EP4630583A1 - Light-enhancing plasmonic nanowell-nanopore biosensor and use thereof - Google Patents

Abstract

Systems for detecting fluorescence from a molecule comprising an ion-impermeable film comprising at least one ion-conducting nanopore and a laser coupling slot; a first and second liquid reservoir separated by the film; a means to induce movement of the molecule from the first reservoir to the second reservoir via the nanopore; a light source capable of exciting the molecule to emit fluorescence, wherein the light source shines into the second reservoir; a metallic layer adhered to the film by an adhesion layer and comprising a nanowell structure located adjacent to the nanopore; and a detector configured to detect the fluorescence emitted by the molecule are provided. Methods of use of the systems are also provided.

Description

LIGHT-ENHANCING PLASMONIC NANOWELL-NANOPORE

BIOSENSOR AND USE THEREOF

CROSS-REFERENCE

[001] This application claims the benefit of priority to U.S Provisional Patent Application No. 63/430,732 filed December 7, 2022 entitled “LIGHT-ENHANCING PLASMONIC NANOWELL-NANOPORE BIOSENSOR AND USE THEREOF”, the contents of which are incorporated herein by reference in their entirety.

FIELD OF INVENTION

[002] The present invention is in the field of nanopore biosensors.

BACKGROUND OF THE INVENTION

[003] The development of nanopore-based biosensors has received considerable attention in the past two decades due to their compatibility with a broad range of analytes, including nucleic acids, proteins, and various small molecules. Particularly, nanopore-based DNA sequencing has recently emerged as a viable alternative to sequencing-by-synthesis approaches, offering a highly portable and affordable solution with high throughput and precision. Currently the most advanced nanopore based sequencing methods are based on protein pores, such as the CsgG or MspA channels, which require a ratcheting enzyme to regulate the transport of a DNA strand. Nevertheless, the development of synthetic nanopores remains a major focus in nanotechnology due to the inherent limitations of the protein pores and the greater flexibility that synthetic nanopores offer in term of the ability to tailor their size, shape and surface properties towards specific sensing applications.

[004] Solid-state nanopores (ssNPs) fabricated in thin inorganic membranes can be crafted with sub-nanometer precision to match the size of the target analyte and are therefore considered to be highly attractive platforms. Moreover, ssNPs are compatible with a variety of single-molecule detection methods (in addition to the ion-current resistive-pulse technique) making them ideally suited for the development of future integrated biological sensors. In particular, because ssNPs are fabricated in essentially 2D, solid membranes, they lend themselves to relatively straightforward implementation of optical sensing, which can provide independent and completely orthogonal information on the analytes. As a result, in the past few years electro-optical sensing in ssNPs has gained growing momentum towards applications such as rapid DNA sequencing, DNA barcoding and epi-genetic modification sensing. Notably, ssNPs can be articulated with plasmonic nanostructures to enhance key features of the nanopore sensing. For example, plasmonic structures have been used to produce local heating in the pore vicinity, hence controlling the translocation speed and capture rate of DNA molecules. Moreover, bow-tie structures fabricated around the nanopore were proposed for rapid DNA sequencing utilizing surface-enhanced Raman scattering from nucleotides passing through the pore.

[005] Despite these major advancements in optical sensing in ssNPs, the detection of individual fluorophores has proven to be challenging due to two competing factors: first, when excited by the laser source, solid dielectric membranes (such as SiNx, SiO2, etc.) emit light through photo-luminescence in wavelengths that overlap with the fluorescence emission. This background noise comes on top of the fluorescence background from molecules residing in the detection volume. Second, the dwell time of the fluorophore in the nanopore is relatively short, hence limiting the photon integration time and diminishing the overall signal. A possible solution for these issues involved the incorporation of molecular quenchers for each fluorophore, but this come at the expense of more complex sample preparation. Nanopore biosensors that are quencher-free, produce a stronger signal and with lower background are much in need.

SUMMARY OF THE INVENTION

[006] The present invention provides methods and systems for detecting fluorescence from a molecule.

[007] According to a first aspect, there is provided a system for detecting fluorescence from a molecule, the system comprising: a. an ion-impermeable film comprising at least one ion-conducting nanopore and a laser coupling slot; b. a first and a second liquid reservoir separated by the film; c. means to induce movement of the molecule from the first reservoir to the second reservoir via the nanopore; d. a light source capable of exciting the molecule to emit fluorescence, wherein the light source emits a laser beam coupled to the laser coupling slot, thus emitting light directly to the nanopore; e. a metallic layer adhered to the film by an adhesion layer, the metallic layer comprising a nanowell structure located adjacent to the nanopore and to and the laser coupling slot; and f. a first detector configured to detect the fluorescence emitted by the molecule.

[008] According to another aspect, there is provided a plasmonic biosensor comprising: a. an ion impermeable film comprising at least one ion-conducting nanopore and a laser coupling slot; and b. a metallic layer adhered to said film by an adhesion layer, said metallic layer comprising a nanowell structure located adjacent to said nanopore and to said laser coupling slot.

[009] According to some embodiments, the system comprises a plasmonic nanowell- nanopore biosensor of the invention.

[010] According to some embodiments, the metallic layer and the adhesion layer comprise a thickness sufficient to block at least 95% of light emitted by the light source. According to some embodiments, the metallic layer and the adhesion layer comprise a thickness sufficient to block at least 50% of light emitted by the light source.

[Oi l] According to some embodiments, the diameter of the nanowell is not greater than half the wavelength of the light emitted by the light source.

[012] According to some embodiments, the molecule comprises at least one fluorescent moiety. According to some embodiments, the fluorescent moiety is a fluorescent tag. According to some embodiments, the fluorescent moiety is Cy5 or CF640R.

[013] According to some embodiments, the molecule is a nucleic acid molecule or a polypeptide. According to some embodiments, the nucleic acid molecule is any one of single- stranded DNA, double-stranded DNA, RNA, and cDNA. According to some embodiments, the nucleic acid molecule is double-stranded DNA. [014] According to some embodiments, the film is a silicon-based membrane. According to some embodiments, the membrane is a silicon nitride (SiNx) membrane. According to some embodiments, the film has a thickness of less than 50 nanometers (nm).

[015] According to some embodiments , the nanopore comprises a diameter not greater than 5 nm. According to some embodiments, the film comprises at least 2 nanopores and the nanopores are separated by at least 1 micrometers (pm).

[016] According to some embodiments, said laser coupling slot has a width of 10 pm to 200 pm and a height of 50 nm to 500 nm.

[017] According to some embodiments, the means to induce movement comprises a negative electrode within the first reservoir, and a positive electrode within the second reservoir and the molecule has a negative charge.

[018] According to some embodiments, the light source produces orange or red light. According to some embodiments, the red light source is a laser having a wavelength in the range 590 to 720 nm. According to some embodiments, the power of the light source is at most 10 microwatts (pW).

[019] According to some embodiments, the metallic layer comprises a metal selected from gold, silver, copper, aluminum and a combination thereof. According to some embodiments, the metal is gold. According to some embodiments, the metallic layer comprises a thickness of between 100 and 150 nm. According to some embodiments, the metallic layer is on the second reservoir- side of the membrane.

[020] According to some embodiments, the adhesion layer comprises a metal oxide According to some embodiments, the adhesion layer comprises chromium, chromium oxide, titanium or titanium oxide. According to some embodiments, the adhesion layer comprises a thickness of between 1 and 20 nm.

[021] According to some embodiments, the nanopore is at the center of the nanowell. According to some embodiments, the nanowell comprises a diameter between 30 and 150 nm.

[022] According to some embodiments, the detecting comprises sub-millisecond (ms) resolution. According to some embodiments, the detecting comprises a high signal to noise ratio. [023] According to some embodiments, the first detector is an active pixel sensor (APS). According to some embodiments, the APS is a complementary metal-oxide semiconductor (CMOS) sensor. According to some embodiments, the first detector is a charge coupled device (CCD) detector. According to some embodiments, the first detector is an Avalanche Photo Diode detector.

[024] According to some embodiments, a system of the invention further comprise a second detector configured to detect ion current flow through the nanopore. According to some embodiments, the second detector is configured to convert the ion current through the nanopore to a measurable electrical current. According to some embodiments, the second detector is a high-gain current amplifier. According to some embodiments, the means to induce movement comprises a first electrode within the first reservoir, and a second electrode within the second reservoir, and the high current amplifier is connected to the first and second electrodes. According to some embodiments, the first and the second detector are synchronized.

[025] According to some embodiments, a system of the invention is for use in sequencing the molecule.

[026] According to another aspect, there is provided a method of detecting fluorescence from a single molecule, the method comprising: a. introducing the molecule into the first reservoir of a system of the invention; b. inducing the molecule to move from the first reservoir to the second reservoir via the nanopore; c. exciting the molecule within the nanopore to emit fluorescence; and d. detecting the fluorescence emitted by the molecule; thereby detecting fluorescence from a single molecule.

