ligand, which is in turn labeled with a fluorescent moiety. Such an analyte detecting moiety can be added to the sample prior to its placement in the nanoslot or it can be immobilized within the nanoslot, such as by covalent or noncovalent attachment to a wall of the void in the first layer. In a preferred embodiment, the device is used for nucleic acid (DNA or RNA) sequencing, by including a polymerase enzyme in the nanoslot, together with a mix of fluorophore-labeled nucleoside triphosphates that allows each base to be identified by its fluorescence emission as it is added to a nascent copy of a nucleic acid molecule in the sample.

The invention also contemplates kits for practicing a method of the invention. Such kits would include one or more nanoslot antenna devices together with one or more reagents and optionally instructions for use of the device in detecting, analyzing, or manipulating an analyte at the single molecule level.

EXAMPLES

Example 1. Numerical Simulations.

A finite-difference time-domain (FDTD) method (www.lumerical.com) was used for numerical simulations. The refractive index of water and the glass substrate was taken as 1.33 and 1.5, respectively. The optical constants of gold were taken from Johnson & Christy (28) and those of aluminum, titanium and chromium were from Palik (29). Autonon-uniform meshing with a finest mesh size of 1 nm was used for balance between accuracy and computational resources. All the structures in the simulations used the same mesh setting to eliminate the effects of mesh size on the final results. Depending on the symmetry of the simulated structures, anti-symmetric or symmetric boundary conditions (www.lumerical.com) were applied to further reduce the simulation times; otherwise, perfectly matched layer (PML) boundaries were used. A plane wave with an electric amplitude of 1 V/m and a wavelength range from 500 to 900 nm was used to illuminate the structure from the glass substrate. The polarization of the plane wave was perpendicular to the long axis of the OSA (x polarized) as shown in Figs. 1A-1 D. A molecule was modeled as a classic dipole in the simulation, and the near field was recorded with a power monitor.

Example 2. Near Field Intensity for a ZMW and an AI-OSA.

The near-field intensity variation as a function of wavelength for the ZMW (monotonically decreasing curve) is shown in Fig. 2A. The ZMW does not show plasmonic resonance and the near field decreases with increasing wavelength. Fig. 2B shows the near-field distribution of the ZMW in the xz plane at λ = 680 nm. The electric filed is well confined at bottom of the ZMW with negligible fields on top. Such a near-field profile shows how the ZMW works for detecting single molecule at high concentrations. Only molecules that diffuse to the bottom of the nanoholes can be excited and detected while leaving other molecules unaffected. The ZMW shows good field confinement, but with very weak field enhancement. The other curve in Fig. 2A (peak at 680 nm) shows the near-field intensity variation as a function of wavelength for an AI-OSA. The AI-OSA shows plasmonic resonance peaked around λ = 680. Its near-field distribution at λ = 680 nm in the xz plane is shown in Fig. 2C. The near-field intensity at the origin of the AI-OSA is 40 times greater than that of the ZMW at λ = 680 nm. The large field enhancement in the AI-OSA will give rise to a higher excitation rate for a single molecule inside the AI-OSA compared to that inside the ZMW. Although the AI-OSA has relatively large field enhancement compared to a ZMW, it is well known that Al is not a good plasmonic material in visible wavelengths where most fluorescence biomarkers are excited. Instead, Al has a more prominent plasmonic effect in the UV region.

Example 3. Near Field Intensity for an Au-OSA and a Heterogeneous Au-AI-OSA.

The Au-OSA was schematically shown in Fig. 1 C with parameters w = 40, I = 1 10 and h = 100 nm. The curves in Fig. 3A show the wavelength dependence of the near-field variation at the origin of the Au-OSA and heterogeneous Au-AI-OSA with water as superstrate. The Au-OSA shows two plasmonic resonant peaks (centered at λ =600 and 750 nm) originated from the plasmonic hybridization between the upper (water-Au interface) and lower (glass-Au interface) mode of the Au-OSA. Fig. 3B shows the near-field distribution of the Au-OSA at the resonant wavelength λ = 750 nm. Clearly, there is a substantial field enhancement at bottom of the Au-OSA with intensity at the origin, 160 times greater than that inside a ZMW. The large field enhancement of the Au-OSA originates from the plasmonic resonance of the slot, which can be easily tuned by changing the size of the slot. The plasmonic resonance of the Au-OSA as a function of its width, length, and depth are shown in Fig. 8. The plasmonic resonant peak redshifts as the length of the slot increases and blueshifts as the width of the slot increases. Decreasing the depth of the slot causes the two plasmonic peaks to separate further. Therefore, one can always keep large field enhancement when tuning the OSA's plasmonic resonance to a desired wavelength. It should be noted that this is hard to achieve with a conventional ZMW. For single molecule detection in solutions at high concentrations, a near-field profile with large field intensity at the bottom while less field intensity at the top of the nanostructure is preferable. Otherwise, the large field intensity on top will also excite molecules and thus increase background noise. Fig. 3B clearly shows that the Au-OSA gives rise to a large field enhancement at bottom as well as on the top. The large field on top is detrimental for detecting single molecule in micromolar solutions, since molecules on the top of the Au-OSA are also excited.