[027] According to some embodiments, the detecting comprises sub-millisecond (ms) resolution. According to some embodiments, the detecting comprises a high signal to noise ratio. [028] According to some embodiments, the system further detects ion current flow through the nanopore and wherein only an event detected simultaneously by fluorescence and electricity is considered detecting fluorescence from the molecule.

[029] According to another aspect, there is provided a method of sequencing a molecule, comprising a method of the invention and further comprising assigning an identity to each detected fluorescence.

[030] According to some embodiments, the identity is a nucleic acid base. According to some embodiments, the identity is an amino acid.

[031] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[032] Figures 1A, IB and 1C are illustrations of a plasmonic biosensor according to some embodiments of the invention.

[033] Figure ID is an illustration of an Au donut shape pattern over silicon nitride membrane can be seen as square behind the gold patch, accoridng to some embodiments of the invnetion.

[034] Figure IE is an illustration of laser entrace angle to the plasmonic biosensor and a Comsol simulations shows effective coupling of the laser with the plasmonic biosensor according to some embodiments of the invention.

[035] Figures IF and 1G are illusrations of a PDMS device with a plasmonic biosensor according to some embodiments of the invention.

[036] Figure 1H is an illustration of system for detecting fluorescence from a molecule using the plasmonic nanowell-nanopore biosensor according to some embodiments of the invention. [037] Figure II is an illustration of the illumination of a single protein chine in the nanopore of the plasmonic biosensor according to some embodiments of the invention.

[038] Figures 2A-2B: Schematic outlines of the full wafer-scale fabrication of PNW-NP chips using negative tone process (2A) and nanopore chips in ultra-thin freestanding SiNx membranes (STD) (2B).

DETAILED DESCRIPTION OF THE INVENTION

[039] The present invention, in some embodiments, provides systems for detecting fluorescence from a molecule, and methods of using those systems, comprising an ion- impermeable film comprising at least one ion-conducting nanopore; a first and second liquid reservoir separated by the film; a means to induce movement of the molecule from the first reservoir to the second reservoir via the nanopore; a light source capable of exciting the molecule to emit fluorescence, wherein the light source shines into the second reservoir; a metallic layer adhered to the film by an adhesion layer and facing the second reservoir, comprising a nanowell structure located adjacent to the nanopore; and a detector configured to detect the fluorescence emitted by the molecule.

Systems

[040] By a first aspect, there is provided a system for detecting fluorescence from a molecule, the system comprising: a. an ion-impermeable film comprising at least one ion-conducting nanopore; b. a first and a second liquid reservoir separated by the film; c. a means to induce movement of the molecule from the first reservoir to the second reservoir via the nanopore; d. a light source capable of exciting the molecule to emit fluorescence, wherein the light source shines into the second reservoir; e. a metallic layer adhered to the film by an adhesion layer, and comprising a nanowell structure located adjacent to the nanopore; and f. a first detector configured to detect said fluorescence emitted by the molecule.

[041] By another aspect, there is provide a plasmonic nanowell-nanopore biosensor: a. an ion impermeable film comprising at least one ion-conducting nanopore and a laser coupling slot; and b. a metallic layer adhered to the film by an adhesion layer, the metallic layer comprising a nanowell structure located adjacent to the at least one nanopore and the laser coupling slot.

[042] By another aspect, there is provided a system for detecting fluorescence from a molecule, the system comprising: a. a plasmonic nanowell-nanopore biosensor of the invention; b. a first and a second liquid reservoir separated by the film; c. a means to induce movement of the molecule from the first reservoir to the second reservoir via the nanopore; d. a light source capable of exciting the molecule to emit fluorescence, wherein the light source coupled into the laser coupling slot and capable of illuminating the molecule only when the molecule is passing via the nanopore; and e. a first detector configured to detect said fluorescence emitted by the molecule.

[043] In some embodiments, illuminating only molecules passing in the nanopore (e.g., a single molecule at the time, as illustrated in Figure IF) may allow the detection the fluorescence from a single molecule at the time. Such arrangement may reduce falls detection of fluorescence from other molecules (e.g., undesired molecules that may enter the second reservoir).

[044] Reference is now made to Figures 1A, IB and 1C which are illustrations of a plasmonic nanowell-nanopore biosensor accoridng to some embodiments of the invention. In some embodiments a biosensor 100 is for use in sequencing the molecule. As used herein, “sequencing” refers to determining the sequence of components that make up the molecule. Nucleotide sequencing is well known in the art and consists of determining the order of bases of nucleic acids in a molecule. When the molecule is a DNA molecule the bases will be adenine, cytosine, guanine and thymine. When the molecule is RNA the fourth base will be uracil and not thymine. In some embodiments, artificial bases can be sequenced. When the molecule is a polypeptide the sequence of amino acids is determined. In some embodiments, the sequencing is of naturally occurring amino acids. In some embodiments, the sequencing comprises artificial amino acids.

[045] In some embodiments, the system detects fluorescence with no background from the first liquid and/or the second liquid reservoir. In some embodiments, the detector is on the second reservoir side of the film. In some embodiments, the laser is coupled to the at least one laser coupling slot such that only the nanopore is illuminated. In some embodiments, the metallic layer comprises a thickness sufficient to block light emitted by the light source. In some embodiments, the adherence layer comprises a thickness sufficient to block light emitted by the light source. In some embodiments, the adherence layer comprises a thickness sufficient to block light shown upon it. In some embodiments, the metallic layer and adhesion layer each comprise a thickness sufficient to block light emitted by the light source. In some embodiments, the metallic layer and adhesion layer each comprise a thickness sufficient to block light shown upon it. In some embodiments, the metallic layer and adhesion layer together comprises a thickness sufficient to block light emitted by the light source. In some embodiments, the metallic layer and adhesion layer together comprises a thickness sufficient to block light shown upon it. In some embodiments, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% of light is blocked. Each possibility represents a separate embodiment of the invention. In some embodiments, at least 95% of light is blocked. In some embodiments, at least 50% of light is blocked.

[046] It will also be understood that as the light source is on the second reservoir side and the nanowell is also on that side the nanopore itself can also act as a barrier for background fluorescence from other molecules. A skilled artisan will understand that detection of the molecule occurs while it is in the nanowell, because the nanopore behind it is narrower than the well and because the molecule itself will be blocking the nanopore, light will not be able, or will be only lowly able, to reach the first reservoir and molecules therein.

[047] In some embodiments, the molecule is an organic molecule. In some embodiments, the molecule is a protein or a nucleic acid molecule. In some embodiments, the molecule is a linearized molecule. In some embodiments, the molecule is a protein. In some embodiments, the molecule is a polypeptide. In some embodiments, the molecule is a nucleic acid molecule. In some embodiments, the nucleic acid molecule is any one of single- stranded DNA, double-stranded DNA, RNA, and cDNA. In some embodiments, the nucleic acid molecule is double-stranded DNA. In some embodiments, the molecule comprises at least one tag. In some embodiments, the molecule comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20 or 30 tags. Each possibility represents a separate embodiment of the invention. In some embodiments, the tags are covalently coupled to specific biochemical groups along the molecule. In some embodiments, the tags are coupled to specific nucleobases along the molecule. In some embodiments, the tags are coupled to specific amino-acids along the molecule. In some embodiment the tags are evenly spaced along the length of the molecule. In some embodiments, there is a tag at least every 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 bases of a nucleic acid molecule. Each possibility represents a separate embodiment of the invention. In some embodiments, there is a tag at least every 4 bases of a nucleic acid molecule. In some embodiments, there is a tag at every nucleic acid of a molecule. In some embodiments, there is a tag at least every 1, 3, 5, 7, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids of a polypeptide. Each possibility represents a separate embodiment of the invention. In some embodiments, there is a tag at least every 3 amino acids of a polypeptide. In some embodiments, there is a tag at every amino acid of a polypeptide. In some embodiments, the tag is a fluorescent tag. In some embodiments, the molecule comprises a fluorescent moiety. In some embodiments, the moiety is a tag. In some embodiments, the fluorescent moiety is CY5 or CF640R. In some embodiments, the fluorescent moiety is CF640R. In some embodiments, the fluorescent moiety is CY5. In some embodiments, the molecule fluoresces only when contacted by light from the light source.

[048] As used herein, the terms “peptide”, "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues. In another embodiment, the terms "peptide", "polypeptide" and "protein" as used herein encompass native peptides, peptidomimetics (typically including non-peptide bonds or other synthetic modifications) and the peptide analogues peptoids and semipeptoids or any combination thereof. In another embodiment, the peptides polypeptides and proteins described have modifications rendering them more stable while in the body or more capable of penetrating into cells. In one embodiment, the terms “peptide”, "polypeptide" and "protein" apply to naturally occurring amino acid polymers. In another embodiment, the terms “peptide”, "polypeptide" and "protein" apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid.

[049] The term “moiety” as used herein refers to a part of a molecule, which lacks one or more atom(s) compared to the corresponding molecule. The term “moiety”, as used herein, further relates to a part of a molecule that may include either whole functional groups or parts of functional groups as substructures. The term “moiety” further means part of a molecule that exhibits a particular set of chemical and/or pharmacologic characteristics which are similar to the corresponding molecule, i.e. fluorescence. In some embodiments, a fluorescent moiety is part or all of a fluorescent molecule that retains the ability to fluoresce.

[050] Biosensor 100 may include an ion impermeable film 110, comprising at least one ion-conducting nanopore 112 and a laser coupling slot 115. In some embodiments, ion impermeable film 110 may be a silicon-based membrane, for example, a silicon nitride (SiNx) membrane. In some embodiments, ion impermeable film 110 may have a thickness of less than 50 nanometers (nm). In some embodiments, ion impermeable film 110 may include an array of ion-conducting nanopores 112, each having a diameter not greater than 5 nm. In some embodiments, each two neighboring pores in the array may be separated by at least 1 micrometers (pm). As used herein, the terms “fdm” and “membrane” are used interchangeably and refer to a thin water-impermeable separation between the first and second reservoirs. In some embodiments, film 120 is ion-impermeable. In some embodiments, film 120 comprises silicon. In some embodiments, film 120 is silicon based. In some embodiments, the film comprises silicon nitride (SiNx). In some embodiments film 120 comprises a metal oxide. In some embodiments, the metal oxide is selected from aluminum oxide (AIO2), titanium oxide (TiOz). silicon oxide (SiOi) and halfnium oxide (HfCh). In some embodiments, film 120 is set in a silicon wafer. In some embodiments, the wafer is a crystal orientation wafer. In some embodiments, the wafer is thicker in regions that lack a nanopore. In some embodiments, the wafer provides stability to the separation between the first and second reservoirs. In some embodiments, the wafer comprises a diameter of at least 1, 10, 50, 75 or 100 mm. Each possibility represents a separate embodiment of the invention. In some embodiments, the wafer comprises a thickness of at least 50, 100, 150, 200, 250, 300, 350 or 400 pm. Each possibility represents a separate embodiment of the invention.

[051] In some embodiments, film 120 has a universal thickness. In some embodiments, the film has a constant thickens across its entire area. In some embodiments, the film has a variable thickness. In some embodiments, film 120 is thinner in the area of the nanopore. In some embodiments, film comprises 120 a thickness of less than 500, 450, 400, 350, 300, 250, 200, 150, 100, 75, 50, 25, 20, 15, 10, or 5 nm. Each possibility represents a separate embodiment of the invention. In some embodiments, film 120 comprises a thickness of less than 100 nm. In some embodiments, the film comprises a thickness of about 25 nm. In some embodiments, the film comprises a thickness of about 10 nm. In some embodiments, the film comprises a thickness of less than 10 nm. In some embodiments, the membrane comprises a thickness of about 25 nm distal to the nanopore and a thickness of about 10 nm proximal to the nanopore. In some embodiments, the membrane comprises a thickness of about 25 nm distal to nanopore 115 and a thickness of less than 10 nm proximal to nanopore 115. In some embodiments, a thin membrane proximal to the pore increases spatial recognition. In some embodiments, a thin membrane proximal to the pore decreases the optical background. In some embodiments, a thin membrane proximal to the pore increases a signal to noise ratio from the molecule. A person skilled in the art will appreciate that the thinner the pore, the fewer the bases in the pore at one instance and thus the greater the spatial recognition of each base of the nucleic acid molecule which also will contribute to decreased background. In some embodiments, the film comprises a thickness that allows light from the light source to pass through the film. In some embodiments, the film allows at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99% or 100% of light to pass through it. Each possibility represents a separate embodiment of the invention.

[052] The production of nanopores (e.g., nanopores 115) in a film is well known in the art. Fabrication of nanopores in thin membranes has been shown in, for example, Kim et al., Adv. Mater. 2006, 18 (23), 3149 and Wanunu, M. et al., Nature Nanotechnology 2010, 5 (11), 807-814. Further, methods of such fabrication of films in silicon wafers, and methods of producing nanopores therein are provided herein in the Materials and Methods section. In some embodiments, the nanopore is produced with a transition electron microscope (TEM). In some embodiments, the nanopore is produced with a high-resolution aberration-corrected TEM or a noncorrected TEM.

[053] In some embodiments, nanopore 115 comprises a diameter not greater than 1, 2, 3, 4, 5, 10, 15, 20, 15, 30, 35, 40, 45 or 50 nm. Each possibility represents a separate embodiment of the invention. In some embodiments, the nanopore comprises a diameter not greater than 5 nm. In some embodiments, the nanopore comprises a diameter of about 5 nm. In some embodiments, the nanopore comprises a diameter between 0.5 and 10, 0.5 and 15, 0.5 and 20, 1 and 10, 1 and 15, 1 and 20, 3 and 10, 3 and 15, 3 and 20, 5 and 10, 5 and 15, or 5 and 20 nm. Each possibility represents a separate embodiment of the invention.

[054] In some embodiments, the film comprises at least one nanopore. In some embodiments, the film comprises at least 2 nanopores 115. In some embodiments, the film comprises a plurality of nanopores 115. In some embodiments, the film comprises an array of nanopores. In some embodiments, the array comprises dimensions of 5 x 5, 5 x l0, 5 x 15, 5 x 20, 5 x 25, 5 x 30, 5 x 35, 5 x 40, 5 x 45, 5 x 50, 10 x 10, 10 x 15, 10 x 20, 10 x 25, 10 x 30, 10 x 35, 10 x 40, 10 x 45, 10 x 50, 15 x 15, 15 x 20, 15 x 25, 15 x 30, 15 x 35, 15 x 40, 15 x 45, 15 x 50, 20 x 20, 20 x 25, 20 x 30, 20 x 35, 20 x 40, 20 x 45, 20 x 50, 25 x 25, 25 x 30, 25 x 35, 25 x 40, 25 x 45, 25 x 50, 30 x 30, 30 x 35, 30 x 40, 30 x 45, 30 x 50, 35 x 35, 35 x 40, 35 x 45, 35 x 50, 40 x 40, 40 x 45, 40 x 50, 45 x 45, 45 x 50, or 50 x 50 pm. Each possibility represents a separate embodiment of the invention. In some embodiments, the array comprises dimensions of 30 pm by 30 pm. In some embodiments, the nanopores are separated by about 1 pm. In some embodiments, the nanopores are separate by at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9 or 10 pm. Each possibility represents a separate embodiment of the invention. In some embodiments, the nanopores are separated by at least 1 pm. In some embodiments, every nanopore will have a corresponding nanowell. In some embodiments, the detector is configured to detect fluorescence at each nanopore -nanowell. In some embodiments, the detector is configured to detect fluorescence at all nanopore- nanowells. In some embodiments, multiple detectors detect fluorescence at multiple wells.

[055] In some embodiments, laser coupling slot 115 has a width of 10 pm to 200 pm and a height of 50 nm to 500 nm. In some embodiments, slot 115 has a rectangle shape having a contour of few millimeters as shown in the illustration of Fig. ID. Laser coupling slot 115 may be fabricated by known lithography etching methods.

[056] In some embodiments, laser coupling slot 115 couples a laser beam 10 to film 110, thus causing film 110 to act as a waveguide. In some embodiments, laser beam 110 may propagate between the upper and lower faces of film 110 to be emitted from the walls of nanopore 112. In the nonlimiting example illustrated in Figs 1A-1C, SiNx film 110 has a higher refractive index (2.2) than neighboring layers SiCh (1.4) and adhesive 125 layer (1.33). Therefore, it will serve as an optical waveguide and directly illuminate nanopore in the center of film 110.

[057] Biosensor 100 may further include a metallic layer 120 adhered to film 110 by an adhesion layer 125. In some embodiments, metallic layer 120 serves serve as a mirror for laser beam 10 propagating inside film 120. In some embodiments, metallic layer 120 comprising a nanowell 128 structure located adjacent to at least one nanopore 112 and to laser coupling slot 115. In some embodiments, the metal for metallic film 120 is selected from gold, silver, copper, aluminum, and any combination thereof. In some embodiments, metallic layer 120 comprises a thickness of between 100 and 150 nm. In some embodiments, adhesion layer 125 comprises, a metal or a metal oxide, selected from, chromium, chromium oxide, titanium or titanium oxide, and the like. In some embodiments, the thickness of adhesion layer 125 is 1 to 20 nm. In some embodiments, nanowell 128 comprises a diameter between 30 and 150 nm. In some embodiments, metallic layer 120 and adhesion layer 125 comprise a thickness sufficient to block at least 50% of light shown thereupon. Reference is now made to Fig. IF which is an illustration of system for detecting fluorescence from a molecule using the plasmonic nanowell-nanopore biosensor accoridng to some embodiments of the invention.