An objective of the invention was to obtain large field enhancement at bottom while decreasing the field intensity on top. The inventors added another metal (Al) layer on top of the Au-OSA to form a heterogeneous OSA as schematically shown in Fig. 1 D. The thickness of the Au and Al films for the present simulation are 100 and 50 nm, respectively. The width and length of the nanoslot is the same with the Au-OSA, except that the nanoslot is over etched into the glass substrate to take the practical fabrication into account. The Au- OSA curve in Fig. 3A shows the wavelength dependence of the near-field intensity at the origin for the heterogeneous OSA with water as superstrate. The original two plasmonic peaks (λ = 600 and 750 nm) of the Au-OSA now degrade into one peak at λ = 680 nm after adding the additional Al layer. According to the plasmonic hybridization shown in Figs. 6A- 6B, the upper (water-Au side) mode is suppressed and only the lower (glass-Au side) mode of the heterogeneous OSA is excited. The plasmonic resonant peak also locates at λ = 680 nm when the thickness of the gold film is 300 nm as shown in Fig. 7. The same plasmonic resonance achieved for these two cases (thicker Au film or Au-AI heterogeneous film) is a direct verification of the mode hybridization. Fig. 3C shows the field distribution of the heterogeneous OSA at resonant wavelength λ = 680 nm. Compared with the field distribution of an Au-OSA shown in Fig. 3B, one can clearly see that the electric field is highly located at the bottom of the heterogeneous OSA. Only when the molecule diffuses deep into the heterogeneous OSA will it be excited, leaving other molecules unaffected. In this way, the heterogeneous OSA enables single molecule detection in micromolar solution just as the ZMW does but with larger field enhancement. Remarkably, the near-field intensity of the heterogeneous OSA is 170 times larger than that of the ZMW. Example 4. Plasmonic Mode Hybridization of an Au-OSA.

Fig. 6A shows the wavelength dependence of the near field variation at the origin of an Au-OSA with water or objective immersion oil as the surrounding medium, respectively. The whole system is symmetric about the gold film when using immersion oil as superstrate. There are two resonant peaks for the Au-OSA with water (centered at λ = 600 and 750 nm) or oil superstrate (centered at λ = 626 and 808 nm). The two resonant peaks, red shifts with superstrate of higher refractive index, are results of plasmonic hybridization between the upper (water- or oil-Au interface) mode w_u and lower (glass-Au interface) mode oj_/ of the Au- OSA as schematically shown in the inset of Fig. 6A. The upper mode w_u and lower mode oj_/ are the intrinsic plasmonic modes at the top and bottom of the Au-OSA when the thickness of the gold film is infinity. As the film thickness decreases, the two modes begin to overlap and interact with each other, resulting in two new modes ω₊ and ω.. The higher energy mode ω₊ corresponds to the resonant peak at λ = 600 nm (626 nm) with water (immersion oil) superstrate. The lower energy mode ω. corresponds to the resonant peak at λ = 750 nm (808 nm) for water (immersion oil) superstrate.