[058] As used herein, the term “layer” refers to a thin flat continuous piece of material. In some embodiments, metallic layer 120 comprises a metallic layer having plasmonic properties. In some embodiments, the metallic layer comprises at least one layer of metal. In some embodiments, the metallic layer comprises more than one layer of metal. In some embodiments, the more than one layer of metal is layered one on top of the other to create one combined metallic layer. In some embodiments, the metallic layer comprises at least one of gold, silver, copper, aluminum and their alloys. In some embodiments, the metallic layer comprises gold. In some embodiments, the metallic layer comprises aluminum. In some embodiments, the metallic layer is a gold layer. In some embodiments, the metallic layer is an aluminum layer. In some embodiment, the metallic layer is composed of multiple layers of metals and/or dielectric materials stacked vertically with respect to the membrane. In some embodiments, the metallic layer is made of gold, silver, copper, aluminum or a combination thereof. In some embodiments, the metallic layer is a homogenous layer. In some embodiments, the metallic layer is a heterogenous layer. In some embodiments, the metallic is a combined layer of gold and aluminum. In some embodiments, a combined layer of gold and aluminum comprises a layer of gold and a layer of aluminum. In some embodiments, the layers of gold and aluminum are stacked vertically with respect to the membrane. In some embodiments, the gold layer is proximal to the membrane. In some embodiments, the aluminum layer is proximal to the membrane. In some embodiments, a combined layer of gold and aluminum is a layer of gold and a layer of aluminum. In some embodiments, the metallic layer is gold. In some embodiments, the metallic layer is aluminum. In some embodiments, the metallic layer is at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or 100% gold. Each possibility represents a separate embodiment of the invention. In some embodiments, the metallic layer is 100% gold. In some embodiments, the metallic layer is at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or 100% aluminum. Each possibility represents a separate embodiment of the invention. In some embodiments, the metallic layer is 100% aluminum. In some embodiments, the metallic layer is at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or 100% gold, aluminum or a combination thereof. Each possibility represents a separate embodiment of the invention. In some embodiments, the metallic layer is 100% gold, aluminum or a combination thereof.

[059] In some embodiments, a combined layer of gold and aluminum is used for an assay with high concentrations of analyte and/or molecules. In some embodiments, a combined layer of gold and aluminum is used for an assay with physiological concentrations of analyte and/or molecules. In some embodiments, a physiological concentration and/or a high concentration is 0.1-100, 0.1-500, 0.1-1000, 0.1-1500, 0.1-2000, 0.1-2500, 0.1-3000, 0.1- 3500, 0.1-4000, 0.1-4500, 0.1-5000, 0.1-10000, 0.1-50000, 0.1-75000, 0.1-100000, 0.1- 500000, 0.5-100, 0.5-500, 0.5-1000, 0.5-1500, 0.5-2000, 0.5-2500, 0.5-3000, 0.5-3500, 0.5- 4000, 0.5-4500, 0.5-5000, 0.5-10000, 0.5-50000, 0.5-75000, 0.5-100000, 0.5-500000, 1- 100, 1-500, 1-1000, 1-1500, 1-2000, 1-2500, 1-3000, 1-3500, 1-4000, 1-4500, 1-5000, 1- 10000, 1-50000, 1-75000, 1-100000, 1-500000, 5-100, 5-500, 5-1000, 5-1500, 5-2000, 5- 2500, 5-3000, 5-3500, 5-4000, 5-4500, 5-5000, 5-10000, 5-50000, 5-75000, 5-100000, 5- 500000, 10-100, 10-500, 10-1000, 10-1500, 10-2000, 10-2500, 10-3000, 10-3500, 10-4000, 10-4500, 10-5000, 10-10000, 10-50000, 10-75000, 10-100000, 10-500000, 50-100, 50-500, 50-1000, 50-1500, 50-2000, 50-2500, 50-3000, 50-3500, 50-4000, 50-4500, 50-5000, 50- 10000, 50-50000, 50-75000, 50-100000, 50-500000, 100-500, 100-1000, 100-1500, 100- 2000, 100-2500, 100-3000, 100-3500, 100-4000, 100-4500, 100-5000, 100-10000, 100- 50000, 100-75000, 100-100000, 100-500000, 500-100, 500-1000, 500-1500, 500-2000, 500-2500, 500-3000, 500-3500, 500-4000, 500-4500, 500-5000, 500-10000, 500-50000, 500-75000, 500-100000, 500-500000, 1000-1500, 1000-2000, 1000-2500, 1000-3000, 1000-3500, 1000-4000, 1000-4500, 1000-5000, 1000-10000, 1000-50000, 1000-75000, 1000-100000, 1000-500000, 2000-2500, 2000-3000, 2000-3500, 2000-4000, 2000-4500, 2000-5000, 2000-10000, 2000-50000, 2000-75000, 2000-100000, 2000-500000, 5000- 10000, 5000-50000, 5000-75000, 5000-100000, 5000-500000, 10000-50000, 10000-75000, 1000-100000, or 1000-500000 nm. Each possibility represents a separate embodiment of the invention. In some embodiments, a combined layer of gold and aluminum generates an increased near-field intensity within the nanowell. In some embodiments, a combined layer of gold and aluminum generates an increased fluorescent emission from the molecule. In some embodiments, the increase is as compared to a metallic layer comprising only one of gold and aluminum. In some embodiments, the increase is at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 times greater than an intensity or fluorescent emission. Each possibility represents a separate embodiment of the invention. In some embodiments, a combined layer of gold and aluminum localizes the nearfield intensity within the nanowell. In some embodiments, the localization is as compared to a metallic layer comprising only one of gold and aluminum. In some embodiments, the localization of a combined layer is at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 170 or 200 times greater than for a single-layer. Each possibility represents a separate embodiment of the invention. In some embodiments, a layer is not composed of freely moving or fixed metallic particles. In some embodiments, the layer is not plasmonic particle within a non-plasmonic substrate. In some embodiments, metallic layer 120 does not contain non-metallic gaps or voids, such as those created between particles. In some embodiments, the metallic layer is a uniformly flat surface. In some embodiments, the metallic layer is a uniform surface. In some embodiments, the metallic layer comprises a thickness of between 50 and 500, 50 and 450, 50 and 400, 50 and 350, 50 and 300, 50 and 250, 50 and 200, 50 and 150, 100 and 500, 100 and 450, 100 and 400, 100 and 350, 100 and 300, 100 and 250, 100 and 200, or 100 and 150 nm. Each possibility represents a separate embodiment of the invention. In some embodiments, the metallic layer comprises a thickness of between 100 and 150 nm. In some embodiments, the metallic layer comprises a thickness of about 130 nm. In some embodiments, the metallic layer comprises a thickness of at least 30, 40, 50, 60, 70, 80, 90 or 100 nm. Each possibility represents a separate embodiment of the invention. In some embodiments, the metallic layer comprises a thickness of at most 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 300, 400 or 500 nm. Each possibility represents a separate embodiment of the invention.

[060] As used herein, an “adhesion layer” is a layer of any material that when deposited on a thin substrate (the membrane of the invention) it allows stable bonding of the substrate to the metallic layer. Adhesion layers are known in the art, and examples of such may be found in Aouani et. al., ACS Nano, 2009, 3 (7):2043-2048 for non-limiting example. In some embodiments, the adhesion layer comprises a metal or metal-oxide dielectric metal. In some embodiments, the adhesion layer comprises a transition metal. In some embodiments, the adhesion layer comprises a metal oxide. In some embodiments, the adhesion layer comprises titanium, chromium, or nickel. In some embodiments, the adhesion layer comprises chromium. In some embodiments, the transition layer comprises titanium. In some embodiments, the adhesion layer comprises chromium or titanium. In some embodiments, the adhesion layer comprises any one of chromium, chromium oxide, titanium, and titanium oxide. In some embodiments, the adhesion layer is configured to ensures the adhesion of the metallic layer to the film. In some embodiments, the adhesion layer comprises a thickness of at most 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nm. Each possibility represents a separate embodiment of the invention. In some embodiments, the adhesion layer comprises a thickness of at most 10 nm. In some embodiments, the adhesion layer comprises a thickness of about 5 nm. In some embodiments, the adhesion layer comprises a thickness of at least 1, 2, 3, 4, or 5 nm. Each possibility represents a separate embodiment of the invention.

[061] It will be understood by one skilled in the art that the adhesion layer is layered directly only the membrane and that the metallic layer is layer directly onto the adhesion layer. The method of performing such is described herein below. In some embodiments, these layers are on the second reservoir side, facing into the second reservoir, which is the side from which the light comes. Regardless of to which side the layers face, the two layers are configured to block light from the light source from reaching the first reservoir.