The mode hybridization was further verified by changing the metal film thickness as shown in Fig. 7. When the gold film thickness is 300 nm, the two modes no longer overlap and only the lower intrinsic mode ω, is excited since the incident light illuminate the structure from glass substrate resulting in one peak centered at λ =680 nm. Fig. 6B shows the near- field distribution for the two modes of the Au-OSA with oil superstrate. The instantaneous current flow direction along x axis J_x is marked by green arrows and the resulting instantaneous charges are also marked as positive (+) or negative (-) depending on the current flow direction. The symmetric mode is defined as the condition where the current flow direction is the same on both surface; otherwise, it is anti-symmetric. The symmetric mode ω. and anti-symmetric mode ω+ of the Au-OSA with oil superstrate are excited at λ = 808 and 626 nm, respectively. The corresponding charges accumulated at the edge of the Au-OSA shown in Fig. 6B are with the same (opposite) signs at top and bottom of the Au- OSA for the symmetric (anti-symmetric) mode. The fields from the two opposite charge oscillations result in destructive interference at middle of the Au-OSA as shown in Fig. 6B. Example 5. Near Field Intensity of Heterogeneous OSA as a Function of Layer Thickness and Metal. The advantage of using a heterogeneous film is two-fold. First, the Au film can greatly enhance the electric field at the bottom of the slot while the Al film can suppress the electric field on top of the slot, which is prerequisite for single molecule detection at high concentrations. Second, it can greatly enhance the excitation and emission rate of a molecule inside the antenna as will be shown below. In practice, the total thickness of the antenna can be further reduced by using either a thinner Au or Al film which is beneficial for fabricating a nanoslot with a high aspect ratio.

Fig. 4A shows the near field intensity recorded at the origin of the heterogeneous OSA for different gold thicknesses. The Al film thickness is kept constant at h2 = 50 nm. The plasmonic resonance blueshifts and the near-field intensity decreases with decreasing gold film thickness. For a heterogeneous OSA with Au and Al thicknesses of 40 and 50 nm, respectively, the plasmonic resonance blueshifts to λ = 645 nm. The near field intensity is still 90 times larger than that inside a ZMW.

The near field intensity of the heterogeneous OSA for three different Al thicknesses is shown in Fig. 9A. The Au film thickness is kept constant at hi = 100 nm. The plasmonic resonance redshifts and the near-field intensity decreases with decreasing Al film thickness. Another effect is that reducing the Al film thickness increases the field intensity on top of the heterogeneous OSA as shown in Fig. 9B and 9C, which should be avoided. Therefore, Al film thicknesses larger than 50 nm are preferred.

The heterogeneous OSA is not limited to the combination of Al and Au films. Other non-plasmonic materials such as, titanium (Ti) or chromium (Cr), can also be used. Fig. 4B shows the near-field intensities of three heterogeneous OSAs with Al, Ti or Cr as the top (second) layer. The thickness of the Au film and the top layer is hi = 100nm and h2 = 50 nm, respectively. Fig. 4B shows the near-field intensity as a function of wavelength for the three heterogeneous OSAs. Varying the materials of the top layer has little effect on the plasmonic resonant peak, since the plasmoinc resonance is mainly determined by the gold layer which is kept the same in the three cases. However, the top metallic layer greatly alters the field enhancement inside the heterogeneous OSA. The top layer with an Al film gives the largest field enhancement compared to the case with a Ti or Cr film as the top layer.

Example 6. Wavelength Dependence of Fluorescence Enhancement with a Heterogeneous OSA.

The large field enhancement of a heterogeneous OSA will result in a significant fluorescence enhancement for molecules inside the antenna. The fluorescence emission rate Y_em is the product of its excitation rate Y_exc and quantum yield η. Thus, the fluorescence enhancement can be expressed as: Yem _ Yexc

Yem Yexc ° where the superscript Ό' indicates the corresponding quantity in the absence of the heterogeneous OSA (i.e., in free space). The excitation rate is proportional to \ρΈ\² if it is not saturated, with p representing the dipole moment and £ being the local electric field. η° is the intrinsic quantum yield in free space and the quantum yield η in the presence of the heterogeneous OSA can be expressed as:

Yr°

Yr + Yabs + Ynr Yr + Yabs + 1 - η°

Yr° η°

Here, the heterogeneous OSA is assumed to have negligible influence on the intrinsic non- radiative rate y_n °. y_r and y° are the radiative decay rates in the presence and absence of the heterogeneous OSA, respectively. y_abs is an additional non-radiative term originated from Ohmic loss in the heterogeneous OSA. The normalized decay rates are calculated as

= and = , respectively, with P_r and P° being the power radiated by a dipole in the presence and absence of the heterogeneous OSA, respectively. P_abs is the dissipated power within the heterogeneous OSA.