[062] As used herein, the term “nanowell” refers to a passage through the metallic layer. A nanowell 128 may also be referred to as a nanoslot or nanoantenna. In some embodiments, the nanowell (e.g., nanowell 128) is circular. In some embodiments, the nanowell is rectangular. In some embodiments, nanowell has a geometric configuration. Geometric configurations include squares, rectangles, circles, ovals, triangles, bowties, rods, cylinders, ellipses, disks, rhombuses and any other shape that may be found by one skilled in the art to confer the plasmonic enhancement to fluorescent imaging of the molecule. In some embodiments, the nanowell also is through the adhesion layer. In some embodiments, the nanowell has a constant diameter. In some embodiments, the nanowell is narrower closer to the nanopore and wider at the surface of the metallic layer. In some embodiments, the nanopore is at the center of the nanowell. In some embodiments, the diameter of the nanowell is not greater than the wavelength of the light emitted by the light source. In some embodiments, the diameter of the nanowell is not greater than half the wavelength of the light emitted by the light source. In some embodiments, the diameter of the nanowell is not greater than half, a third, or a quarter of the wavelength of the light emitted by the light source. Each possibility represents a separate embodiment of the invention. A person skilled in the art will appreciate that by having a diameter that is less than the wavelength the nanowell amplifies the fluorescence from the molecule. This is achieved owing to the close proximity of the edges of the nanowell that enhance plasmon resonance between them, and the attenuation of light within the waveguide aperture which results in more localized excitation. In some embodiments, the nanowell comprises a diameter between 10 and 100, 10 and 150, 10 and 200, 20 and 100, 20 and 150, 20 and 200, 30 and 100, 30 and 150, 30 and 200, 40 and 100, 40 and 150, 40 and 200, 50 and 100, 50 and 150 or 50 and 200 nm. Each possibility represents a separate embodiment of the invention. In some embodiments, the nanowell comprises a diameter of between 30 and 150 nm. In some embodiments, the nanowell comprises a diameter of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nm. Each possibility represents a separate embodiment of the invention. In some embodiments, the nanowell comprises a diameter of at most 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nm. Each possibility represents a separate embodiment of the invention.

[063] In some embodiments, the nanowell is configured to excite a specific plasmonic resonance. In some embodiments, the nanowell is configured to excite at least one plasmonic resonance. In some embodiments, the nanowell is configured to excite a plurality of plasmonic resonance. It will be understood by a skilled artisan that exciting a specific plasmonic resonance will enhance fluorescence from the molecule at a particular wavelength. Thus, by configuring the nanowell to excite at a given plasmonic resonance the nanowell with enhance fluorescence at a desired wavelength. In some embodiments, the configured nanowell enhances fluorescence at or about the wavelength of the fluorescent moiety of the molecule. In some embodiments, the enhancement is single spectral. In some embodiments, the enhancement is multispectral. In some embodiments, the nanowell comprises a reflective layer. In some embodiments, the configuring is modifying the reflective layer. In some embodiments, the configuring is modifying the adhesion layer.

[064] Different fluorochromes have distinct excitation ranges and emission ranges and the nanowell can be configured to enhance specific fluorochromes. Some non-limiting examples of fluorochromes and their maximum excitation and emission wavelengths (nm) include: 7- AAD (7- Aminoactinomycin D) 546, 647; Acridine Orange (+DNA) 500, 526; Acridine Organe (+RNA) 460, 650; Allophycocyanin (APC) 650, 660; Aniline Blue 370, 509; BODIPY® FL 505, 513; CF640R 642, 662; Cy5® 649, 670; Cy5.5® 675, 694; Cy7® 743, 767; DAPI 358, 461; EGFP 489, 508; Fluorescein (FITC) 494, 518; Pacific Blue 410, 455; PE (R-phycoerythrin ) 480 and 565, 575; PE-Cy5 480 and 650, 670; PE-Cy7 480 and 743, 767; Propidium Iodide (PI) 536, 617; and YFP (Yellow Fluorescent Protein) 513, 527. Spectra for fluorochromes can also be found at the following websites: probes.com/servlets/spectra/ and clontech.com/gfp/excitation.shtml as well as many others known to those skilled in the art.

[065] Configurations of nanowells to enhance excitation at specific or multiple plasmonic resonances are well known in the art and comprise using particular geometries, dimensions, materials, refractive indecies or a combination thereof. Examples of these geometries, materials and dimensions can be found in Fermamdez-Garcia, et al., Design Considerations for Near-filed Enhancement in Optical Antennas, Contemporary Physics, 2014, and may include for example rod, ellipsoid, bowtie, disk and square geometries; gold, silver aluminum and copper nanowells; as well as diameters measuring about 40, 30, 20, 10 and 5 nm.

[066] In some embodiments, all nanowells 128 of the system comprise the same configuration. In some embodiments, the configuration is a geometric configuration. In some embodiments, a plurality of nanowells of the system comprise the same configuration. The nanowells of the system may be designed such that each nanowell has a different configuration, such that all nanowells have the same configuration or any combination in between. Thus, a first proportion of the nanowells may have a first confirmation, a second proportion of the nanowells have a second confirmation, a third proportion of the nanowells have a third confirmation and so on for as many types of configurations as are desired.

[067] In some embodiments, metallic layer 120 comprises a plurality of nanowells 128 in a proximity to one another sufficient to generate inter-nanowell plasmonic resonance. In some embodiments, at least 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nanowells are in a proximity to one another sufficient to generate inter-nanowell plasmonic resonance. Each possibility represents a separate embodiment of the invention. In some embodiments, a proximity sufficient to generate inter-nanowell plasmonic resonance is between 10-1000 nm. In some embodiments, the proximity is less than 10000, 9000, 8000, 7000, 6000, 5000, 4000, 3000, 2500, 2000, 1500, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100 or 50 nm. Each possibility represents a separate embodiment of the invention. In some embodiments, the proximity is between 1-5000, 1- 4000, 1-3000, 1-2000, 1-1500, 1-1000, 1-900, 1-800, 1-700, 1-600, 1-500,5-5000, 5- 4000, 5-3000, 5-2000, 5-1500, 5-1000, 5-900, 5-800, 5-700, 5-600, 5-500, 10-5000, 10- 4000, 10-3000, 10-2000, 10-1500, 10-1000, 10-900, 10-800, 10-700, 10-600, 10-500, 20-5000, 20- 4000, 20-3000, 20-2000, 20-1500, 20-1000, 20-900, 20-800, 20-700, 20-600, 20-500, 50-5000, 50- 4000, 50-3000, 50-2000, 50-1500, 50-1000, 50-900, 50-800, 50-700, 50-600, or 50-500 nm. Each possibility represents a separate embodiment of the invention. In some embodiments, enhanced inter-nanowell plasmonic resonance enhances fluorescence of a molecule within at least one nanowell in the plurality of nanowells within the proximity. In some embodiments, fluorescence is enhanced in a plurality of nanowells in the proximity. In some embodiments, fluorescence is enhanced in all nanowells in the proximity. In some embodiments, the enhanced fluorescence is at a particular wavelength or plurality of wavelengths. In some embodiments, the enhancement is single spectrum or multispectral.

[068] In some embodiments, the plurality of nanowells in a proximity are evenly spaced apart. In some embodiments, the plurality of nanowells in a proximity are asymmetrically spaced. In some embodiments, the plurality of nanowells in a proximity are spaced in a geometric shape. Examples of possible geometric shapes include, but are not limited to, a circle, a square, a triangle, a rectangle, an oval, a pentagon, a hexagon, a bowtie, an ellipse, and a line. Further examples, of spatial configuration of a plurality of nanowells can be found in Gopinath, et al., Photonic-Plasmonic Scattering Resonance in Deterministic Aperiodic Structures, Nano Letters, 2008 and Langguth, et al., Plasmonic Band Structure Controls Single-Molecule Fluorescence, ACS Nano, 2013.

[069] In some embodiments, biosensors 100 may further include a chip 130 consisting of four layers. In some embodiments, the chip size of 10-30 mm3 (e.g., 4 mm X 4mm). In the nonlimiting example illustrated in Figs. 1A-1C a first layer 132 of chip 130 may include silicon nitride (SiNx), having between 10 to 80 nm (e.g., 50 nm) thickness. A second layer 134 which includes SiO2, having a thickness of between 400 to 600 nm (e.g., 500 nm), followed by a third layer 136 made from Si having a thickness of between 300 to 400 pm (e.g., 360 pm). A fourth layer 138 is made from SiO2, having a thickness of 400 to 600 nm (e.g., 500 nm).

[070] In some embodiments, a free standing film 110 (e.g., a 20 pm SiNx film) is created on top of layer 138 using lithography. Later, metallic layer 120 (e.g., a 50 nm layer of gold (Au)) is deposited in a donut shape on the free side of the film 110.

[071] Reference is now made to Figures IF and 1G which are illustration of polydimethylsiloxane (PDMS) device comprising the biosensor, enclosed inside a sample chamber accoridng to some embodiments of the invention.

[072] In some embodiments, biosensor 100 may be attached to a PDMS device 140, such that film 110 is splitting PDMS device into two liquid reservoirs, a first liquid reservoir 142 and a second liquid reservoir 144. First liquid reservoir 142 is on the cis side of biosensor 100 and second liquid reservoir 144 is in the trans side of biosensor 100, as illustrated in Figure IF. In some embodiments, PDMS device 140 is also attached to a coverslip 146, for example, the glass coverslip illustrated. In some embodiments, first reservoir 142 is suitable to receive a sample comprising the molecule to be detected. In some embodiments, second reservoir 144 is suitable for the molecule to pass into after detection. In some embodiments, the reservoirs are the same size. In some embodiments, the first reservoir is larger than the second. In some embodiments, the second reservoir is larger than the first. In some embodiments, the second reservoir is attached to a drainage system for emptying the reservoirs. In some embodiments, the first reservoir holds a volume such that the concentration of molecules in reservoir is not too dilute that molecules infrequently contact the nanopore and not too concentrated that there is crowding and/or blockage of the nanopore. In some embodiments, the first reservoir is configured such that the concentration of molecules in the reservoir is between 1 femtomole and 1 micromole.

[073] In some embodiments, the means to induce movement comprises a means of inducing an electrical current from the first reservoir to the second reservoir, as illustrated in Fig. II. In some embodiments, the means to induce movement comprises a negative electrode within the first reservoir and a positive electrode in the second reservoir and wherein the molecule has a negative charge. In some embodiments, the means to induce movement comprises a positive electrode within the first reservoir and a negative electrode in the second reservoir and wherein the molecule has a positive charge. In some embodiments, the molecule is treated with a substance that provides a charge to the molecule before addition to the first reservoir.

[074] In some embodiments, the metallic layer is on the second reservoir side of the membrane. In some embodiments, the metallic layer is on the first reservoir side of the membrane. In some embodiments, the metallic layer is on the first or second reservoir side of the membrane.

[075] In some embodiments, biosensor 100 attached to PDMS 140 may be held in sample chamber 150, between base 150A and lid 150B, as illustrated in Figure IF.

[076] Reference is now made to Figure IE which is an illusration of laser entrace angle to the plasmonic biosensor and a Comsol simulations shows effective coupling of the laser with the plasmonic biosensor accoridng to some embodiments of the invention. Laser beam 10 incident film 110 (e.g., silicon nitride) water interface (from the side of the second reservoir) in the slot at an angle of incidence 2° <ii <84° (considering RI of glass = 1.5 and RI of Buffer= 1.33). This may create an angle of refraction 1.2°< r <37.2° (considering RI of Buffer= 1.33 and SiNx = 2.2) inside the SiNx membrane.

[077] In some embodiments, light may incident on interface between film 110 and layer 138 (e.g., SiNx-SiC interface) at an angle i2=90°-r (88.8 >12 >52.8°), which will always be greater than the critical angle of film 110/layer 138 interface (e.g., SiNx-SiOi interface) (i.e., 39.6°, considering RI of SiNx = 2.2 and RI of SiO2= 1.4). As a result, the light may bounce back and forth inside the film 110 (e.g., SiNx membrane), and the membrane may serve as a waveguide for the beam. The travel path of the laser may illuminate the pore or multiple pores from the inside of the membrane (e.g., film 110).

[078] In some embodiments, the main advantage of our technique is it may couple laser 10 inside film 110 (e.g., thin SiNx membrane), unlike the other techniques, which illuminates pore directly from outside. Simultaneously a metal (Au) layer 120 at the bottom may perform multiple roles. (I) it may completely block the unwanted PL coming from film 110 (e.g., SiNx membrane) illumination, (ii) It may serve as a flat mirror to strongly reflect and guide the laser in the membrane, and (iii) Since gold is in close proximity to the pore, it may create a hotspot and play a role in the plasmonic enhancement of the passing fluorophore-labeled single analyte molecules. This may significantly reduce the background level and increase SNR. It is the first kind of attempt to couple laser in a 50 nm membrane.

[079] In a nonlimiting example, optically flat layers of SiNx and Au may be created for the process. In this example, the laser will be coupled to the waveguide “off the center” and at an angle that will remove the possibility of a background. This chip may efficiently fit inside a developed disposable microfluidic PDMS device suitable for simultaneous electro-optical measurements.

[080] For the nonlimiting example, an optical simulation was performed to prove the concept by designing the same device with mentioned refractive index profile of the materials, showing the successful coupling of a 560 nm laser (10 pm diameter) beam to a 50 nm thick SiNx membrane through a coupling slot with a range of incident angles.

[081] Reference is now made to Figure 1H which is an illustration of a system for detecting fluorescence from a molecule accoridng to some embodiments of the invention. A system 1000 may include biosensor 100 attached to PDMS 140 and held inside sample chamber 150. System 1000 may further include a light source 200 capable of exciting the molecule to emit fluorescence. In some embodiments, light source 200 produces laser beam 10 which is coupled to laser coupling slot 112.

[082] In some embodiments, light source 200 emit laser beam 10 into slot 115.

[083] In some embodiments, the light source produces coherent light. In some embodiments, the light source produces collimated light. In some embodiments, the light source produces coherent and collimated light. In some embodiments, the light source produces a coherent and collimated light beam. In some embodiments, the light source is a laser or light emitting diode (LED). In some embodiments, the light source is a laser. In some embodiments, the light source is a monochromatic light source. In some embodiments, the light source produces red light. In some embodiments, the light source produces light having a wavelength between 640 and 650 nm.

[084] In some embodiments, the power of the light source is at most 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 25, 20, 15, 10 or 5 microwatts (pW). Each possibility represents a separate embodiment of the invention. In some embodiments, the power of the light source is at most 10 pW. A person skilled in the art will appreciate that since the system of the invention increases the fluorescence of the molecule light with a lower power may be used. This may result in decreased background.

[085] In some embodiments, the system further comprises a quencher, not illustrated. In some embodiments, the quencher is a dye. In some embodiments, the quencher is at a plasmonic hotspot of a nanowell. In some embodiments, the quencher is outside of the plasmonic hotspot. In some embodiments, the quencher reduces background fluorescence. In some embodiments, the quencher reduces fluorescence outside of the plasmonic hotspot. In some embodiments, the molecule to be analyzed comprises the quencher. In some embodiments, the quencher is proximal to the fluorescent moiety on the molecule. In some embodiments, the quencher increases quantum efficiency. Quenchers in plasmonics are well known in the art. An example of a possible quencher includes but is not limited to methyl viologen, and further examples of quenchers can be found in Wenger et al., Fluorescence enhancement factors on optical antennas: enlarging the experimental values without changing the antenna design, Internal. J. of Optics, 2012, herein incorporated by reference.

[086] In some embodiments, system 1000 may further include a first detector 300 configured to detect the fluorescence emitted by said molecule. In some embodiments, first detector 300 is an active pixel sensor (APS). According to some embodiments, the APS is a complementary metal-oxide semiconductor (CMOS) sensor. According to some embodiments, first detector 300 is a charge coupled device (CCD) detector. According to some embodiments, first detector 300 is an Avalanche Photo Diode detector.

[087] In some embodiments, the first detector is configured to detect the fluorescence from at least 2 molecules in at least 2 nanowell-nanopores. In some embodiments, the first detector is a fluorometer. In some embodiments, the first detector is a photo detector. In some embodiments, the first detector is an Avalanche Photo Diode detector. In some embodiments, the first detector is configured to also detect ion current flow through the nanopore.

[088] In some embodiments, the systems of the invention further comprise a second detector 400. In some embodiments, the second detector is configured to detect ion current flow through the nanopore. In some embodiments, the second detector is configured to convert the ion current through the nanopore to a measurable electric current. In some embodiments, the second detector is a high-gain current amplifier. In some embodiments, the means to induce movement comprises a first electrode with the first reservoir and a second electrode with the second reservoir and the high current amplifier is connect to the first and second electrodes. In some embodiments, the first and second detectors are synchronized. In some embodiments, one of the detectors and/or both detectors are configured to coordinate a fluorescent signal with an electrical signal.

[089] In some embodiments, the detecting comprises sub-millisecond (ms) resolution. In some embodiments, the detection comprises a high signal to noise ratio. In some embodiments, the detecting comprises the detecting only fluorescence that occurs in close temporal proximity to a change in ion current flow through the nanopore. In some embodiments, the fluorescence and change in ion current flow occur within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 or 50 ms of each other. Each possibility represents a separate embodiment of the invention. In some embodiments, the fluorescence and change in ion current flow occur within 20 ms of each other. In some embodiments, the fluorescence and change in ion current flow occur simultaneously.