A spectral analysis was performed for the fluorescence enhancement for a molecule located at x = 0, y = 0, z = 10 nm of the system. For simplicity, the dipole moment of the molecule was assumed to be oriented along the x axis so that p_y = p_z = 0 and η° ~ 1. This is justified since the x component of the electric field £_x dominates at this location, and only dipoles with x component p_x will be effectively excited. In this case, the normalized excitation rate can be simply calculated with the local electric field enhancement as:

where £_χ and Ε ° are the x component of the local electric field in the presence and absence of the heterogeneous OSA, respectively. The dotted curve showing a peak at 680 nm in Fig. 5A shows the wavelength dependence of the normalized excitation rate, and the other curve shows the quantum yield. The molecule obtained an excitation enhancement factor of 108 at λ = 680 nm. The wavelength dependence of the normalized radiative y_r and nonradiative decay rate y_abs is shown in Fig. 10. Both y_r and y_abs are enhanced around the plasmon resonance wavelength. However, y_r dominates giving rise to a maximum quantum yield η =

0.65 at λ = 700 nm. It is interesting that there is a second nonradiative peak around λ = 550 nm. This peak originated from the longitudinal plasmonic mode of the heterogeneous OSA which can be excited by an incident light with polarization along the long axis of the slot (y polarized). The final wavelength dependence of the fluorescence enhancement is shown in Fig. 5B, with a maximum enhancement factor of 70 at λ = 680 nm. These results strongly indicate that the heterogeneous OSA has superior ability to enhance fluorescence emission and reduce the background noise over the conventional ZWM design. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. Any recitation herein of the term "comprising", particularly in a description of components of a composition or in a description of elements of a device, can be exchanged with "consisting essentially of" or "consisting of".

While the present invention has been described in conjunction with certain preferred embodiments, one of ordinary skill, after reading the foregoing specification, will be able to effect various changes, substitutions of equivalents, and other alterations to the

compositions and methods set forth herein.

This application claims the priority of U.S. Provisional Application No. 62/008,571 , filed 06 June 2014 and entitled "Single-molecule Detection and Radiation Control in Solutions at High Concentrations via a Heterogeneous Optical Slot Antenna", which is hereby incorporated by reference.

All patent and non-patent references cited herein are hereby incorporated by reference.

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Claims

CLAIMS What is claimed is:

1. An optical slot antenna device comprising:

an optically transparent substrate;

a second layer deposited onto the first layer, the second layer comprising a nonplasmonic material;

wherein the first and second layers each comprise an essentially rectangular void, wherein the voids in the first and second layers are aligned so as to create a nanoslot having nanoscale width, length, and height and having said substrate as floor, and wherein light entering the nanoslot through the substrate is substantially confined to a lower portion of the nanoslot.

2. The optical slot antenna device of claim 1 , wherein the substrate comprises an optically transparent material having a refractive index in the range from about 1.5 to about 2.0.

3. The optical slot antenna device of claim 2, wherein the optically transparent material is selected from the group consisting of glass, quartz, silicon dioxide, silicon nitride, and optically transparent polymer materials.

4. The optical slot antenna device of claim 3, wherein the substrate is glass.

5. The optical slot antenna device of claim 1 , wherein the first layer comprises Au, Ag, or Al.

6. The optical slot antenna device of claim 1 , wherein the first layer consists essentially of Au, Ag, or Al.

7. The optical slot antenna device of claim 1 , wherein the first layer consists essentially of Au.

8. The optical slot antenna device of claim 1 , wherein the thickness of the first layer is in the range from about 50 nm to about 300 nm.

9. The optical slot antenna device of claim 1 , wherein the second layer comprises Al, Cr, or Ti, with the proviso that if the first layer comprises Al, the second layer does not comprise Al.

10. The optical slot antenna device of claim 1 , wherein the second layer consists essentially of Al, Cr, or Ti, with the proviso that if the first layer comprises Al, the second layer consists essentially of Cr or Ti.

1 1 . The optical slot antenna device of claim 1 , wherein the thickness of the second layer is about 20 nm or greater.

12. The optical slot antenna device of claim 1 , wherein the width of the nanoslot is in the range from about 10 nm to about 50 nm.

13. The optical slot antenna device of claim 1 , wherein the length of the nanoslot is in the range from about 50 nm to about 200 nm.

14. The optical slot antenna device of claim 1 , wherein the height of the nanoslot is in the range from about 70 nm to about 500 nm.