Methods of use

[090] By another aspect, there is provided a method for detecting fluorescence from a single molecule, the method comprising: a. introducing the molecule into the first reservoir of any of the systems of the invention; b. inducing the molecule to move from the first reservoir to the second reservoir via the nanopore; c. exciting the molecule within the nanopore to emit fluorescence; and d. detecting the fluorescence emitted by the molecule; thereby detecting fluorescence from a single molecule.

[091] By another aspect, there is provided of sequencing a single molecule, the method comprising: a. introducing the molecule into the first reservoir of any of the systems of the invention; b. inducing the molecule to move from the first reservoir to the second reservoir via the nanopore; c. exciting the molecule within the nanopore to emit fluorescence; d. detecting the fluorescence emitted by the molecule; and e. assigning to each fluorescence emitted by the molecule an identity; thereby sequencing a single molecule.

[092] In some embodiments, the first reservoir contains a solution suitable for receiving the molecule. In some embodiments, the molecule is dissolved in a solution. In some embodiments, the dissolving solution and the reservoir solution are the same solution. In some embodiments, the dissolving solution and the reservoir solution are different solutions. In some embodiments, the solution is configured to keep the molecule in a linear state. In some embodiments, the solution is configured to retard and/or reduce the formation of secondary structure in the molecule. In some embodiments, the solution is ionic. In some embodiments, the solution is Tris-EDTA (TE) buffer. TE buffer is well known in the art, and any standard TE buffer may be used. In some embodiments, the reservoir solution is a salt solution. In some embodiments, the reservoir solution is saline. In some embodiments, the reservoir solution comprises potassium chloride.

[093] In some embodiments, the method of sequencing a molecule comprises any of the methods of detecting fluorescence of the invention and further comprises assigning an identity to each detected fluoresce. In some embodiments, the molecule is a nucleic acid and the identity is a nucleic acid base. In some embodiments, the identity is a naturally occurring base. In some embodiments, the identity is an artificial base. In some embodiments, the molecule is DNA and the identity is one of adenine, cytosine, guanine and thymine. In some embodiments, the molecule is RNA and the identity is one of adenine, cytosine, guanine and uracil. In some embodiments, the molecule is a polypeptide and the identity is an amino acid. In some embodiments, the identity is a naturally occurring amino acid. In some embodiments, the identity is an artificial amino acid. In some embodiments, the identity is one of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[094] As used herein, the term "about" when combined with a value refers to plus and minus 10% of the reference value. For example, a length of about 1000 nanometers (nm) refers to a length of 1000 nm+- 100 nm.

[095] It is noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polynucleotide" includes a plurality of such polynucleotides and reference to "the polypeptide" includes reference to one or more polypeptides and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements or use of a "negative" limitation.

[096] In those instances where a convention analogous to "at least one of A, B, and C, etc." is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B." [097] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[098] Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

[099] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

Material and Methods

Wafer-scale fabrication of PNW-NP and STD devices

[0100] The wafer-scale fabrication process of PNW-NP devices is presented schematically in Figure 2A and can be broken down into three main processes. These are: (I) Fabrication of gold nanowell array, (II) Fabrication of freestanding SiNx membrane, and (III) nanopore drilling.

[0101] (I) Fabrication of gold nanowell array was carried out using 100 mm diameter, < 100> crystal orientation, 350 um thick silicon wafers as the underlying substrate (Virginia Semiconductor). The wafers were coated with 25 nm low-stress silicon nitride (SiNx) from both sides using Low Pressure Chemical Vapor Deposition (LPCVD- Tystar). Before any use, the wafers were cleaned in acid and base baths to remove any organic contamination, followed by oxygen plasma for 2 min at 100 W (Plasma-Therm) to remove any remaining moisture on the surface (Fig. 2A, step 1). Following the cleaning process, a 300 nm layer of Ma-N 2403 negative-tone electron beam lithography resist (Micro Resist Technology) was

Z1 spun on the wafer at 3000 rpm for 45 sec and baked for 1 min at 90 °C on a hot plate. A pattern of nanowell arrays and alignment marks were defined on the resist using electronbeam lithography system (Raith 150) at 30kV accelerating voltage (Fig. 2A, step 2). The nanowells were exposed as single dots and their sizes were defined by the total dose irradiated onto the resist. After a post-exposure baking at 90 °C for 2 min, the patterns were developed in Ma-D 525 developer (Micro Resist Technology) for 45 seconds, leaving behind arrays of cylindrical columns “pillars” on the surface of the wafer, as illustrated in Figure 2A, step 3. Then a thin chromium film of 5 nm thick followed by a gold film of 130 nm thick were deposited on the resist pattern by e-beam evaporation system (CHA). Finally, the resist pillars were lifted-off and dissolved away in an 1165 resist strip bath, leaving behind the imprint of the pillars and forming nanowell arrays in the gold film (Fig. 2A, step 4).

[0102] (II) For the fabrication of freestanding SiNx membranes, a standard UV photolithography was used to pattern squares and dice lines openings on the other side of the wafer (Fig. 2A, step 5), through which the nitride was etched using reactive ion etch of CHF3 and 02 plasma (Fig. 2A, step 7). The squares were aligned to the pre-fabricated nanowell arrays using a mask aligner (Karl Suss MA6). Finally, the photoresist was stripped, and an anisotropic KOH etch resulted in ~250 chips, each with a ~30 pm * 30 pm freestanding SiNx membrane, supports the gold nanowell array (Fig. 2A, step 8).

[0103] (III) For nanopore drilling, the 25 nm thick SiNx membranes were thinned down to improve signal-to-noise ratio by controlled buffered oxide etch (BOE), leaving a sub 10 nm thick free-standing membrane in the nanowell base (Fig. 2A, step 10), where pores were later fabricated. Nanopores were drilled using a high-resolution aberration-corrected TEM (Titan 80-300 FEG-S/TEM, FEI) or a noncorrected TEM (JEOL 2010F).

Wafer-scale fabrication of STD devices

[0104] For the control experiments, non-metallic nanopore (STD) devices were fabricated. Briefly, 50 nm thick SiNx membranes were locally thinned down to improve the electrical and optical signal-to-noise ratio by controlled reactive ion etching (RIE) in ~1.5 pm diameter circular regions patterned by full-wafer optical lithography, leaving a sub 10 nm thick wells in which pores were later fabricated using a transmission electron microscope. The wafer fabrication process of the STD devices is illustrated schematically in Figure 2B.

Experimental setup [0105] Figure 1H displays schematically our optical-electro measurement system. For the synchronous optical and electrical measurements, the nanochip is mounted on a closed-loop XYZ piezo nanopositioner (Physik Instrumente, P 561.3) with sub-nm accuracy on top of a high-NA objective (Olympus Plan Apochromat 60x/1.2) in a custom built confocal setup. 3 solid state lasers were used, 720 nm, 640 nm and 560 nm solid state lasers for the excitation, and the intensity is adjusted using natural density (ND) filter wheel (Thorlabs FW212CNEB). The laser beam is expanded to completely fill the back aperture of the objective using a custom-made telescope. Emitted light is collected by the same objective and filtered using the appropriate long pass and notch filters (Semrock) and then focused using a single 20 cm focal length lens onto either a EMCCD camera (ANDOR, iXon 887) or to a 50 pm pinhole (Thorlabs) in confocal mode. Light passing through the pinhole is collimated using a 10 cm lens and focused using additional 2.5 cm focal length achromatic doublet lense onto APD (Perkin Elmer SPCM-AQR-14). Back reflection was continuously measured using a photodiode (Thorlabs) to monitor and correct stage or sample drift during the experiment. All lenses were obtained from Thorlabs. The ion current is synchronously measured using two Ag/AgCl electrodes connected to an Axon Axopatch 200B patch-clamp and filtered at 10 KHz. The entire apparatus was shielded from external electromagnetic noise by a Faraday cage. For data acquisition we used National Instruments NI-6211 DAQs for analog signals (sampled at 125 KHz) and NI-6602 for photon counting (sampled at 500 KHz). The two cards were triggered and synchronized via a common hardware connection and were fully controlled by a custom Lab VIEW (National Instrument) program.

DNA labeling and sample preparation

[0106] For DNA labeling, the M.TaqI was used to attach a red fluorophore CF640R (Biotium, CA, USA) with excitation and emission peaks at 642 nm and 662 nm, to adenine residue in a four base pair sequence TCGA. Labeling is achieved in a single step by feeding the enzyme with a synthetic cofactor containing a fluorophore at the transfer position. A total of 2.5 pg of 5 kbp DNA containing 7 M.TaqI sites (No Limit, Thermo Scientific), was treated with 2.5 pg of M.TaqI and 40 pM of AdoYnCF640R in labeling buffer (20 mM Tris/HOAc, 50 mM KOAc, pH 7.9, 10 mM MgOAc₂, 1 mM DTT, 0.01 % by volume Triton X-100, 100 pg/ml BSA) in a total reaction volume of 25 pl at 65°C for 2 h. The labeled DNA was then reacted with 40 pg of protein kinase K (Thermo Scientific) at 45°C for 1 hr to disassemble protein and DNA aggregates. The reaction was cleaned by ethanol precipitation: 62.5 pl of cold absolute ethanol and 9.6 pl of sodium acetate 3M were added to the reaction, the mixture was incubated for 12 hours at -20°C followed by centrifugation at 20k RCF for 1 hr at 4°C. The pellet was washed 5 times in 70% absolute ethanol, vacuum dried and dissolved in TE buffer for UV-Vis absorption quantification and nanopore translocation experiments.

Numerical Simulation Methods

[0107] A finite-difference time-domain (FDTD) method was used for the numerical simulations in commercial software, FDTD Solutions, from Lumerical Solutions (Vancouver, Canada). The refractive index of water and SiN_x was taken as 1.33 and 2.1, respectively. The optical constants of gold are known in the art. A uniform mesh size of 1 nm was used to resolve field enhancement of nanometer- sized structures in the simulations. Depending on the symmetry of the simulated structures, we applied anti-symmetric or symmetric boundary conditions to further reduce the simulation times; otherwise, perfectly matched layer (PML) boundaries were used. A plane wave with amplitude of 1 V/m and wavelength range from 400 to 800 nm was used to illuminate the structure by a total-field- total-scattering source. Polarization of the plane wave was perpendicular to the long axis of the NW. A fluorophore was modeled as a classic dipole in the simulation. The near field was recorded with a power monitor. Decay rate and quantum efficiency were calculated by power flow into box monitors surrounding the dipole and PNW-NW structures, by taking the mean of three independent simulations results, wherein a single dipole at the center of the PNW was polarized along the x-, y-, and z-axes respectively.

[0108] The fluorescence rate y_em of a single molecule can be expressed as a product of excitation rate y_eXc and quantum yield q.^' ^8]. The fluorescent enhancement is then

Yem/yem⁰ = Yexc/yexc⁰ q/q°, (Equation 1) where superscript ‘o’ indicates the corresponding free-space quantity, in absence of the PNW. Below saturation, the quantum mechanical decay rate in an inhomogeneous environment is related to the classical power radiated by a dipole in the same environment as y/y° = P/P°, (Equation 2) and the quantum efficiency of an imperfect emitter in a homogenous environment is q° = Yr° / ( ° + Ynr⁰), (Equation 3) where y_r and y_m are the radiative and intrinsic non-radiative decay rates. The presence of the PNW structure introduces an additional decay rate yioss due to metallic absorption, and Q Yr (Y^r "I" Y"' ⁺ Yloss), (Equation 4) assuming intrinsic decay rate is unchanged by environment, y_nr = Ynr°, we can use (Equation 3) and (Equation 4) to express the quantum efficiency enhancement (Equation 1) as q = Pr/Pr° / [ (l-q°) + q°(Pr/Pr° + Pioss/Pr°), (Equation 5)

[0109] The PNW-NP’s excitation enhancement is localized to the illuminated gold nanowell. The Au nanowell rapidly attenuates light beyond the aperture, and the planar metallic layers entirely block light from reaching the non-illuminated cis side. The excitation enhancement at the center of aperture is 3.6. Enhancement at this location in our current device of 120 nm diameter NW is near-optimal.

[0110] The CF640R (ex./em. 642/662nm) fluorophore is assumed to have an intrinsic quantum efficiency of q°=0.3. Taking this value and results from power flow through the box monitors placed at the center of the PNW into (Equation 5) give a quantum efficiency enhancement at 662 nm of 1.6. It should be noted that if q° is reduced with a quencher the eta of the nanowell is increased according to equation 1. This increase in quantum efficiency of the fluorophore (q/q°) is achieved without reducing the excitation enhancement which improves the relative fluorescence enhancement of the nano well.

[0111] Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

Claims

CLAIMS:

1. A plasmonic biosensor comprising: a. an ion impermeable film comprising at least one ion-conducting nanopore and a laser coupling slot; and b. a metallic layer adhered to said film by an adhesion layer, said metallic layer comprising a nanowell structure located adjacent to said at least one nanopore and to said laser coupling slot.

2. The biosensor of claim 1, wherein said film is a silicon-based membrane.

3. The biosensor of claim 2, wherein said membrane is a silicon nitride (SiNx) membrane.

4. The biosensor of any one of claims 1 to 3, wherein said film has a thickness of less than 50 nanometers (nm).

5. The biosensor of any one of claims 1 to 4, wherein said nanopore comprises a diameter not greater than 5 nm.

6. The biosensor of any one of claims 1 to 5, wherein said film comprises at least 2 nanopores and said nanopores are separated by at least 1 micrometers (pm).

7. The biosensor of any one of claims 1 to 5, wherein said laser coupling slot has a width of 10 pm to 200 pm.

8. The biosensor of claim 7, wherein the slot has a height of 50 nm to 500 nm.

9. The biosensor of any one of claims 1 to 8 wherein said metal is selected from gold, silver, copper and aluminum.

10. The biosensor of any one of claims 1 to 9 wherein said metallic layer comprises a thickness of between 100 and 150 nm.

11. The biosensor of any one of claims 1 to 10, wherein said adhesion layer comprises at least one of: a. a metal oxide; b. chromium, chromium oxide, titanium or titanium oxide; and c. a thickness of between 1 and 20 nm. The biosensor of any one of claims 1 to 11, wherein said nanowell comprises a diameter between 30 and 150 nm. The biosensor of any one of claims 1 to 12, wherein said metallic layer and said adhesion layer comprise a thickness sufficient to block at least 50% of light shown thereupon. A system for detecting fluorescence from a molecule, the system comprising: a. a biosensor of any one of claims 1 to 13; b. a first and a second liquid reservoir separated by said film; c. a means to induce movement of said molecule from said first reservoir to said second reservoir via the nanopore; d. a light source capable of exciting said molecule to emit fluorescence, wherein said light source is configured to emit a laser beam coupled to said laser coupling slot; and e. a first detector configured to detect said fluorescence emitted by said molecule. The system of claim 14, wherein the diameter of the nanowell is not greater than half the wavelength of said light emitted by said light source. The system of claim 14 or 15, wherein said molecule comprises at least one fluorescent moiety, said molecule is a nucleic acid molecule or a polypeptide, or both. The system of claim 16, wherein said nucleic acid molecule is any one of singlestranded DNA, double-stranded DNA, RNA, and cDNA. The system of any one of claims 14 to 17, wherein said means to induce movement comprises a negative electrode within said first reservoir, and a positive electrode within said second reservoir and said molecule has a negative charge. The system of any one of claims 14 to 18, wherein a power of said light source is at most 10 microwatts (pW). The system of any one of claims 14 to 19, wherein said metallic layer is on the second reservoir- side of said membrane. The system of any one of claims 14 to 20, wherein said detecting comprises submillisecond (ms) resolution. The system of any one of claims 14 to 21, wherein said first detector is selected from an active pixel sensor (APS), a charge coupled device (CCD) detector and an Avalanche Photo Diode detector. The system of any one of claims 14 to 22, further comprising a second detector configured to detect ion current flow through said nanopore. The system of claim 23, wherein said second detector is configured to convert said ion current through said nanopore to a measurable electrical current, is a high-gain current amplifier or both. The system of claim 23 or 24, wherein said means to induce movement comprises a first electrode within said first reservoir, and a second electrode within said second reservoir, and said high current amplifier is connected to said first and second electrodes. The system of any one of claims 23 to 24, wherein said first and said second detector are synchronized. The system of any one of claims 14 to 25, for use in sequencing said molecule. A method of detecting fluorescence from a single molecule, the method comprising: a. introducing said molecule into said first reservoir of the system of any one of claims 14 to 27; b. inducing said molecule to move from said first reservoir to said second reservoir via said nanopore; c. exciting said molecule within said nanopore to emit fluorescence; and d. detecting said fluorescence emitted by said molecule; thereby detecting fluorescence from a single molecule. The method of claim 28, wherein said detecting comprises sub-millisecond (ms) resolution. The method of any one of claims 28 to 29, wherein said nanowell enhances fluorescence from said molecule by at least 5 -fold. The method of any one of claims 28 to 30, wherein said system further detects ion current flow through said nanopore and wherein only an event detected simultaneously by fluorescence and electricity is considered detecting fluorescence from said molecule. A method of sequencing a molecule, comprising the method of any one of claims 28 to 31 and further comprising assigning an identity to each detected fluorescence. The method of claim 32, wherein said identity is a nucleic acid base or an amino acid.