15. The optical slot antenna device of claim 1 , wherein the first layer consists essentially of Au, the second layer consists essentially of Al, the thickness of the first layer is about 100 nm, the thickness of the second layer is about 50 nm, the width of the nanoslot is about 40 nm, the length of the nanoslot is about 1 10 nm, and the height of the nanoslot is about 150 nm.

16. The optical slot antenna device of claim 1 , further comprising one or more additional nanoslots formed by one or more additional overlapping voids in the first and second layers.

17. The optical slot antenna device of claim 16, wherein said nanoslots form a two- dimensional array of nanoslots.

18. The optical slot antenna device of claim 1 , further comprising a light source for illuminating the nanoslot through the substrate.

19. The optical slot antenna device of claim 1 , further comprising a detector for light emitted from the nanoslot.

20. The optical slot antenna device of claim 1 , which is capable of detecting a single analyte molecule present in a solution disposed in the nanoslot by means of fluorescence, surface plasmon resonance, Raman spectroscopy, or a nonlinear optical property.

21 . The optical slot antenna device of claim 1 , wherein fluorescence of an analyte molecule disposed in the lower portion of the optical slot is enhanced about 70-fold compared to fluorescence of said analyte molecule in free space.

22. The optical slot antenna device of claim 1 , capable of detecting an analyte molecule present in a solution disposed in the nanoslot at a concentration in the range from about 10^"12 molar to 10^"6 molar.

23. The optical slot antenna device of claim 1 , which does not act as a zero mode waveguide.

24. The optical slot antenna device of claim 1 , configured for use with a fluorescence microscope.

25. The optical slot antenna device of claim 1 which is capable of confining light ranging from about 300 nm to about 2000 nm in wavelength in said lower portion of said nanoslot.

26. The optical slot antenna device of claim 1 , further comprising a biomolecule immobilized in said lower portion of the nanoslot.

27. The optical slot antenna device of claim 26, wherein the biomolecule is a DNA polymerase or a fluorescent biomolecule.

28. A method of detecting a single analyte molecule in a sample, the method comprising the steps of:

(a) providing the optical slot antenna device of claim 1 , the device comprising a sample suspected of containing said analyte disposed in and above said nanoslot;

(b) irradiating the nanoslot through the substrate of the device with light; and (c) detecting an optical signal from a single molecule of said analyte in the nanoslot from below.

29. The method of claim 28, wherein the optical signal is single molecule fluorescence, a Raman signal, or a nonlinear optical signal such as second or higher order harmonic generation.

30. The method of claim 28, wherein the analyte is fluorescent or is a material or particle with a nonlinear optical property.

31 . The method of claim 28, wherein the sample is an aqueous solution containing said analyte.

32. The method of claim 31 , wherein the aqueous solution further comprises an enzyme.

33. The method of claim 32, wherein the enzyme is a DNA polymerase, and the method is used for nucleic acid sequencing.

(a) depositing a first layer comprising a plasmonic material on an optically

transparent substrate;

35. The method of claim 34, wherein the first layer comprises Au, Ag or Al.

36. The method of claim 34, wherein the first layer consists essentially of Au, Ag, or Al.

37. The method of claim 36, wherein the first layer consists essentially of Au.

38. The method of claim 34, wherein the second layer comprises Al, Cr, or Ti, with the proviso that if the first layer comprises Al, the second layer comprises Cr or Ti.

39. The method of claim 34, wherein the second layer consists essentially of Al, Cr, or Ti.

40. The method of claim 39, wherein the second layer consists essentially of Al.

41 . The method of claim 34, wherein the width of the nanoslot is in the range from about 10 nm to about 50 nm, the length of the nanoslot is in the range from about 50 nm to about 200 nm, and the height of the nanoslot is in the range from about 70 nm to about 500 nm, preferably in the range from about 100 to 150 nm.

42. The method of claim 34, wherein the total thickness of the first and second layers is in the range from about 100 nm to about 150 nm.

43. The method of claim 34, wherein a plurality of said nanoslots are formed, and said nanoslots form a two-dimensional array of nanoslots.

44. The method of claim 34, further comprising immobilizing a biomolecule in a lower portion of the nanoslot or nanoslots.

45. A kit comprising the nanoslot antenna device of claim 1 and one or more reagents or biomolecules, or comprising instructions for detecting a single analyte molecule using the device.

46. A microfluidic device comprising the nanoslot antenna device of claim 1 , wherein said microfluidic device comprises one or more channels for delivery of a fluid sample or other liquids to the nanoslot of the nanoslot antenna device.